From the imagined vantage point of the year 2020, the author recounts the problems and deficiencies of health care in the 1990s and describes how academic medicine's leaders successfully confronted them. A key part of their strategy was to work together to form a coordinated network of medical schools, teaching hospitals, and academically oriented health systems, along with their staffs and a variety of community-based partners. In this way, they set a national agenda, pursued common goals, freely shared information and best practices, and cooperated to optimize their effectiveness in education, research, and clinical care. A major outcome of this new network was the Collaborative Care model of health care, based on the premise that a basic purpose of the health care system is to achieve measurable improvements in the health of individuals and communities in ways that are cost-conscious, quality-driven, evidence-based, and patient-, family-, and community-oriented. Academic institutions formed strong partnerships with many stakeholders (e.g., purchasers of health care services) to make the Collaborative Care approach work. In addition, there were several other important keys to Collaborative Care's success, such as the full integration of clinical research with clinical care and the restoration of trust in the health care enterprise. The author returns to reality and the 1990s. He challenges academic medicine to pursue the Collaborative Care vision, saying that "we should not accept without challenge what we know to be abominable just because it appears to be inevitable.... Our choice is to continue to struggle for survival as the environment around us gets harsher and harsher ... or to fix the environment" using the power of collaboration to unleash academic medicine's unlimited creativity and wisdom.
In 1990, a collaborative project was launched to determine what the people of Ontario expect of their physicians and how the programs that prepare future physicians should be changed in response. The project, called Educating Future Physicians for Ontario (EFPO), brought together the five Ontario medical schools, the Council of Ontario Faculties of Medicine (COFM); a nonprofit, charitable organization, Associated Medical Services (AMS); and the Ontario Ministry of Health. The first phase ran for five years and was described in the November 1998 issue of Academic Medicine. After an external review, the project was continued for a second phase (EFPO II) for four more years until December 1998; that second phase is the topic of this article. EFPO II (1) focused more on residents' education; (2) emphasized four of the EFPO I-created physician roles in project activities; (3) maintained the province-wide, inter-institutional medical education framework of phase I, but fostered greater involvement of the seven sites (five medical schools and two regional health centers) in project activities; (4) stressed five project components (e.g., needs assessment and community partnerships) and worked for collaboration among components at all sites; (5) enhanced the original EFPO I Fellowship Program by adding residents and community fellows to the existing fellowships and by initiating leadership development activities, all of which bode well for the future leadership of medical education in Ontario. Students and residents played a vital role in EPFO II. Most of EFPO II's objectives were met, but the overall view of external reviewers was that the project was less successful than EFPO I. For example, the impact on clinical education, especially residency education, was less than anticipated. On the other hand, the project helped encourage the wide adoption of the eight physician roles that originated in EFPO I and advanced faculty development and assessment activities based on these roles. A third phase of EFPO concerning continuing medical education was planned, but support was not available. However, one of the funders will continue to support the successful fellowship and leadership program and the provincial education network for the next three years. Overall, the two phases of EFPO substantially modified medical education in Ontario to make it more responsive to evolving social needs.
The ethics of caring, though the subject of much recent discussion by philosophers, has hardly been applied to medical ethics and medical education. Based on receptivity (that is, empathy and compassion) toward and taking responsibility for other persons, the ethics of caring has particular relevance to medicine. Caring guides the physician always to remain the patient's advocate and to maintain the therapeutic relationship when dealing with and resolving ethical dilemmas. This article discusses the philosophy behind the ethics of caring and then explores three issues that arise within its context: receptivity, taking responsibility, and creating an educational environment that fosters caring.
Physician leaders are crucial as never before to ensure the proper integration of good care and cost containment; such integration is both a reasonable expectation of patients and essential for the survival of health care delivery systems. In today's health care environment, a critical mass of physician leaders must be developed in a systematic fashion so that physicians may truly lead the health care enterprise. The authors (1) describe, with examples, the various types and levels of physician leadership training programs currently being offered; (2) explain the costs and benefits of each program type; and (3) offer a program rationale and model (using a program at their medical school), which they analyze using traditional management concepts such as strategic planning, net present value, and make-versus-buy. The authors emphasize that physician leadership training should be local, offer long-term instruction, and be led by physicians. They conclude by stating that the concept of physician leadership will not and should not be taken seriously by non-physician health care executives until the physician community becomes as serious about leadership and management training as it is about clinical training.
Though few question the importance of incorporating professionalism and humanism in the training of physicians, traditional residency programs have given little direct attention to the processes by which professional and humanistic values, attitudes, and behaviors are cultivated. The authors discuss the underlying philosophy of their primary care internal medicine residency program, in which the development of professionalism and humanism is an explicit educational goal. They also describe the specific components of the program designed to create a learner-centered environment that supports the acquisition of professional values; these components include a communication-skills training program, challenging-case conferences, home visits with patients, a resident support group, and a mentoring program. The successful ten-year history of the program shows how a residency program can enable its trainees to develop not only the requisite excellent diagnostic and technical tools and skills but also the humane and professional attributes of the fully competent physician.
PURPOSE: Despite efforts to increase the numbers of underrepresented minorities (URMs), only 3.9% of medical school faculty are URMs. The authors compared the specialty choices, compensation, and career satisfaction of minority faculty with those of their majority counterparts to determine whether there were differences that might affect the recruitment and retention of minority faculty. METHOD: In 1995, the authors mailed a self-administered survey to a stratified random sample of 3,013 eligible full-time salaried faculty in 24 randomly selected medical schools. Those schools, which had at least 200 faculty, did not include the Puerto Rican or historically black medical schools. RESULTS: Of the eligible faculty surveyed, 1,807 (60%) responded; 1,463 were majority faculty, 195 were URM faculty, and 149 were other-minority faculty. Similar proportions of the three groups were in the primary care specialties. Only 11% of the URM respondents were in basic science departments. There was no significant difference in adjusted mean compensation between majority, URM, and other-minority faculty. However, URM faculty were significantly less satisfied with their careers (adjusted scores: 60 versus > 65; p = .001) and more often considered leaving academic medicine within five years (58% versus < 45%). CONCLUSION: Given the demographic changes of the U.S. population, these issues should be addressed by deans and department heads in order to enhance recruitment and facilitate retention of URM faculty in academic medicine.
PURPOSE: Instruments that rate teaching effectiveness provide both positive and negative feedback to clinician-educators, helping them improve their teaching. The authors developed the Clinical Teaching Effectiveness Instrument, which was theory-based and generic across their entire academic medical center, The Cleveland Clinic Foundation. They tested it for reliability, validity, and usability. METHOD: In 1997, using an iterative qualitative development process involving key stakeholders, the authors developed an institution-wide instrument to routinely evaluate clinical faculty. The resulting instrument has 15 questions that use a five-point evaluation scale. The instrument, which was administered to medical students, residents, and fellows over a 20-month period, produced data that were rigorously tested for instrument characteristics, reliability, criterion-related and content validity, and usability. RESULTS: This instrument, implemented in all departments across the institution, produced data on a total of 711 clinician-educators. Correlation coefficients among the items were high (.57 to .77). The scores were reliable (g coefficient of 0.935), and the instrument had both content and criterion-related validity. CONCLUSIONS: The Cleveland Clinic's Clinical Teaching Effectiveness Instrument is reliable and valid, as well as usable. It can be used as an evaluation tool for a wide variety of clinical teaching settings.
PURPOSE: To compare the performances of three evaluation methods in detecting deficiencies of professionalism among third-year medical students during their ambulatory care and inpatient ward rotations of a core internal medicine clerkship. METHOD: From 1994 to 1997, 18 students at The Uniformed Services University of the Health Sciences failed to satisfactorily complete their core 12-week third-year internal medicine clerkship due to deficiencies in professionalism. Three evaluation methods had been used to assess all students' professionalism during the two rotations of their clerkship: standard checklists, written comments, and comments from formal evaluation sessions. Using qualitative methods and the information obtained by the three evaluation methods, the authors abstracted the record of each student concerning his or her clerkship behavior in terms of the six domains of professionalism used on the standard checklist. A detection index, which is the percentage of all instructors' less-than-acceptable ratings of a student across the six professionalism domains, was calculated for each evaluation method for each of the two clerkship settings. RESULTS: For each evaluation method, deficiencies in professionalism were twice as likely to be identified during the ward rotation as during the ambulatory care rotation (p < .002 for all). Formal evaluation session comments had the highest detection index in both clinical settings. Although the numbers of written and formal evaluation session comments per evaluator and per cited professionalism domain were similar, nearly a fourth of the instructors made identifying comments at the evaluation sessions only. CONCLUSION: In the clerkship studied, deficiencies in professionalism of such magnitude as to require remediation were more likely to be identified in the inpatient than in the ambulatory care setting. Of the three evaluation methods studied, the face-to-face, formal evaluation sessions significantly improved the detection of unprofessional behavior in both clerkship settings. Further efforts at such an interactive evaluation process with ambulatory care clerkship instructors may be essential for improving the identification of unprofessional behavior in that setting.
PURPOSE: Noticing that moderately to severely incompetent physicians (as measured by a standardized assessment of physician competence) did not improve after traditional remedial continuing medical education (CME), the authors investigated the effects of a polyvalent, intensive, prolonged educational intervention on five physicians' competence. METHOD: The five physicians participated in a CME program that lasted three years and consisted of individualized review, ongoing small-group and evidence-based discussions, simulated patients and role playing, formal chart review, and peer review. At the end of the program, the physicians were reassessed. RESULTS: Only one physician improved; another remained the same, and three deteriorated. CONCLUSION: Successful remediation of severely incompetent physicians is uncertain at best, even with prolonged, intensive CME that incorporates modalities thought to be effective in changing physicians' behaviors. Alternative educational techniques may need to be developed for this select population. Conversely, there may be reasons that preclude improvement even with optimal techniques.
PURPOSE: Remediation of some incompetent physicians has proven difficult or impossible. The authors sought to determine whether physicians with impaired competency had neuropsychological impairment sufficient to explain their incompetence and their failure to improve with remedial continuing medical education (CME). METHOD: During a one-year period, 1996-97, all 27 participants in the Physician Review Program (PREP) conducted at McMaster University, a physician competency assessment program, undertook a detailed neuropsychological screening battery. RESULTS: Nearly all physicians assessed as competent also performed well on the neuropsychological testing. However, a significant number (about one third) of the physicians who performed poorly on the competency assessment had neuropsychological impairments sufficient to explain their poor performances. The difficulties were more marked in elderly physicians. CONCLUSION: A significant minority of incompetent physicians have cognitive impairments sufficient to explain both their incompetence and, probably, their failure to improve with remedial CME. Testing physicians for these impairments is important: to detect and treat reversible conditions, to manage irreversible conditions that preclude successful educational intervention, and to facilitate compensation in this instance. Serious consideration should be given to the incorporation of neuropsychological screening in all intensive physician review programs.
Medical diagnosis is a categorization task that allows physicians to make predictions about features of clinical situations and to determine appropriate course of action. The script concept, which first arose in cognitive psychology, provides a theoretical framework to explain how medical diagnostic knowledge can be structured for diagnostic problem solving. The main characteristics of the script concept are pre-stored knowledge, values acceptable or not acceptable for each illness attribute, and default values. Scripts are networks of knowledge adapted to goals of clinical tasks. The authors describe how scripts are used in diagnostic tasks, how the script concept fits within the clinical reasoning literature, how it contrasts with competing theories of clinical reasoning, how educators can help students build and refine scripts, and how scripts can be used to assess clinical competence.
When they became aware that many of their internal medicine residents were not being routinely exposed to a representative range of medical illnesses in pregnancy, the authors set out to develop and implement a brief practical curriculum on the medical problems of pregnancy. They began with a retrospective chart review of 562 consultations with pregnant women and used their findings to develop nine 15-minute lectures that covered a majority of the concepts essential to the care of the medically compromised pregnant woman. Topics included hypertension in pregnancy, the febrile pregnant woman, and renal disease in pregnancy. The authors also created a learner handout, a teaching script, teaching cases, and a bibliography for each lecture. Residents have responded well to the curriculum, and their mean pre- and posttext scores have shown that the lectures improved their knowledge of obstetric medicine. This brief-lecture format may be adapted to other special topics in residency training and readily integrated into already-crowded training schedules.
The integrative sciences, which are of great practical and conceptual interest, investigate complex systems (cells, multicellular organisms, families, institutions, communities, nations, etc.) at the higher levels of organization. Academic medical centers are intrinsically integrative because of their mission and thus are likely places for integrative study to flourish. They also possess vast resources of the kind needed to implement integrative studies. They can thus begin to address, from a scholarly point of view, a variety of integrative issues: how myriad parts (molecules, cells, organisms) form stable, complex, living wholes; the dynamics of health, illness, healing, dying, and death; problems of integration relating to patient care, health care delivery systems, and medical education. If academic medical centers undertake this mission they can be springboards for scholarly advances that will potentially affect all areas of thought. The authors describe how an integrative studies program can be started, and share the experiences of the University of Kentucky's Office of Integrative Studies.
Increasingly, medical educators are looking for ways to train residents and medical students in outpatient medicine. One novel idea, outpatient morning report, draws upon the concept of inpatient morning report and applies a similar conference format to the outpatient setting. The authors describe outpatient morning report and comment on its successful use in their institution.
A national panel on medical education was appointed as a component of the AAMC's Mission-based Management Program and charged with developing a metrics system for measuring medical school faculty effort and contributions to a school's education mission. The panel first defined important variables to be considered in creating such a system: the education programs in which medical school faculty participate; the categories of education work that may be performed in each program (teaching, development of education products, administration and service, and scholarship in education); and the array of specific education activities that faculty could perform in each of these work areas. The panel based the system on a relative value scale, since this approach does not equate faculty performance solely to the time expended by a faculty member in pursuit of a specific activity. Also, a four-step process to create relative value units (RVUs) for education activities was developed. This process incorporates quantitative and qualitative measures of faculty activity and also can measure and value the distribution of faculty effort relative to a school's education mission. When adapted to the education mission and culture of an individual school, the proposed metrics system can provide critical information that will assist the school's leadership in evaluating and rewarding faculty performance in education and will support a mission-based management strategy in the school.
Many lawmakers have embraced the idea of bringing more competition to the Medicare program as a way to achieve greater cost efficiency and provide more choice for beneficiaries. Advocates of this strategy believe Medicare should move away from its historical administrative pricing approach toward a competitive bidding process similar to those used by many private purchasers. Yet, despite support for the concept, every effort to test it in the marketplace has been met with strong opposition. Most recently, competitive bidding demonstration projects set to take place in Phoenix and Kansas City have been delayed by congressional action. Nevertheless, it is clear that those participating in the demonstration projects-notably private purchasers, consumers, health plans, and the Health Care Financing Administration-have already learned a great deal about the types of issues that must be considered if Medicare is truly to pay health plans in a competitive manner. This article explores the lessons learned so far in Phoenix and Kansas City, including design considerations such as plan participation, the standard benefit package, the bidding process, and the government contribution to premiums. It also examines the reasons for opposition to the project and their implications for broader Medicare reform efforts.
Evidence-based medicine (EBM) is an important new paradigm of the medical profession. While the quantitative approach of EBM has its place, clinical medicine must take into account many subtleties that EBM fails to consider. In this article, the authors describe three caveats to this quantitative approach: (1) the detection of "maybe disease" (physiologic, anatomic, or histologic abnormalities that may not ever be overtly expressed in the patient's lifetime) inflates apparent diagnostic test performance; (2) probability revision is valuable primarily as an exercise to gain qualitative insights; and (3) patients are likely to be interested more than just central tendencies in making treatment decisions. They then consider some challenging questions facing clinician-educators: how do they prepare students for situations where there is an absence of rigorous evidence? Should they teach students that the burden of proof lies in demonstrating efficacy or in demonstrating ineffectiveness? And what should they tell students about when to seek evidence to aid diagnostic and treatment decisions?
The author recounts an incident of cheating by two first-year medical students, and how it was handled. One of the students, George, had waited until the last minute to write what he called a "stupid" paper that was required as the final examination in a health policy course. His classmate Ellen offered to write the paper for him, and other students also offered to help; no one pointed out that this would be unethical. After some hesitation, George was persuaded to accept Ellen's offer, and he turned in the paper as his own. The course director deduced the deception, and when the students were confronted, they immediately admitted what they had done, blamed only themselves, and said they had been "foolish." Subsequent events showed that the faculty saw the incident as a clear-cut case of cheating, whereas many students felt that George and Ellen's transgression was trivial when compared with plagiarizing a research paper or falsifying lab results on a patient's chart. Also, the faculty chose a more severe and long-lasting punishment, one that many students did not agree with. The author believes that the faculty's refusal to give George and Ellen a clean slate after a reasonable time reflected a lack of forgiveness that is antithetical to the compassionate, forgiving role of physician-healer that medical education promotes. She concludes by explaining how this incident illustrates complex generational and cultural differences in moral reasoning and the selection of punishment, and the great emphasis that medical education places on knowing the facts rather than working creatively with ideas.
The seventh and final meeting of the Association of American Medical Colleges' (AAMC's) Forum on the Future of Academic Medicine began December 4, 1998, with a talk by William W. Stead, MD, associate vice-chancellor for health affairs at Vanderbilt University Medical Center and director of its informatics center. Dr. Stead envisions a future in which informatics and information technology will place the consumer squarely in the center of the system, empowered with greater knowledge of health care; he gave three short scenarios to illustrate future typical interactions of consumers with the system. He then discussed the implications for academic medicine. For example, academic medical centers (AMCs) could become the information providers and quality assurance hubs of their regions. Various participants questioned some of the speaker's claims (one asserting that there would be serious complications if clinical information were made available to patients). The second speaker, Valerie Florance, PhD, director of the AAMC's better-health@here.now program, discussed her program, whose purpose is to explore the ways medical schools and teaching hospitals can best use information technology and the Internet in the coming decade to improve individual and community health. Nothing in the ensuing discussion indicated that the participants believed that academic medical centers would be spared painful dislocations if they were to embark on a road of institutional reform to respond to the pressures of the new and more competitive global economy. Greater awareness of this not-necessarily-welcomed message may be one of the lasting legacies of the forum.
The aging of the U.S. population has led many organizations to call for an increase in the amount of clinical geriatrics training in medical education. A subcommittee of the American Geriatrics Society's Education Committee was assigned the task of defining core competencies for geriatrics education in medical schools. The subcommittee reviewed the available literature, surveyed selected programs in geriatrics education, and sought input from experts in geriatrics education. They then defined the core knowledge, attitudes, and skills students must develop to care for older people. This article summarizes these core competencies, which medical educators may find useful in developing new curricula on aging or in evaluating existing curricula.
PURPOSE: This article provides a critical overview of problem-based learning (PBL), its effectiveness for knowledge acquisition and clinical performance, and the underlying educational theory. The focus of the paper is on (1) the credibility of claims (both empirical and theoretical) about the ties between PBL and educational outcomes and (2) the magnitude of the effects. METHOD: The author reviewed the medical education literature, starting with three reviews published in 1993 and moving on to research published from 1992 through 1998 in the primary sources for research in medical education. For each study the author wrote a summary, which included study design, outcome measures, effect sizes, and any other information relevant to the research conclusion. RESULTS AND CONCLUSION: The review of the literature revealed no convincing evidence that PBL improves knowledge base and clinical performance, at least not of the magnitude that would be expected given the resources required for a PBL curriculum. The results were considered in light of the educational theory that underlies PBL and its basic research. The author concludes that the ties between educational theory and research (both basic and applied) are loose at best.
PURPOSE: To compare four standard-setting procedures for an objective structure clinical examination (OSCE). METHODS: A 12-station OSCE was administered to 84 students in each of the final (fourth-) year medical classes of 1996 and 1997 at Dalhousie University Faculty of Medicine. Four standard-setting procedures (Angoff, borderline, relative, and holistic) were applied to the data to establish a cutoff score for a pass/fail decision. RESULTS: The procedures yielded highly inconsistent results. The Angoff and borderline procedures gave similar results; however, the relative and holistic methods gave widely divergent results. The Angoff procedure yielded results reliable enough to use in decision making for a high-stakes examination, but would have required more judges or more stations. CONCLUSIONS: The Angoff and borderline procedures provide reasonable and defensible approaches to standard setting and are practical to apply by non-psychometricians in medical schools. Further investigation of the other procedures is needed.
PURPOSE: The personal health care of medical students is an important but neglected issue in medical education. Preliminary work suggests that medical student-patients experience special barriers to health care services and report problematic care-seeking practices that merit further inquiry. METHOD: A self-report questionnaire was piloted, revised, and distributed to students at nine medical schools in 1996-97. The survey included questions regarding access to health services, care-seeking practices, and demographic information. RESULTS: A total of 1,027 students participated (52% response rate). Ninety percent reported needing care for various health concerns. Fifty-seven percent did not seek care at times, in part due to training demands, and 48% had encountered difficulties in obtaining care. A majority had received treatment at their training institutions, and students commonly pursued informal or "curbside" care from medical colleagues. Almost all participants (96%) were insured. Differences in responses were associated with level of training, gender, and medical school. CONCLUSION: Medical schools shoulder the responsibility not only of educating but also of providing health services for their students. Students encounter barriers to care and engage in problematic care-seeking practices. Greater attention to issues surrounding medical student health may benefit students and their future patients.
PURPOSE: To determine the influence of the quality of attending physicians and residents on the specialty choices of excellent medical students, who actually have a broad choice of specialties. METHOD: In 1993-94 and 1994-95, 169 third-year students at the University of Kentucky College of Medicine were randomly assigned to two one-month rotations on general medicine inpatient wards. At the end of each rotation, the students confidentially evaluated the attending physician and the supervising resident (different for each rotation) with whom they had worked. Data were collected for 62 attending physicians and 89 residents. The authors analyzed the influences of the "best" and "worst" clinical instructors (those rated in the top and the bottom 20% by all students with whom they had worked over the two years) on "excellent" medical students (the 52 students whose USMLE I scores were in the top 30% of their class). RESULTS: Using regression approaches from the general linear model, the authors found that independent predictors of internal medicine residency choice for excellent medical students were exposure to highly rated internal medicine attendings (p = .02) and residents (p = .03). Nine of 29 (30%) of the excellent students who worked with a "best" medicine clinical instructor chose an internal medicine residency, while none of the 23 excellent medical students who did not work with a "best" medicine clinical instructor did so. The authors found no correlation in students' ratings of their pairs of attendings and residents, suggesting that rater bias did not explain the results. CONCLUSION: Better medical students who work with the best internal medicine attending physicians and residents in their internal medicine clerkship are more likely to choose an internal medicine residency.
PURPOSE: To assess attitudes of female faculty about career progress, resources for career development, and values related to academic success and recognition. METHOD: In 1997, the authors surveyed all faculty at Virginia Commonwealth University School of Medicine and its associated Veterans Affairs Medical Center. RESULTS: Of 918 faculty, 567 (62%) responded to the survey; 33% of the respondents were women. Compared with men, women faculty were less likely to be tenured or at the level of professor, spent more time in clinical activities, had less time for scholarly activity, and reported slower career progress. Women were more likely to report that promotion and tenure criteria had not been reviewed with them. Significant differences were found between female physicians and non-physician faculty; female physicians reported the least time for scholarly activities and poorest understanding of promotion and tenure criteria. When the authors asked faculty how they valued certain indicators of career success, women were less likely to value leadership than were men. Female physicians were less likely to value scholarship and national recognition as indicators of their career success. CONCLUSION: This survey found important differences in career progress of male and female faculty, with women reporting less time for career development. In addition, there were differences in values related to career success and recognition, which were most pronounced for female physicians. These differences may have an important impact on promotion for women in general and particularly for female physicians.
The chief complaint is an important element of the medical history. Over a one-month period in 1999, the author observed 55 presentations of house officers and medical students on an inpatient ward at Miami Valley Hospital, an affiliate of Wright State. House officers noted the chief complaint in 49% of their presentations; students, in 61% of theirs. Educators need to stress the importance of the chief complaint during verbal case presentations.
Standardized evaluation criteria would be useful for sorting through the huge volume of medical educational information now available on the Internet. The authors developed and pilot-tested a simple rating instrument. Using this instrument, students identified preferred Web sites and indicated that speed was particularly important in their assessments.
The authors describe and discuss clinical problem analysis (CPA), an approach to solving complex clinical problems. They outline the five steps of the CPA model and the essential elements of each step. Next, they discuss the value of CPA's content-independent (methodical) approach and argue that teaching students to use CPA will enable them to avoid some common diagnostic reasoning errors and pitfalls. Finally, they compare CPA with two existing approaches to clinical problem solving.
With growing pressures to consolidate and reorganize health care delivery systems, graduate medical education (GME) consortia can draw faculty from affiliated members to assemble educational programs. The authors report on consortium-based research education seminars of a quality that many residency programs would be unable to develop and support on their own. Drawing a diverse faculty from consortium members and area universities, the OHEP Center for Medical Education's annual Research Workshop Series focuses on the design of research projects; data analysis and hypothesis testing; and written and oral presentation of scientific research. Each spring, OHEP sponsors a research forum in which the best research projects from consortium members are presented by the resident-researchers, who compete for recognition and prize money. Further, of the 128 presentations made thus far at the annual OHEP Research Forum, 25% were subsequently published. The consortium's research education program has been well received by residents, is cost-effective, and is an integral component of the research curricula of many area residency programs. Including research training in GME provides residents an opportunity to become more competitive for fellowship, faculty, and leadership positions.
The authors surveyed all 125 allopathic medical schools to determine the number of schools that had implemented a formal curriculum in managed care and how many had a substantial interest in a Web-based clearinghouse for managed care curricular resources. They describe the results of their survey and the Web site they developed, the Managed Care Education Clearinghouse.
The authors of this article, who were the members and staff of a research panel formed by the AAMC as part of its mission-based management initiative, reflect on the growing interest in quantitative information in the management of the research mission of medical schools. They note the serious limitations of any such system of measures for research, particularly its inability to represent directly the quality of the research effort. Despite these concerns, the authors acknowledge that leaders in academic medicine have always used quantitative measures in one form or another to compare performance or assess progress. Two factors appear to be driving increases in this practice: (1) the need to demonstrate to institutional stakeholders that resources are being used wisely and that the school's performance justifies continued investment in the research mission; and (2) the need to fashion an economic strategy to manage precious institutional resources, particularly research space. Given these realities, the authors offer guidelines for the proper development and use of measures to assess contributions by faculty, departments, and institutions to the research mission. They also comment on the measures most commonly used in four areas: grants and other revenue-generating activities; publications; faculty members' research reputation and contributions to the national research enterprise; and support to the general research mission of the school. The authors conclude that quantitative information can help institutional leaders in important management decisions. However, the potential for misuse is great. The key is always to regard this information as an aid to judgment, not a substitute for it.
Limited proteolysis of most large protein precursors is carried out in vivo by the subtilisin-like pro-protein convertases. Many important biological processes such as peptide hormone synthesis, viral protein processing and receptor maturation involve proteolytic processing by these enzymes, making them potential targets for the development of novel therapeutic agents. However, the efficient development of such molecules requires a better understanding of the molecular mechanisms of proteolytic protein processing. Herein, we review the most recent findings on the molecular aspects of subtilisin-like convertase activity, such as the structural analysis of the proteases, the mechanisms of enzyme/substrate specificity, their interaction with other proteins such as 7B2, and the comparative tissue and cellular distribution of the enzymes and their substrates. These data are then used as a background for the review of the known biological functions of subtilisin-like pro-protein convertases, the reported clinical cases involving proteolytic processing defects and, finally, the ongoing development of new therapeutic inhibitor molecules based on this knowledge.
Upregulation of the steroidogenic acute regulatory protein (StAR) is implicated in the rapid synthesis and secretion of steroidogenic cells to produce steroids in response to stimulation by trophic hormones of the gonadal and stress axes. In the present study, we have assessed the kinetics of both StAR gene transcription and protein biosynthesis in primary cell cultures of bovine adrenocortical and ovarian theca cells, under conditions of acute stimulation by corticotrophin (ACTH) and luteinizing hormone (LH), respectively. In both cell systems, detectable upregulation of StAR gene transcription occurred within 1-2 h, reaching maxima at 4 h (theca cells) or 6 h (adrenocortical cells). mRNA levels returned rapidly to baseline, by 12 h or 24 h, respectively. Specific StAR protein levels were assessed by western blotting using a novel antibody raised against a bovine StAR peptide, and showed a similar fast upregulation, albeit delayed by 1-2 h compared with the mRNA. The response of the cultured theca cells was more acute than that of the adrenocortical cells, possibly reflecting the propensity of the LH receptor to desensitize rapidly, unlike the ACTH receptor. The primary bovine theca cell cultures were also used for fully homologous transfection studies using various deletion promoter-reporter constructs of the bovine StAR gene. Kinetic analysis of the results indicated that the acute transcriptional response resides within the proximal (-315 bp) promoter region, which includes two putative responsive elements for the steroidogenic factor-1. More distal promoter regions may be involved in modulating the specificity of expression by combining enhancer and inhibitory functions.
We have investigated thyroid hormone deiodination in the liver, kidney and thyroid of the domestic cat. Affinity labelling with (125)I-bromoacetyl reverse T(3) (125)(I-BrAc-rT(3) demonstrated that liver and kidney, but not the thyroid, express type I iodothyronine deiodinase (IDI), results that were confirmed by measuring the activity of the IDI using (125)I-rT(3) and T(4) as substrate. Feline hepatic and renal IDI metabolised rT(3) at approximately 0.2% of the rate of rat hepatic IDI under identical assay conditions. The K(m) of the feline enzyme was at least 500-fold greater than that of rat IDI. However, feline and rat hepatic IDI metabolised T(4) at a similar rate and had similar K(m) values (1.35 microM and 2.25 microM, respectively). This study demonstrates that cats and rats express IDI in the liver and kidney in similar concentrations; however, the feline enzyme appears unable to utilise rT(3) as a substrate under physiological conditions.
The epididymal epithelial cells of the lizard (Lacerta vivipara) produce large amounts of specific proteins under androgenic control. Amongst them, a major protein family that binds to the head of spermatozoa, the lizard epididymal secretory protein (LESP) family, has been identified as a member of the lipocalin superfamily. LESPs are composed of 9 elements that present an identical molecular mass of 18 000 Da but have a large range of pHi (3.5 to 9). The structural analysis of this protein family was performed by the determination and comparison of both the aminoterminal sequence of each element and the complete sequence of three specific LESP cDNA clones. When not identical, LESP elements present randomly dispatched nucleotide and amino acid substitutions, indicating the existence of at least five LESP mRNA populations encoded by a multigenic family. We determined that these LESP genes are differentially expressed during the annual epididymal cycle.
In the endometrium two enzymes are known to convert estradiol to its inactive metabolite estrone: microsomal 17beta-hydroxysteroid dehydrogenase type 2 (17beta-HSD2) and peroxisomal 17beta-HSD4. In order to elucidate the particular function of each of these two different enzymes, the human endometrial epithelial cell lines HEC-1-A and RL95-2 were examined with respect to the expression of 17betaHSD isozymes. They were compared with human endometrium in vivo. Non-radioactive in situ hybridization revealed both enzymes in glandular epithelial cells of human endometrium. The two cell lines were screened for mRNA expression of 17beta-HSD 1-4 by RT-PCR and Northern blot. 17beta-HSD2 and 4 could be detected by either method, 17beta-HSD1 only by RT-PCR, 17beta-HSD3 not at all. Both cell lines were proven to have no receptor for progesterone which is known as a physiological inducer of several 17beta-HSD isozymes. To study the regulation of 17beta-HSD2 and 17betaHSD4, the concentration of fetal calf serum in the cell culture media was reduced stepwise to 0.3% by dilution with a defined serum replacement. This treatment led to an inhibition of 17beta-HSD2 mRNA expression and an increase in the mRNA expression of 17beta-HSD4. Concomitantly, distinct morphological changes were observed, such as a decrease in the number and length of microvilli and a decrease in the formation of domes on top of the monolayers. The endometrial epithelial cell lines HEC-1-A and RL95-2 represent a suitable in vitro model for further studies of the differential expression of the major endometrial HSD isozymes, independent of the effect of progesterone.
Oestrogens exert their actions via specific nuclear protein receptors that are members of the steroid/thyroid receptor superfamily of transcription factors. Recently, a second oestrogen receptor (ERbeta) has been cloned, and using reverse transcription-PCR and immunohistochemistry it has been shown to have a wide tissue distribution in the rat that is distinct from the classical oestrogen receptor, ERalpha. Using commercial polyclonal antisera against peptides specific to human ERbeta, we have determined the sites of ERbeta expression in archival and formalin-fixed human tissue and compared its expression with that of ERalpha. ERbeta was localised to the cell nuclei of a wide range of normal adult human tissues including ovary, Fallopian tube, uterus, lung, kidney, brain, heart, prostate and testis. In the ovary, ERbeta was present in multiple cell types including granulosa cells in small, medium and large follicles, theca and corpora lutea, whereas ERalpha was weakly expressed in the nuclei of granulosa cells, but not in the theca nor in the copora lutea. In the endometrium, both ERalpha and ERbeta were observed in luminal epithelial cells and in the nuclei of stromal cells but, significantly, ERbeta was weak or absent from endometrial glandular epithelia. Epithelial cells in most male tissues including the prostate, the urothelium and muscle layers of the bladder, and Sertoli cells in the testis, were also immunopositive for ERbeta. Significant ERbeta immunoreactivity was detected in most areas of the brain, with the exception of the hippocampus - a tissue that stained positively for ERalpha. In conclusion, the almost ubiquitous immunohistochemical localisation of ERbeta indicates that ERbeta may play a major role in the mediation of oestrogen action. The differential expression of ERalpha and ERbeta in some of these tissues suggests a more complex control mechanism in oestrogenic potential than originally envisioned.
DAX1 is an unusual member of the orphan nuclear receptor family of transcription factors. Mutations in human DAX1 cause X-linked adrenal hypoplasia congenita, while abnormal duplication of the gene is responsible for male-to-female dosage-sensitive sex reversal. Based on these and other observations, DAX1 is thought to play a role in adrenal and gonadal development in mammals. As DAX1 has not previously been described in any other vertebrate, a putative avian DAX1 clone was isolated from an embryonic chicken (Gallus domesticus) urogenital ridge cDNA library. The expression profile of this cDNA was then examined during gonadogenesis. The clone included the conserved 3' ligand-binding motif identified in humans and mice but the 5' region lacked the repeat motif thought to specify a DNA-binding domain in mammals. Southern blot analysis and fluorescence in situ hybridisation mapping showed that the gene is autosomal, located on chromosome 1q. Sequence comparisons showed that the putative chicken DAX1 protein has 63 and 60% identity with the human and mouse proteins respectively over the region of the conserved ligand-binding domain. However, stronger identity (74%) exists with a putative alligator DAX1 sequence over the same region. Northern blotting detected a single 1.4 kb transcript in late embryonic chicken gonads, while RNase protection assays revealed expression in the embryonic gonads of both sexes during the period of sexual differentiation. Expression increased in both sexes during gonadogenesis, but was higher in females than in males. This is the first description of a DAX1 homologue in a non-mammalian vertebrate.
The pathophysiological role for the expression of human chorionic gonadotrophin (hCG) in malignant neoplasms is currently speculative. We investigated the overall expression of genes hCG-beta 5, 3, 8 and 7 in breast carcinoma (n=214), fibroadenoma (n=37) and macromastia (n=10) by quantitative reverse transcriptase-PCR. Eighty (37.4%) of the breast cancer samples revealed positive hCG-beta mRNA expression and the mean value was 67. 9 copies per 200 ng total RNA (range: 0-1743; 95% confidence interval (CI) for mean: 44-92). Fibroadenomas had more frequently detected (56.8%) and greater hCG-beta copy numbers (mean 86.9; range: 0-845; 95% CI for mean: 35-138). Macromastia probes yielded no positive hCG-beta mRNA. The hCG-beta mRNA expression was significantly different in the three histological subgroups (P=0. 006). Among breast carcinomas, a positive correlation was detected between hCG-beta mRNA copy numbers and progesterone receptor (PgR) values (P<0.001). No significant differences were seen regarding disease-free (P=0.87) and overall survival (P=0.20) depending on hCG-beta mRNA status. Finally, our findings do not support a role for hCG-beta in malignant transformation of human breast cells and indicate a possible involvement of hCG-beta in benign breast disease. The relationship with PgR expression may suggest that progestins regulate the expression of hCG in breast epithelial cells.
Using site-directed mutagenesis, we have undertaken a study of a potential IGF-binding site in the C-terminal domain of rat IGFBP-5, lying close to or within a previously described heparin-binding domain (residues 201-218) in this protein. After analysis of binding activity using three different methods - ligand blotting, solution phase equilibrium binding and biosensor measurement of real-time on- and off-rates - we report that the mutation of two highly conserved residues within this region (glycine 203 and glutamine 209) reduces the affinity of the binding protein for both IGF-I and IGF-II, while having no effect on heparin binding. In addition, we confirm that mutation of basic residues within the heparin-binding domain (R201L, K202E, K206Q and R214A) results in a protein that has attenuated heparin binding but shows only a small reduction in affinity for IGF-I and -II. Previous findings have described the reduction in affinity of IGFBP-5 for IGFs that occurs after complexation of the binding protein with heparin or other components of the extracellular matrix (ECM) and have postulated that such an interaction may result in conformational changes in protein structure, affecting subsequent IGF interaction. Our data suggesting potential overlap of heparin- and IGF-binding domains argue for a more direct effect of ECM modulation of the affinity of IGFBP-5 for ligand by partial occlusion of the IGF-binding site after interaction with ECM.
To purify novel ligands for the corticotrophin-releasing factor binding protein (CRF-BP) from ovine brain, whole brain was homogenised in methanol and the supernatant extracted on Sep-pak C18 cartridges followed by a preliminary HPLC step. Three peaks of ovine CRF-BP ligand activity were detected in the HPLC fractions, the first two of which were also detected by a specific corticotrophin-releasing factor two-site immunoradiometric assay, the third peak being detected by a human CRF-BP ligand assay, which will not detect ovine CRF. Human CRF-BP ligand-containing fractions were further purified by affinity chromatography on a human recombinant CRF-BP column with two additional HPLC steps. The human CRF-BP ligand was found to: (a) possess a molecular mass of 4707 Daltons, (b) have an N-terminal amino acid sequence (5 residues) identical to rat urocortin, (c) be detected by a specific urocortin radioimmunoassay, (d) have high affinity for both the human and ovine CRF-BPs and (e) be present in many regions of the ovine brain. Additionally, a 300 bp cDNA fragment sharing 83% homology with the rat urocortin gene was cloned from ovine brain, the product of which was predicted to have an identical amino acid sequence to that of rat urocortin. These pieces of information confirmed the identity of the human CRF-BP ligand as an ovine urocortin. The specially developed CRF-BP ligand assays showed that the rank orders of affinity of the CRF family members for human CRF-BP were: carp urotensin-1>>human CRF=rat/ovine urocortin>human urocortin>>frog sauvagine>>ovine CRF, and those for the ovine CRF-BP were: carp urotensin-1> human CRF=rat/ovine urocortin>human urocortin> frog sauvagine>>ovine CRF. This study describes a successful technique for the purification and detection of peptide ligands for the CRF-BP. We conclude that urocortin is the principal ligand for the CRF-BP in ovine brain and we could find no evidence for a centrally located mammalian sauvagine-like peptide.
The present study was designed to evaluate the regulation of nitric oxide (NO) synthesis in porcine oocytes during follicular development. Cumulus-oocyte complexes were obtained by aspirating the small follicles of immature porcine ovaries and cultured at 39 degrees C for 24-72 h with FSH in a serum-free medium. The oocyte-surrounding cumulus cells markedly proliferated and expressed LH receptor mRNA in response to FSH. The endothelial type of NO synthase (eNOS) (130 kDa) was detected in the oocyte, but not in the proliferated cumulus cells, by immunoblotting. The amount of oocyte eNOS did not significantly alter during culture, but measurement of nitrite and nitrate revealed FSH suppression of NO synthesis by approximately 50%. NO-releasing agents were added to the cultures to examine the effect of NO on the growth of cumulus cells. NO-releasing agents showed inhibitory effects on proliferation of the cumulus cells and expression of LH receptor mRNA. Thus, synthesis of eNOS-derived NO is suppressed in the porcine oocyte during development with no change in the enzyme amount, and it is suggested that it has an inhibitory function in the growth of cumulus cells.
Mutations in HSD3B2, the gene for 3beta-hydroxysteroid dehydrogenase type II (3beta-HSD II) have been detected and activities analysed through the in vitro expression of mutant cDNAs. Two full sibs with male pseudohermaphroditism were found to be double heterozygotes: N100S/266DeltaA. This genotype leads to the most profound loss of 3beta-HSD II enzyme activity (1.3% of normal) described to date in cases without severe salt-loss. One sib (N100S/266DeltaA) is the first reported male case of type II deficiency affected with premature adrenarche. Three apparently independent kindreds had propositi affected with the HSD3B2 mutation A82T/A82T, which is associated with a non salt-losing phenotype with variable expressivity in females. These three families had the same extended HSD3B haplotype and are likely to have inherited the same ancestral mutation. The significance of this finding is discussed in the light of the presence of A82T mutation at a homologous position in pseudogene varphi5 that is present in the HSD3B cluster.
Retinoic acid (RA) and sodium butyrate (NaB) are regulators of cell growth and differentiation. We studied their effect on normal (SVC1) or v-Ki-ras-transformed (Ki-SVC1) rat seminal vesicle (SV) epithelial cell lines. The treatment of these cells with 10(-((7( M RA did not produce significant changes in the morphological and biochemical parameters analyzed. When RA was used in combination with 2 mM NaB, the treatment induced substantial morphological changes, apoptosis-independent growth arrest, up-regulation of tissue transglutaminase (tTGase), and down-regulation of beta and gamma RA receptor (RAR) mRNA expression. The same cells did not express RAR alpha either before or after NaB/RA treatment. A similar treatment did not change the amount of mRNA coding for the protein SV-IV (a typical differentiation marker of the SV epithelium) in normal or ras-transformed cells nor the level of v-Ki-ras mRNA in Ki-SVC1 cells. These findings suggest that a defective RA/RARs signaling pathway is probably the biochemical condition that underlies the unresponsiveness to RA of our in vitro culture system, and indirectly points to the possibility that the NaB/RA-induced effects were brought about by a cooperation at the transcription level between the histone deacetylase inhibitory activity of NaB and the ability of RA/RAR to modulate the expression of various genes involved in the control of cell growth and differentiation.
The prolactin (PRL) family is comprised of a group of hormones/cytokines that are expressed in the anterior pituitary, uterus, and placenta. These proteins participate in the control of maternal and fetal adaptations to pregnancy. In this report, we have identified two new nonclassical members of the rat PRL family through a search of the National Center for Biotechnology Information dbEST database. The cDNAs were sequenced and their corresponding mRNAs characterized. Overall, the rat cDNAs showed considerable structural similarities with mouse proliferin-related protein (PLF-RP) and prolactin-like protein-F (PLP-F), consistent with their classification as rat homologs for PLF-RP and PLP-F. The expression of both cytokines/hormones was restricted to the placenta. The intraplacental sites of PLF-RP and PLP-F synthesis differed in the rat and the mouse. In the mouse, PLF-RP was expressed in the trophoblast giant cell layer of the midgestation chorioallantoic and choriovitelline placentas and, during later gestation, in the trophoblast giant cell and spongiotrophoblast layers within the junctional zone of the mouse chorioallantoic placenta. In contrast, in the rat, PLF-RP was first expressed in the primordium of the chorioallantoic placenta (ectoplacental cone region) and, later, exclusively within the labyrinth zone of the chorioallantoic placenta. In the mouse, PLP-F is an exclusive product of the spongiotrophoblast layer, whereas in the rat, trophoblast giant cells were found to be the major source of PLP-F, with a lesser contribution from spongiotrophoblast cells late in gestation. In summary, we have established the presence of PLF-RP and PLP-F in the rat.
PROBLEM: The influence of anti-sperm (ASA), anti-phospholipid (APA), and antizonal (AZA) antibodies on in vitro fertilization (IVF) results and the need for intracytoplasmic sperm injection (ICSI) were assessed. METHOD OF STUDY: Forty-four couples with infertility of immunologic origin were investigated. ASA in serum and ovulatory mucus were studied by a tray agglutination test (TAT) and indirect mixed anti-globulin reaction test (MAR) test, AZA were studied by passive hemagglutination and commercial enzyme-linked immunosorbent assay (ELISA; BioGen, Germany), and APA were tested by ELISAs in immunoglobulin isotypes IgG and IgM. RESULTS: Because of failed or very low fertilization after standard IVF in the previous cycle, ICSI had to be used in five out of 15 cases with ASA (33.3%), in 16 out of 18 couples with AZA (89.4%), and in only one case if APA were present (9%). Clinical pregnancy rate was 60% in cases with ASA, 38.5% with AZA, and 27.3% per embryo transfer (ET) if APA were detected. CONCLUSIONS: Immunologic infertility can be treated by IVF with very good results. The most important group are women with AZA, in whom IVF ICSI without any delay is recommended.
PROBLEM: The aims of this study were to evaluate the presence of insulin-like growth factor (IGF)-I, platelet-derived growth factor (PDGF), and epidermal growth factor (EGF) in pre-ovulatory follicular fluid (FF) in patients undergoing ovarian hyperstimulation for intra-cytoplasmic sperm injection (ICSI) treatment, to determine the differences between the concentrations of these cytokines in relation to ovarian stimulation regimens, and to find the relationship between these parameters and estradiol 17-beta, progesterone, and luteinizing hormone (LH) concentration in serum, as well as ICSI outcome. METHOD: IGF-I and PDGF were measured in the FF of 85 patients. The IGF-I levels were measured by radioimmunoassay, whereas the concentrations of PDGF and EGF were measured by enzyme-linked immunosorbent assay technique, using commercially available kits. RESULTS: IGF-I (0.42 +/- 0.09 ng/mL), PDGF (307.3 +/- 274.5 pg/mL), and EDF (8.88 +/- 6.4 pg/mL) were present in pre-ovulatory FF in patients undergoing ovarian hyperstimulation for ICSI treatment. The mean concentration of IGF-I in the follicle-stimulating hormone (FSH) group was significantly higher (P = 0.036) than that found in the human menopausal gonadotrophin (hMG)/FSH group, whereas no significant difference in the mean concentrations of PDGF (P = 0.58) and EGF was shown between all investigated groups. CONCLUSION: Controlled ovarian stimulation regimens affect only IGF-I levels in FF and the cytokine concentrations of all investigated groups, in turn, showed no correlation either with steroid hormones in serum or ICSI outcome.
PROBLEM: To assess the relationship between serum cancer antigen (CA)-125 concentrations and the likelihood of pregnancy after in vitro fertilization/embryo transfer (IVF ET). METHOD OF STUDY: A prospective longitudinal follow-up study was conducted in 44 IVF patients receiving luteinizing hormone-releasing hormone (LHRH) suppression, followed by human menopausal gonadotrophin (hMG). There were no indications of endometriosis. Progesterone or human chorionic gonadotrophin (hCG) were given as luteal support. Serum samples were taken just before oocyte pick-up (OPU), 14 days after ET and, after the establishment of a clinical pregnancy, on day ET + 21. Samples were stored at -80 degrees C until analysis. CA-125 was measured using a commercially available enzyme immunoassay (IMx CA-125; Abbott, North Chicago, IL, USA). The signed rank (paired samples) and Wilcoxon tests were used for statistical analysis. RESULTS: There were no differences in CA-125 concentrations at the time of OPU between pregnant (n = 18) and non-pregnant (n = 26) IVF patients. After OPU. the CA-125 concentrations rose both in pregnant (P<0.0001) and in non-pregnant (P <0.001) patients. This rise was greater after successful implantation, and 14 days after ET, higher CA-125 concentrations were found in the pregnant patients (P< 0.01). CONCLUSIONS: Differences in serum CA-125 concentrations between pregnant and non-pregnant IVF patients appear to be related to the likelihood of successful implantation.
PROBLEM: To compare changes in serum vascular endothelial growth factor (VEGF) levels during normal and in vitro fertilization (IVF) cycles. METHOD OF STUDY: Ten healthy women with ovulatory cycles and 37 infertile women participating in an IVF program were followed by frequent serum samples and with VEGF measurements throughout their cycles. RESULTS: Serum VEGF remained unchanged during the normal menstrual cycle, whereas the IVF program participants showed elevations in serum VEGF in the luteal phase of the cycle. When data from controls and patients were pooled, redundant midluteal VEGF level correlated with progesterone and with peak follicular phase estrogen level. The midluteal VEGF level in the IVF cycles was associated with body mass index (P < 0.01) and progesterone level (P < 0.05) by multiple regression. The 14 women conceiving tended to have higher VEGF levels than those failing to become pregnant. CONCLUSIONS: The IVF program was associated with increased synthesis of VEGF either in the ovaries, endometrium, or at other sites and this may be of significance for the outcome of IVF.
PROBLEM: To examine whether the occurrence of activated protein C resistance (APCR) and factor V Leiden mutation differs in women with first- compared to women with second-trimester unexplained recurrent pregnancy loss. METHOD OF STUDY: Seventy eight consecutive women with two or more unexplained post-embryonic recurrent pregnancy losses and 139 fertile women with at least one successful pregnancy and no abortions were prospectively investigated for APCR and the factor V Leiden mutation. No women were pregnant at the time of investigation. APCR was defined as APC sensitivity ratio (APC SR) of < or = 2.0. All patients with an APC SR < or = 2.4 were investigated for the factor V Leiden mutation. Women in this study were divided into three groups. Group A included only women with a history of recurrent first-trimester embryonic loss (37 women) and Group B included women with second-trimester abortions with or without additional first-trimester abortions (41 women). Group C included the controls (139 women). RESULTS: APCR and factor V Leiden mutations were significantly more prevalent in all recurrent pregnancy loss patients in this study as compared to controls. 38%(30/78) and 19%(15/78) in contrast to 8% (11/139) and 6% (8/139), respectively. All three groups in the study were comparable regarding age, parity, and number of living children, whereas Groups A and B were also comparable regarding gravidity. Mean APC SRs were significantly higher in Group C as compared to Groups A and B. The incidence of APCR was significantly higher in Groups A and B, as compared to controls, 27 and 49% in contrast to 8%, respectively. Moreover, the incidence of the factor V Leiden mutation was significantly higher in Groups A and B as compared to Group C, 16 and 22% as distinct from 6%, respectively. The incidence of APCR was higher in Group B as compared to Group A, 49% in contrast to 27%, with borderline significance: however, the factor V Leiden mutation did not significantly differ between the two groups. CONCLUSIONS: APCR and factor V Leiden are associated with unexplained recurrent pregnancy loss. The occurrence of APCR and factor V Leiden seems to be linked to post-embryonic first-trimester as well as second-trimester recurrent pregnancy loss. The significance of acquired, non-heritable APCR in recurrent fetal loss patients, especially in the second-trimester aborters, is still to be determined.
PROBLEM: The study was conducted to investigate the efficacy of prednisone and aspirin in autoantibody seropositive patients with repeated in vitro fertilization embryo transfer (IVF ET) failure. METHODS OF STUDY: The study group comprised 52 consecutive patients seropositive for non-organ-specific autoantibodies, i.e., anti-cardiolipin antibodies (ACA), anti-nuclear antibodies (ANA), anti-double-stranded (ds) DNA, rheumatoid factor (RF), and lupus anti-coagulant (LAC). These patients were treated with prednisone, 10 mg per day, and aspirin, 100 mg per day, starting 4 weeks before induction of ovulation in 52 IVF cycles. RESULTS: The clinical pregnancy rate per cycle was 32.7% (17/52). No increased incidence of pregnancy complications, including premature labor, gestational diabetes mellitus, and pregnancy-induced hypertension, were found. CONCLUSIONS: Combined treatment of prednisone for immunosuppression and aspirin as an anti-thrombotic agent, starting before ovulation induction, may improve pregnancy rate in autoantibody seropositive patients who have had repeated IVF-ET failures.
PROBLEM: To examine the capacity of sperm cells from fertile and infertile men to secrete interleukin (IL)-6, and the involvement of serum factors and lipopolysaccharide (LPS) in the regulation of IL-6 and IL-1 production by sperm cells. METHODS: Swim-up sperm cells from fertile (donors) and oligoteratoasthenospermic (OTA)-infertile men were incubated with or without 5% fetal calf serum (FCS) and LPS (10 microg/mL) for 2-24 hr. After incubation, IL-6 and IL-1 bioactivities were measured in supernatants and lysates by specific bioassays (B9 cell proliferation assay and 1A-5 system, respectively). RESULTS: IL-6- and IL-1-like activities were observed to be produced by swim-up sperm cells from both study groups. Stimulation of swim-up sperm cells with either LPS or FCS or both together did not affect their capacity to produce IL-1. However, LPS, but not serum increased the secretion levels of IL-6 by swim-up sperm cells. CONCLUSIONS: Swim-up sperm cells from both study groups constitutively produce IL-6 and IL-1, and serum components did not affect this capacity. However, LPS was shown to increase the capacity of swim-up sperm cells of both study groups to secrete IL-6, but not IL-1. Cytokines may be involved in the physiology and pathophysiology of sperm functions and, thus, may affect male fertility.
PROBLEM: The aim of this study was to clarify the physiological effects of interferon (IFN)-gamma on secretion of vascular endothelial growth factor (VEGF) by endometrial stromal (ES) cells. METHOD OF STUDY: ES cells were obtained from human uterine endometrium by enzymic digestion and filtration. The effects of IFN-gamma on production of VEGF by ES cells were examined by analyzing VEGF mRNA expression with Northern blotting analysis and by assaying VEGF protein. RESULTS: IFN-gamma inhibited VEGF mRNA and protein expression by ES cells in a dose-dependent manner. In ES cells treated with IFN-gamma, VEGF production was not significant until 6 hr of incubation and was significantly affected after 6 hr of incubation, but decreased significantly after 12 to 48 hr. IFN-gamma also suppressed VEGF mRNA expression by ES cells. CONCLUSIONS: ES cells produce VEGF, which may contribute to endometrial neovascularization and proliferation. IFN-gamma may play an important role in regulating VEGF production by ES cells.
PROBLEM: The pathogenesis of uterine leiomyomata is still unclear. Recently it has been suggested that mac25 plays a tumor-suppressive role in various tumors. The aims of this study were to evaluate a possible involvement of mac25 in the growth of leiomyoma and in the mechanism of a gonadotropin-releasing hormone agonist (GnRHa) inducing shrinkage of leiomyoma. METHODS OF STUDY: Mac25 mRNA transcript was measured by Northern blot in total RNA extracted from the paired specimens of leiomyoma and adjacent myometrium from untreated patients (n = 25) and from leiomyoma specimens from GnRHa-pretreated patients (n = 10). RESULTS: Mac25 mRNA expression was significantly lower in large leiomyoma (more than 150 cm3 in volume) than in adjacent myometrium and small leiomyoma (less than 120 cm3 in volume) from untreated patients. There was no difference in this expression between the proliferative and secretory phases of the menstrual cycle. Leiomyoma from GnRHa-pretreated patients had mac25 gene expression levels similar to myometrium and small leiomyoma from untreated patients. CONCLUSIONS: Mac25 may be involved in the growth of uterine leiomyoma and the action of GnRHa may, in part, be mediated by mac25.
PROBLEM: To determine if interleukin (IL)-1 produced by autologous endometrial coculture (AECC) was associated with outcome in patients with a history of multiple in vitro fertilization (IVF) failures. METHOD OF STUDY: The conditioned media (CM) from AECC cells exposed or non-exposed to human embryos was analyzed for IL-1. RESULTS: Embryos grown on AECC demonstrated a significant improvement in number of blastomeres and fragmentation (frag) when compared to embryos grown in conventional media without ECC (6.4 +/- 1.3 vs. 5.5 +/- 1.2 blastomeres and 14.6 +/- 9.3%, vs. 18.4 +/- 9.8% frag; P< 0.008 and 0.003, respectively). When IL-1alpha and IL-1beta were undetectable in the CM, the embryos grown in ECC were of improved quality as compared to the embryos grown only in conventional media. Conversely, IL-1ra levels in the CM were positively associated with embryo quality. Exposure or non-exposure to an embryo did not result in differing levels of IL-1alpha, IL-1beta, or IL-1ra in the CM. IL-1beta levels were negatively associated with clinical pregnancy outcome (3.3 pg/mL (pregnant, n = 12) vs. 27.1 pg/mL (not pregnant, n = 17); P = 0.008, Mann Whitney U-test). IL-1alpha and IL-1ra levels were not associated with outcome. CONCLUSIONS: We have demonstrated a significant improvement in blastomere number and frag with ECC. The presence of IL-1beta in the CM was negatively associated with embryonic development and clinical pregnancy. The presence of IL-1alpha in the CM was negatively associated with embryonic development and the presence of IL-1ra in the CM was positively associated with embryonic development. Whether IL-1beta itself interferes with successful outcome after embryo transfer or if it is a marker for undetected endometritis in the biopsy specimens remains to be determined.
The allene oxide synthase (AOS) was purified from corn (Zea mays) seeds to homogeneity and characterized partially. The corn AOS was a hemoprotein cytochrome P450 with a molecular weight and pI of 53,000 and 6.0, respectively. The corn AOS was found to be irreversibly inactivated by a substrate, 13-hydroperoxyoctadienoic acid. The rate of the enzyme inactivation was higher at low pHs.
Urease was purified from leaves of mulberry (Morus alba, L.) by ammonium sulfate fractionation, acetone fractionation and sequential column chromatography including Q-Sepharose HP, Phenyl-Sepharose HP, Superdex 200 HR and Mono Q. The enzyme was purified 5700-fold to apparent homogeneity with a recovery of 3.6%. The molecular mass of the enzyme was determined to be 90.5 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis and 175 kDa by gel filtration, indicating that the enzyme was a homodimer. In the western blot analysis, 90.5 kDa subunit of the mulberry leaf urease cross-reacted with antiserum raised against jack bean seed urease. The N-terminal sequence of the first 20 residues of the enzyme revealed that it has a high similarity (80-90%) to ureases from other plant sources, suggesting that the mulberry leaf urease is closely related to other plant ureases. However, the mulberry leaf enzyme showed an optimum pH for activity of 9.0, while the optimum pH of most ureases isolated from plants and bacterial is neutral. In addition, the K(m) value for urea was 0.16 mM, which is lower than those of ureases from other sources. It is also proposed that urease activity ingested by browsing silkworm releases ammonia that is subsequently used in silkworm protein synthesis.
Feeding experiments using 13C labelled precursors and NMR spectroscopic studies revealed general biosynthetic incorporation of phenylalanine and variable incorporation of cinnamic acid, p-coumaric acid, caffeic acid and ferulic acid into phenylphenalenones in root cultures of Anigozanthos preissii. Evidence was obtained for parallel pathways of phenylphenalenone biosynthesis, with respect to the left phenylpropanoid unit, and a sequence involving utilisation of p-coumaric acid with late generation of an intermediate catechol moiety in the right phenylpropanoid unit.
The growth and droserone content of callus cultures of Triphyophyllum peltatum grown in liquid 1/5 Linsmaier and Skoog medium was studied. During a lag phase in growth, droserone concentrations in the medium reached a value of 2.1 mg g-1 fr. wt. After this maximum value the concentration decreased slightly to 1.8 mg g-1 fr. wt., while the growth of the calli was enhanced (25% increase in fr. wt. within 7 days). Plumbagin and isoshinanolone were likewise present in the medium. By feeding 13C2-labelled acetate to the cultures the biosynthesis of droserone was elucidated. The incorporation of whole C2-units unambiguously shows its acetogenic origin and fits well in the biosynthetic scheme suggested for the structurally--and biogenetically--related naphthylisoquinoline alkaloids.
Shoot organ cultures were established from callus derived from anthers of Hypericum perforatum flowers and the effect of elicitors on hypericin and pseudohypericin production in shoot organ cultures was investigated. Mannan stimulated pseudohypericin production up to four fold (0.82 mg/g dry wt) and hypericin production up to two fold (0.04 mg/g dry wt.) beta-1,3-glucan and pectin slightly stimulated pseudohypericin production (ca. two fold), but had no effect on hypericin production. On the other hand, yeast extract showed no stimulatory effect, on either hypericin or pseudohypericin production.
An anthracenone analogue of abscisic acid (ABA) was synthesized as a potential photoaffinity reagent and tested for biological activity. Reaction between 10,10'-dimethoxy-9-anthrone with two equivalents of the lithiated dianion of cis-3-methylpent-2-en-4-yn-1-ol afforded an acetylenic alcohol key intermediate. Subsequent reduction of the triple bond, functional group manipulation of the side chain alcohol and deprotection of the dimethoxy protected anthrone provided anthracenone ABA analogue 7 as a potential photoaffinity reagent for ABA-binding proteins. The effect of natural ABA and the potential photoaffinity anthracenone ABA 7 on corn cell growth was determined at various concentrations. The results show that anthracenone ABA 7 is perceived as ABA-like, although producing less inhibition than ABA itself. For example, 7 at 33 microM produces approximately the same inhibition as ABA at 10 microM.
Adventitious root cultures of Rhus javanica L. produced large amounts of galloylglucoses (gallotannins) and an anthocyanidin, riccionidin A, formerly found only in liverworts. Production of both galloylglucoses and riccionidin A in the adventitious root culture system was suppressed by light. The Rhus root culture showed the highest productivity for those secondary metabolites in a modified Linsmaier-Skoog (LS) liquid medium containing 30 mM NH4+ and 30 mM NO3- as nitrogen sources in the presence of 10(-6) M 3-indoleacetic acid (IAA).
We earlier reported that when phenylalanine ammonia-lyase (PAL) activity in radish seedlings was inhibited by the competitive inhibitor 2-aminoindan-2-phosphonic acid (AIP), soluble sinapoyl esters carried over from the seed were converted to wall-bound esters in young cotyledons. We now report that these soluble sinapoyl esters may also be converted into lignin in the cotyledons. When radish seedlings were grown in the presence of 100 microM AIP, lignin formation (determined as lignothioglycolic acid) was inhibited ca. 74% in the cotyledons and ca. 80% in hypocotyls plus roots. The syringyl to guaiacyl (S/G) ratio in the lignin of AIP-grown plants, as determined by alkaline cupric oxidation and from Fourier-transform infrared (FT-IR) spectra, was higher in cotyledons, but lower in hypocotyls plus roots, as compared to plants grown on distilled water. These results support the view that soluble sinapoyl esters preformed in seeds may contribute to the syringyl moiety of lignin in cotyledons during early seedling development and that there is no appreciable transport of soluble sinapoyl esters from cotyledons to the hypocotyls and roots.
Two new triterpenoids, 6 alpha-O-acetyl-7-deacetylnimocinol [24,25,26,27-tetra-norapotirucalla-(apoeupha)-6 alpha-acetoxy-7 alpha-hydroxy-1,14,20,22-tetraen-21,23-epoxy-3-one] (1) and meliacinol [24,25,26,27-tetranorapotirucalla-(apoeupha)-1 alpha-trimethylacryloxy-21,23-6 alpha,28-diepoxy-16-oxo-17-oxa-14,20,22-trien-3 alpha,7 alpha-diol] (2) were isolated from the methanolic extract of the fresh leaves of Azadirachta indica (neem). Their structures have been elucidated through spectral studies, including 2D-NMR (COSY-45, NOESY, HMQC and HMBC). The bioactivity of these as well as of nimocinol, reported earlier from the same source, is reported. The first compound and nimocinol showed toxicity on fourth instar larvae of mosquitoes (Aedes aegypti) with LC50 values of 21 and 83 ppm, respectively. The second compound had no effect upto 100 ppm.
[3H, 14C] Ochratoxin A, prepared biosynthetically, was applied in dilute NaHCO3 solution to the soil in which coffee plants had grown to four pairs of leaves. Three weeks later the compound, isolated from dilute NaHCO3 extract of leaves by immunoaffinity chromatography, was detected by scintillation counting as a 1-2 ppm component of leaf dry weight, greatly exceeding the trace (ppb) occurrence of ochratoxin A in some green coffees, which therefore might arise in the field directly from fungal activity in soil rather than from fungal infection of cherries or processed green coffee.
Adenophora triphylla var. japonica (Campanulaceae) yielded two new alkaloids, the 6-C-butyl derivative of 2R,5R-bis(hydroxymethyl)-3R,4R-dihydroxypyrrolidine (DMDP) and alpha-1-C-ethyl-fagomine, together with the known alkaloids 1,4-dideoxy-1,4-imino-D-arabinitol, 1-deoxynojirimycin, and 1-deoxymannojirimycin. 6-C-Butyl-DMDP showed inhibitory activity toward almond beta-glucosidase (IC50 = 68 microM), whereas alpha-1-C-ethyl-fagomine inhibited bovine liver beta-galactosidase (IC50 = 29 microM).
The needles of several yew species and cultivars were analysed by high-pressure liquid chromatography for paclitaxel, 10-deacetylpaclitaxel, cephalomannine, baccatin III, 10-deacetylbaccatin III and brevifoliol. About 750 samples were collected from five different locations in the Netherlands and the UK. The results of this screening show a large variation in taxane content between the different species and cultivars. The content of paclitaxel and 10-deacetylbaccatin III varied from 0 to 500 micrograms/g and 0 to 4800 micrograms/g dried needles, respectively. Brevifoliol was found in a very high concentration in Taxus brevifolia. 10-Deacetylpaclitaxel, cephalomannine and baccatin III were found in concentrations ranging from 0 to 500 micrograms/g dried needles.
Several catechin compounds were examined for their ability to induce apoptosis in human histiocytic lymphoma U937 cells. Catechins with a pyrogallol-type structure in a B-ring induced apoptosis and a 3-O-gallate group in cis-relationship to the B ring enhanced the activity. Catechins without a pyrogallol-type structure in a molecule lacked activity. These data suggest the important role of the 5'(3')-hydroxyl group in the B-ring and that a pyrogallol-type structure in a molecule is a minimum requirement for apoptosis induction by catechin compounds.
Incubation of 18-hydroxy-9-epi-ent-pimara-7,15-diene with the fungus Gibberella fujikuroi gave the compounds 18-hydroxy-7 alpha,8 alpha-epoxy-9-epi-ent-pimara-15-ene, 18-hydroxy-7-oxo-ent-pimara-15-ene, 6 beta, 18-dihydroxy-7 alpha, 8 alpha-epoxy-9-epi-ent-pimara-15-ene, 9 beta,18-dihydroxy-7 alpha, 8 alpha-epoxy-ent-pimara-15-ene and 6 beta, 14 alpha, 18-trihydroxy-9-epi-ent-pimara-7,15-diene. Oxidation of C-19, which is characteristic of the biosynthesis pathway of the gibberellins is not produced.
Three new chalcanol glucosides have been isolated from the seeds of Trifolium alexandrinum, of which the first two are alpha'-chalcanol-alpha, beta-epoxides and the third one is an alpha, beta-dihydroxy-alpha'-chalcanol. The structures of the isolated compounds were verified by means of MS and 2D NMR spectral analyses.
Two novel phenolic compounds from the leaves of Cornus controversa (Cornaceae) were characterized as (-)-2,3-digalloyl-4-(E)-caffeoyl-L-threonic acid and (-)-2-galloyl-4-(E)-caffeoyl-L-threonic acid, using spectroscopic methods.
Six tropane alkaloids were isolated from the Sri Lankan endemic plant Erythroxylum zeylanicum O.E. Schulz (Erythroxylaceae) and structurally elucidated by NMR and MS measurements. Three of them, erythrozeylanines A [1R,3R,5S,6R-6-acetoxy-3-(3',4',5'-trimethoxybenzoyloxy)tropane], B [cis-3 beta-(cinnamoyloxy)tropane], and C [cis-6 beta-acetoxy-3 alpha-(cinnamoyloxy)tropane] are new, whereas the others have already been found in other Erythroxylum species. For the first time, the absolute configuration of a tropane alkaloid (erythrozeylanine A) has been determined by quantum chemical CD calculations.
Two novel diglycosylated steroidal alkaloids of 5 delta-pregnene nucleus, named obtusine-20(R)-O-[beta-thevetopyranosyl-(1-->4)-beta-cyma ropyranoside] and obtusolactam-20(R)-O-[beta-thevetopyranosyl-(1-->4)-beta- cymaropyranoside], together with the known beta-sitosteryl-3-O-beta-glucopyranoside were isolated from the roots of Cryptolepis obtusa N. E. Br.
Psychrophilic enzymes produced by cold-adapted microorganisms display a high catalytic efficiency and are most often, if not always, associated with high thermosensitivity. Using X-ray crystallography, these properties are beginning to become understood, and the rules governing their adaptation to cold appear to be relatively diverse. The application of these enzymes offers considerable potential to the biotechnology industry, for example, in the detergent and food industries, for the production of fine chemicals and in bioremediation processes.
The increased awareness of the differences in biological activity of the two enantiomers of a chiral drug has raised the demand for enantiomerically pure products, particularly in the pharmaceutical industry. Simulated moving-bed chromatography can be used for the separation of the two enantiomers of a chiral molecule, which is feasible at all production scales, from laboratory to pilot to production plant. The use of non-enantioselective synthesis of racemic mixtures and simulated moving-bed enantiomer separation might make the development process of a new chiral drug substantially shorter and cheaper.
Gene therapy promises to revolutionize medicine by treating the causes of disease rather than the symptoms. We are nearing the end of the first decade of gene therapy, and this article summarizes the approaches taken, results achieved, lessons learned and important recent developments. The early results on the clinical efficacy of gene therapies were disappointing, largely because the available gene-transfer vectors proved to be inadequate. Recently, however, clinical benefit has been clearly demonstrated and great progress made in selecting and improving vectors. There is now every prospect that the second decade will see gene therapy live up to its enormous potential.
At present, the assignment of function to novel genes uncovered by the systematic genome-sequencing programmes is a problem. Many studies anticipate that this can be achieved by analysing patterns of gene expression via the transcriptome, proteome and metabolome. Thus, functional genomics is, in part, an exercise in pattern classification. Because many genes have known functional classes, the problem of predicting their functional class is a supervised learning problem. However, most pattern classification methods that have been applied to the problem have been unsupervised clustering methods. Consequently, the best classification tools have not always been used. Furthermore, the present functional classes are suboptimal and new unsupervised clustering methods are needed to improve them. Better-structured functional classes will facilitate the prediction of biochemically testable functions.
The application of gene-transfer technology to domestic animals provides a way for the introduction of genes encoding biochemical pathways that are currently nonfunctional in these animals. This might provide a mechanism for increasing the availability of specific substrates that currently limit certain production characteristics, such as the production of wool. The progress and problems associated with recent attempts to transfer a cysteine biosynthetic pathway and a glyoxylate cycle to sheep are discussed, in addition to the extension of this concept to other biochemical pathways.
BACKGROUND: The creation of osseous defects in the upper and lower jaws in children is an uncommon occurrence. It is therefore likely that a head and neck reconstructive surgeon will accumulate only limited experience in restoring such defects. We have reviewed 7 pediatric bone-containing microvascular free flap reconstructions in 6 patients for reconstruction of the upper or lower jaws. Three patients were available for long-term follow-up to evaluate the effect of osseous free flap reconstruction on function and growth and development of the donor site. DESIGN: Retrospective review. SETTING: Academic tertiary referral center for otolaryngology. PATIENTS AND METHODS: Six pediatric patients ranging in age from 8 to 16 years underwent 2 fibular, 4 scapular, and 1 iliac free flap procedure for restoration of 2 maxillary and 5 mandibular defects from 1992 to 1997. Three of the 6 patients were available for long-term follow-up to assess the postoperative donor site function in an effort to determine the effect of this surgery on long-term donor site morbidity and development. RESULTS: Two patients were lost to follow-up, and 1 died secondary to complications related to distant metastatic disease. Three of 6 patients were observed for 2 years 6 months, 4 years, and 4 years 2 months, respectively. Two of the 3 patients who were observed long term have undergone full dental rehabilitation and currently maintain a regular diet and deny pain with mastication or deglutition. One patient did not require dental rehabilitation. All 3 patients demonstrate gross facial symmetry and normal dental occlusion. Assessment of the fibular donor site demonstrated normal limb length and circumference. The patients denied pain or restriction to recreational activity. Scapular donor sites demonstrated normal range of motion, strength, and shoulder stability. CONCLUSIONS: Free flap reconstruction of the pediatric maxilla and mandible requires harvesting bone from actively growing donor sites. We have found no evidence of functional deficit after bone harvest from the fibular or scapular donor sites. Patients demonstrate normal growth at the donor sites, and symmetry of the mandible and maxilla is preserved.
OBJECTIVE: To report our experience in a case series of 5 posterior scalping flaps. DESIGN: Retrospective review of a case series. SETTING: A tertiary academic care otolaryngology-head and neck surgery referral center. PATIENTS: Five patients having undergone posterior scalping flap reconstruction of cutaneous midface defects. METHODS: Reconstruction was performed for 4 cheek defects, 1 of which included the lateral third of the upper and lower lips, and 1 combined midfacial and lateral nasal wall defect. RESULTS: All 5 patients had excellent cosmetic and functional results. The only complication was a single case of partial-thickness distal flap necrosis. CONCLUSION: The posterior scalping flap offers a reliable source of skin with appropriate color and texture and minimal donor-site morbidity.
BACKGROUND: The prognostic importance of vascular invasion has not been extensively studied in patients with papillary thyroid cancer. OBJECTIVE: To determine whether the presence of vascular invasion in papillary thyroid carcinoma, even within the thyroid gland, is associated with more aggressive disease at diagnosis and a higher incidence of tumor recurrence. PATIENTS AND METHODS: We identified 410 patients who had been diagnosed with papillary thyroid cancer since 1986 who had a follow-up period of longer than 1 year (median follow-up, 5.5 years). Pathology reports were reviewed and patients were separated into 3 groups: no vascular invasion, intrathyroidal vascular invasion, and extrathyroidal vascular invasion. MAIN OUTCOME MEASURES: Statistical comparison was performed by univariate and multivariate analysis. RESULTS: Patients with intrathyroidal vascular invasion were more likely to have distant metastasis at the time of diagnosis (26.1% vs 2.2%, P = .001). Similarly, patients with extrathyroidal vascular invasion had a higher incidence of distant metastases at diagnosis (40% vs 4.4%, P = .02). Patients with tumors identified to have intrathyroidal vascular invasion were more likely to develop distant recurrence (20% vs 3%, P = .002). CONCLUSIONS: These associations were found to be independent by multiple regression analysis. Patient age, sex, palpable or fixed lymph nodes, radiation exposure, and race did not differ between the patient group with and those without vascular invasion. Preliminary analysis of our data suggests that the presence of vascular invasion in papillary, thyroid carcinoma, even within the thyroid gland, is associated with more aggressive disease at diagnosis and with a higher incidence of tumor recurrence.
OBJECTIVE: To describe a clinical experience with sentinel lymph node biopsy (SLNB) of head and neck nodal basins for clinical stage I melanomas draining to these areas. DESIGN: Consecutive clinical case series with a mean follow-up of 10.7 months. SETTING: University tertiary care referral medical center. PATIENTS: Seventy patients with clinical stage I cutaneous melanoma who underwent SLNB of cervical and/or parotid lymph node basins. INTERVENTIONS: Patients underwent same-day preoperative technetium Tc 99m lymphoscintigraphy followed by SLNB using gamma probe and blue dye (66 patients) and blue dye alone (4 patients). Patients with histological evidence of tumor (here in after "positive") according to SLNB results underwent modified cervical completion lymph node dissection, including parotidectomy, as appropriate. Patients without histological evidence of tumor (hereinafter "negative") according to SLNB results were followed up clinically without undergoing completion lymph node dissection. MAIN OUTCOME MEASURES: The rates of SLNB success, SLNB positivity, completion lymph node dissection positivity, complications, and SLNB false-negative results were determined by clinical follow-up. RESULTS: Locations of melanomas in the 70 patients were the face (n = 20), neck (n = 14), ear (n = 9), scalp (n = 9), and upper thorax (n = 18). Locations of basins that underwent biopsy (n = 104) were in the cervical (n = 68), parotid (n = 19), and axillary (n = 17) regions. The mean Breslow thickness was 2.1 mm (range, 0.4-12.0 mm). Sentinel lymph node biopsy was successful in 103 basins (99%). The mean number of sentinel lymph nodes per basin was 2.5 (range, 1.0-8.0). Positive sentinel lymph nodes were found in 12 patients (17%) and 15 basins (14%). Sentinel lymph node biopsy results correlated with the American Joint Committee on Cancer tumor stage (P = .05) and a Breslow thickness of 1.23 mm or greater (P = .03). Additional tumor-containing nodes were noted in 5 (42%) of the 12 patients who underwent completion lymph node dissection, and these results correlated with the presence of multiple positive sentinel lymph nodes (P = .01). There were complications in 3 patients (4%) (seromas in 2 patients and temporary spinal accessory nerve paresis in 1 patient). One nodal recurrence in a basin that was negative according to SLNB results (SLNB with blue dye only) was noted (false-negative rate, 2%). The results of SLNB were accurate in 69 patients (99%). CONCLUSIONS: Sentinel lymph node biopsy using lymphoscintigraphy and blue dye to manage cutaneous melanomas draining to the head and neck nodal areas is reliable and safe. Sentinel lymph node biopsy results correlated with a Breslow thickness of 1.23 mm or greater and the American Joint Committee on Cancer tumor stage. Completion lymph node dissection is recommended after determining positive SLNB results.
OBJECTIVE: To assess the impact of clinical pathways on the practice of head and neck oncologic surgery in an academic center. DESIGN: Cross-sectional study. SETTING: Cancer treatment center. PATIENTS: The study population consisted of 3 groups of patients who underwent unilateral neck dissection and were treated in the Department of Head and Neck Surgery of the University of Texas M. D. Anderson Cancer Center, Houston. Additional procedures which may have been performed were direct laryngoscopy, rigid esophagoscopy, and/or dental extractions. Ninety-six patients treated during 1993-1994 prior to the implementation of the clinical pathway (historical control group) were compared with 94 patients treated during 1996-1998, 64 who were not (contemporaneous nonpathway group) and 30 who were managed on the clinical pathway (pathway group). Patients from 1995 were excluded since the pathway was in the planning stages then. MAIN OUTCOME MEASURES: Median length of stay; median total costs of care. RESULTS: The median length of hospital stay of the historical control, contemporaneous nonpathway, and pathway groups decreased from 4.0 to 2.0 days (P<.001). The total median costs of care were less in the pathway group as compared with the historical control group ($6,227 and $8,459, respectively, P<.001) and also less in the contemporaneous nonpathway group compared with the historical control group (S6885 and $8,459, respectively, P<.001). Mean and median length of hospital stay and costs were lower in the pathway group as compared with the nonpathway group but not significantly (P = .11 and P = .07, respectively) The contemporaneous nonpathway and pathway groups did not differ in complications or readmissions. CONCLUSIONS: Development and implementation of this clinical pathway played a statistically significant role in decreasing length of hospital stay and total costs of care associated with neck dissection between nonpathway and pathway patients. Thus, a more cost-effective practice environment has resulted for all of our patients.
OBJECTIVES: To summarize our quality-of-life (QOL) research findings for patients with head and neck cancer, to suggest areas for future productive QOL research, and to discuss how to undertake QOL studies in a cost-effective manner. DESIGN: Review of previously published analyses of advanced larynx cancer, advanced oropharynx cancer, and neck-dissection cases and current data from the complete set of patients. PATIENTS: From January 1, 1993, through December 31, 1998, data on 549 patients were entered in our head and neck database. Of these patients, 364 met additional criteria for histologic findings (squamous cell carcinoma) and the restriction of their cancer to 4 major anatomical sites (oral, oropharynx, hypopharynx, or larynx). Of these, 339 patients were more than 1 year beyond initial treatment. Complete baseline TNM staging and QOL data were obtained for 260 of these patients, of whom 210 presented with an untreated first primary tumor (index cases) to the University of Washington, Seattle. INTERVENTION: Pretreatment QOL was assessed with an interviewer-supervised self-administered questionnaire. Subsequent self-administered tests were completed at 3, 6, 12, 24, and 36 months. Other data collected on each patient included cancer site, stage, treatment, histologic findings, type of surgical reconstruction, and current disease and vital status. RESULTS/CONCLUSIONS: It is difficult to achieve "statistically significant" results in a single-institution setting. The "composite" QOL score may not be a sufficiently sensitive tool. Analysis of separate domains may be more effective.
OBJECTIVE: To determine the efficacy of concurrent preoperative cisplatin chemotherapy and radiotherapy (CT/RT) for patients with advanced head and neck cancer and cervical metastatic disease. DESIGN: Retrospective analysis. SETTING: University hospitals. PATIENTS: Eighty-eight patients with operable stage III and IV squamous cell carcinoma of the head and neck and palpable cervical lymphogenous metastases received preoperative concurrent CT/RT followed by planned neck dissection. INTERVENTIONS: All patients undergoing CT/RT received concomitant continuous infusions of cisplatin (20 mg/m2) on days 1 to 4 and 22 to 25 of CT/RT. Thirty-nine patients underwent single-fraction (1.8-Gy) radiotherapy to 45.0 Gy, and 49 patients received 10 single-fraction (1.8-Gy) treatments, which were hyperfractionated (1.2-Gy twice a day) to 46.8 Gy. MAIN OUTCOME MEASURES: The 71 patients for whom complete post-CT/RT data were available were evaluated for clinical response in addition to survival. Histologic complete response (HCR) was confirmed from planned neck dissection specimens (n = 48) after clinical complete response (CCR) from initial CT/RT. Kaplan-Meier statistical analysis for disease-specific survival and overall survival was performed on all 88 patients who received CT/RT. RESULTS: A CCR and an HCR were noted in 78% (18/23) and 59% (10/17) of patients with N1 lesions, respectively, and in 60% (29/48) and 45% (14/31) of patients with N2-3 lesions, respectively. The percentage of patients with CCR who also had HCR was 67% (10/15) for patients with N1 lesions and 54% (14/26) for patients with N2-3 lesions. With a median follow-up of 18.5 months, the Kaplan-Meier disease-specific survival rate at 54 months (n = 88) was 70% (21/30) for patients with N1 lesions, 60% (24/40) for patients with N2 lesions, and 39% (7/18) for patients with N3 lesions. The overall survival and disease-specific survival rates at 5 years for all nodal groups combined were 36% (32/88) and 59% (52/88), respectively. CONCLUSIONS: A CCR to CT/RT was achieved in nearly two thirds of patients with head and neck cervical lymphogenous metastases, independent of nodal tumor load. Most patients (59% [24/41]) with CCR were pathologically tumor free before neck dissection.
OBJECTIVES: To test the efficacy of a natural cytokine mixture (IRX-2), cyclophosphamide, indomethacin, and zinc to induce immune regression of squamous cell carcinoma (SCC) of the head and neck (H&N) prior to conventional therapy and to characterize the responses. PATIENTS AND DESIGN: A phase 2 trial was performed in 15 adults with recently diagnosed, biopsy-confirmed H&N SCC (3 with stage II disease, 6 with stage III disease, and 6 with stage IV disease). The patients were treated with 20 days of perilymphatic injections of IRX-2 (administered subcutaneously at the base of the skull) in combination with contrasuppression consisting of a low-dose infusion of cyclophosphamide (300 mg/m2), and daily oral indomethacin and zinc (StressTabs) in a 21-day cycle before surgery and/or radiotherapy. Tumor dimensions, toxic effects, and disease-free survival were monitored. The tumor sections were histologically examined after surgery, and tumor reduction, fragmentation, and lymphoid infiltration were assessed. RESULTS: All 15 patients responded clinically to the 21-day IRX-2 protocol: 1 with a complete response, 7 with a partial response, and 7 with a minor response. All 15 patients responded pathologically with tumor reduction (mean, 42%) and fragmentation (mean, 50%) in the histological section and increased lymphoid infiltration. The adverse effects of the IRX-2 protocol were negligible except for an allergic skin rash (n = 1) and parotiditis (n = 1). Indomethacin caused gastritis in 1 patient. Reduction of pain and ulceration and bleeding were observed in 8 and 4 patients, respectively. Four of 5 patients with lymphopenia showed increased CD3, CD4, and CD8 cell counts. After surgery (n = 13) and/or radiotherapy (n = 10) and with a mean follow-up of 17 months, 3 patients have had recurrences, 1 patient has died of disease, 1 patient has been re-treated with immunotherapy and has no evidence of disease, and 1 patient is alive with disease. Two patients died of other causes with no evidence of disease. CONCLUSIONS: The IRX-2 immunotherapy induced lymphocyte mobilization and infiltration in H&N SCC associated with clinical and histological tumor responses indicative of immune regression in all 15 patients. Minimal toxic effects were observed, and overall survival may have been improved. A phase 3 trial seems warranted.
BACKGROUND: Lieutenant General James Longstreet was arguably the finest corps commander on either side during the Civil War. He was severely wounded at the Battle of the Wilderness in Virginia on May 6, 1864, after a successful flank attack that nearly routed the Union army. DESIGN: A thorough review of the firsthand accounts of the events leading up to and following Longstreet's wounding was made. In addition, all articles listed in the medical literature describing Longstreet's care and numerous recent texts and articles about Longstreet have been researched. RESULTS: After being wounded on May 6, Longstreet received appropriate care by John Syng Dorsey Cullen, MD. Cullen controlled the hemorrhage from Longstreet's wound, helped evacuate him from the battlefield, and diligently cared for him during his convalescence. CONCLUSIONS: Longstreet was wounded by "friendly fire." The bullet's trajectory and the location of the gunshot wound suggest a posterior wound of entry rather than an anterior one as has been previously assumed.
BACKGROUND: Management of the clinically negative neck among patients with oral and oropharyngeal squamous cell carcinoma at the Royal Prince Alfred Hospital, Sydney, Australia has been based on the site and stage of the primary cancer, the likely incidence of microscopic nodal involvement, the treatment modality used for the primary cancer, and whether the neck will be entered during resection or reconstruction. This report analyzes the results of treatment when patients are allocated to either treatment or observation of the neck based on these clinical factors. METHODS: This is a prospectively documented series of 162 consecutively treated patients with squamous cell carcinoma of the oral cavity and oropharynx and clinically negative necks, treated by 1 surgeon (C.J.O.). There were 128 oral cavity and 34 oropharyngeal cancers clinically staged at T1 for 62 patients, T2 for 61, T3 for 16, and T4 for 23 patients. Management of the neck consisted of elective neck dissection (END) in 96 patients (12 bilateral), elective radiotherapy in 8, and observation in 58. Neck treatment correlated with the T stage in a statistically significant way. Forty-six patients underwent postoperative radiotherapy, which was directed to the neck in 22 patients because of pathological findings following neck dissection. Free-flap reconstruction was used in 90 patients. RESULTS: Metastatic squamous cell carcinoma was identified in 32 of 108 neck dissections (30%). There was 1 positive node in 15 necks, 2 positive nodes in 11 necks, and 3 or more positive nodes in 6 necks. Extracapsular spread was present in 8 of 32 positive END specimens (25%). Regional control rates in the neck at 3 years were 94% for END, 100% for elective radiotherapy, and 98% for patients initially observed and then treated by therapeutic neck dissection. Death with uncontrolled disease in the neck occurred in 4 of 96 patients (4%) after END and 1 of 58 patients (2%) after neck observation. Overall disease-specific survival was 83%, comprising an 86% rate for patients with pathologically negative necks and 68% if pathologically positive. Disease-specific survival was 86% at 3 years for patients having END, 67% following radiotherapy, and 94% for the observation group. CONCLUSIONS: Elective neck dissection was performed in most patients, and occult metastatic disease was found in nearly 30% of neck dissections. Observation was most frequently used for patients with early stage disease, and subsequent development of neck metastases was uncommon (9%) in this group. Selective treatment of the clinically negative neck based on the primary tumor site and stage led to a high rate of regional disease control in this series.
OBJECTIVE: To evaluate the diagnostic efficacy of computed tomography (CT)-guided needle biopsies of head and neck lesions. DESIGN: All CT-guided needle biopsies of head and neck lesions performed between September 1994 and February 1999 were included. Cytopathologic and histologic records, along with patient clinical records, were reviewed. SETTING: A tertiary care medical center. PATIENTS: Patients referred for evaluation of lesions inaccessible to routine methods of needle biopsy. RESULTS: Thirty-seven patients underwent 42 CT-guided biopsies. There were included 12 lesions in or adjacent to the skull base and 9 lesions around the pharyngoesophageal or laryngotracheal complex; the other lesions were located in the deep lobe of the parotid gland (n = 7), deep neck area (n = 12), and thyroid gland (n = 2). Diagnostic cytologic biopsy specimens were obtained in 38 (91%) of 42 needle biopsy procedures. The results were supported histologically and/or clinically in 36 cases (95%). Eighteen patients underwent open surgical procedures. Histologic confirmation was found in 86% of cases. Nineteen patients (51%) avoided an open surgical procedure: 11 with benign disease and 8 with recurrent malignancy. There were no false-positive or false-negative results, and no complications were identified. CONCLUSIONS: Computed tomography-guided needle biopsy is a safe and reliable minimally invasive technique for the diagnosis of poorly accessible or deep-seated lesions of the head and neck. Diagnostic needle biopsies allow improved preoperative planning and patient counseling in surgical patients and avoidance of open surgical procedures in patients with benign disease or recurrent malignant neoplasms.
OBJECTIVE: To define the site-specific swallowing dysfunctions of patients with head and neck cancer with respect to tumor site and stage by, videofluoroscopic oropharyngeal motility (OPM) study prior to initiation of treatment. DESIGN: Retrospective survey. SETTING: Academic university institution. PATIENTS: A consecutive sample of 79 patients with stage III or IV head and neck cancer without prior treatment or tracheotomy. Patients were divided into groups according to tumor site: oral cavity (n = 7), oropharynx (n = 27), larynx (n = 24), and hypopharynx (n = 10). Patients with sinonasal, nasopharyngeal, and unknown primary carcinomas served as the comparison group (n = 11). INTERVENTION: All patients underwent OPM study prior to treatment. MAIN OUTCOME MEASURES: Parameters of swallowing function, including oral impairment, pharyngeal impairment, cervical esophageal impairment, aspiration, and Swallowing Performance Status Scale (SPSS) score (a global measure of swallowing function) were extracted from the pretreatment OPM study and analyzed with reference to tumor site, T stage, and overall stage. The relations between tumor site and area or degree of dysfunction, and between stage of disease and area or degree of dysfunction were analyzed using chi2 and Fisher exact tests. RESULTS: Aspiration status, cervical esophageal impairment, and pharyngeal impairment examined as a function of disease site showed statistically significant differences between groups, with laryngeal and hypopharyngeal sites revealing the most severe dysfunctions. The SPSS score did not correlate with tumor site, T stage, or overall stage. Other OPM parameters analyzed as a function of T stage and overall stage revealed no consistent patterns. CONCLUSIONS: Hypopharyngeal and laryngeal disease sites have a high degree of pretreatment functional impairment. The SPSS score is a good global measure of swallowing dysfunction. In addition, significant site-specific dysfunctions are found when the OPM study is analyzed via its separate parameters. It is therefore critical that posttreatment function is compared with baseline pretreatment dysfunction.
BACKGROUND: The swallowing deficits that result from oral or oropharyngeal resections vary considerably depending on the site, extension of the resection, and type of reconstruction. Most patients will experience some degree of dysphagia despite the reconstructive effort. Furthermore, a glossectomy is frequently associated with voice and speech difficulties. OBJECTIVES: To characterize swallowing in patients who underwent a glossectomy and to define the limits and the compensatory movements using video fluoroscopic analysis. DESIGN AND SETTING: Video fluoroscopic evaluation of 15 patients who underwent glossectomies at the Centro de Tratamento e Pesquisa Hospital do Cancer A. C. Camargo, S*ao Paulo, Brazil. PATIENTS: We examined 15 patients: 5 who underwent a partial glossectomy, 2 who underwent a subtotal glossectomy, and 8 who underwent a total glossectomy with laryngeal preservation and reconstruction with myocutaneous flaps (9 pectoralis major flaps and 1 latissimus dorsi flap). The 15 patients were enrolled in a program that included voice, speech, and swallowing rehabilitation. RESULTS: All patients who underwent a partial glossectomy had difficulties with formation and anteroposterior propulsion of the bolus in the oral cavity and an increase in oral transit time, which was more evident with materials of thicker consistencies. All patients who underwent a total or subtotal glossectomy with laryngeal preservation had an increase in oral transit time and stasis of food in the oral cavity, the pharynx, and the superior esophageal sphincter. Of the 15 patients, 2 had moderate and asymptomatic aspiration. These 2 patients had swallowing compensations, such as increased buccal, mandibular, pharyngeal, and laryngeal activity and voluntary protection of the larynx during swallowing. CONCLUSIONS: This study demonstrates the effectiveness of swallowing in patients who were enrolled in voice, speech, and swallowing rehabilitation after undergoing a partial or total glossectomy. An increase in oral transit time was detected in all patients. Only 2 of the 10 patients who underwent a total glossectomy had persistent asymptomatic aspiration.
OBJECTIVE: To evaluate the long-term effects on swallowing function of concomitant continuous infusion hydroxyurea and hyperfractionated radiation therapy used to treat advanced head and neck carcinoma. DESIGN: A prospective evaluation of swallowing function was performed on an inception cohort by analyzing posttreatment videoflouroscopic swallow function studies using radiological descriptors for pharyngeal transport abnormalities and temporal measures of structural movements, as well as by conducting patient interviews to assess alimentation, more than 1 year after tumor treatment (range, 52-124 weeks; median, 70 weeks). SETTING: Academic tertiary care referral medical center. PATIENTS: Ten patients, aged 44 to 71 years, with stage III and IV squamous cell carcinoma of the oral cavity, oropharynx, or hypopharynx. MAIN OUTCOME MEASURE: Radiographic and temporal swallow abnormalities, as well as functional status, were documented and compared with published norms and results of earlier swallowing studies when possible. RESULTS: Pharyngeal transport dysfunction and anterior segment abnormalities, manifested by epiglottic dysmotility, vallecular residue, laryngeal penetration, or aspiration, were evident in all 10 patients. Posterior segment abnormalities, such as pharyngeal stasis, constrictor dysmotility and piriform residue were documented in 8 patients. Three patients developed late aspiration, and the majority of patients showed persistent or worsened delay in laryngeal movement compared with their earlier posttreatment evaluations. Also, 3 patients developed a hypopharyngeal stricture, and 6 patients continued to require gastrostomy tube supplementation beyond 1 year. There was no association between site of primary, duration to swallowing evaluation, and severity of dysfunction. CONCLUSION: Prolonged and debilitating functional swallowing abnormalities may occur after this aggressive concomitant chemotherapy and radiotherapy regimen.
OBJECTIVE: To evaluate the benefits and difficulties encountered when using various 3-dimensional (3-D) navigation systems in head and neck procedures. DESIGN: Five different navigation systems were used for preoperative planning and intraoperative 3-D navigation in procedures at the paranasal sinuses, the frontal and lateral skull bases, and the petrous bone. INTERVENTION: Intraoperative 3-D localizing systems (position-sensitive mechanical arms, infrared cameras, etc) demand reliable patient fixation on the operating table. We achieved this by developing a noninvasive head holder. Other systems allow patient movements by using magnetic digitizing technology (ARTMA System) and sophisticated programming. RESULT: Having surpassed an initial learning curve, we now achieve an accuracy of 1 to 2 mm regularly. Especially in paranasal and frontal basal surgery, all navigation systems used provide valuable positioning information during surgery. In particular for revision or tumor surgery, decisive benefits resulted from use of these systems: shorter overall operation time; safer manipulation near delicate structures; and reliable identification of the skull base even in patients with bleeding, scarring, or missing anatomical landmarks. CONCLUSIONS: We performed approximately 250 operations with different systems and introduced navigation at the lateral skull base and the petrous bone with mechanical, optic, and magnetic digitizers. In these anatomical areas, navigation was used successfully; the technical challenge is greatest at the lateral skull base, however.
OBJECTIVE: To report on 8 years of experience with 156 titanium mesh and porous polyethylene implants used for craniofacial reconstruction after skull base surgery in 100 patients. DESIGN: Cohort study with a mean follow-up of 5 years. SETTING: Population based. PATIENTS: A consecutive sample of 100 patients treated for skull base tumors or craniofacial trauma who underwent reconstruction with 156 3-dimensional titanium mesh and/or porous polyethylene implants. A retrospective review of the Skull Base Program database, along with photographic and imaging documentation, was undertaken. MAIN OUTCOME MEASURES: Rate of complications as well as the degree of functional and esthetic reconstruction. INTERVENTION: The reconstructive technique focused primarily on the substitution of removed craniofacial skeleton for oncologic reasons or soft tissue defects. RESULTS: After completion of follow-up (mean, 5 years), all 100 patients remained healed except for 7 patients (7%) with 8 implants (5%). Overall, excellent craniofacial symmetry and stability were achieved with both types of implants. CONCLUSIONS: Immediate craniofacial skeletal reconstruction and soft tissue augmentation is feasible with 3-dimensional titanium mesh and porous polyethylene implants. The reviewed 8-year evolution in the use of these technologies (156 implants in 100 patients) highlights the excellent tolerance of these implants (5% implant complication rate) in 100 patients (7% complication rate). The few encountered complications were judged to be primarily related to the quality of the overlying soft tissue and not to the implants themselves. The advantages of using these implants for immediate 3-dimensional skeletal and soft tissue substitution, including availability, easy contouring, stability, primary healing, and tolerance of adjuvant therapy, translate to an improved function and esthetic appearance, with a better quality of life for patients.
OBJECTIVE: To determine whether levels of PH-20, a hyaluronidase similar to that found in human sperm, are elevated in laryngeal cancer tissue. DESIGN: In this case-control study. reverse transcription polymerase chain reaction was used to measure levels of PH-20 messenger RNA in tissue taken from laryngectomy specimens. SETTING: A university medical center. PATIENTS: We compared tissue samples taken from 11 patients with laryngeal cancer, and from 2 metastatic lymph nodes, with samples of normal, healthy laryngeal tissue and prostate cancer tissue (positive control). MAIN OUTCOME MEASURE: PH-20 complementary DNA expression as quantified by densitometric analysis. RESULTS: Expression of PH-20 was significantly higher in nonirradiated laryngeal cancer specimens than in normal laryngeal tissue (P<.01). Metastatic lymph nodes also had higher levels of PH-20 expression than did primary laryngeal cancer tissue (P = .11) and normal laryngeal tissue (P<.01). Irradiated laryngeal cancer specimens had PH-20 levels comparable to normal. CONCLUSIONS: We report the first data on PH-20 expression in laryngeal cancer tissue. PH-20 expression is significantly elevated in primary laryngeal cancer tissue and seems to be even higher in metastatic lesions compared with normal laryngeal tissue. PH-20 may be a useful tumor marker and prognostic tool for laryngeal cancer.
OBJECTIVE: While clinical observation has suggested an association between gastroesophageal reflux and laryngeal carcinoma, the nature of this relationship has yet to be defined. The purpose of this study is to determine the carcinogenic potential of acid and pepsin mixtures in the hamster cheek pouch animal model. DESIGN: A blinded intervention study. SUBJECTS: One hundred male Syrian hamsters aged approximately 5 weeks. INTERVENTIONS: A control group of 20 hamsters received application of the carcinogen 9,10-dimethyl-1,2-henzanthracene (DMBA) to their cheek pouch mucosa. One experimental group (n = 20) received applications of DMBA plus hydrochloric acid, and another (n = 20) received DMBA plus an acid and pepsin solution. Latency to squamous cell tumor production, size of tumors, and numbers of tumors were compared among groups. RESULTS: Latency to tumor production and size of tumor were similar among groups, with both experimental and control groups developing tumors of comparable size after 12 weeks of chemical application. However, the number of tumors produced was significantly higher in the DMBA/acid and DMBA/acid/ pepsin groups than in the DMBA only group at 18 weeks, with 23, 27, and 10 tumors in these groups, respectively (P<.02). Likewise, a cumulative dysplasia score was different among groups at 18 weeks with the DMBA/acid and DMBA/acid/pepsin groups scoring higher degrees of dysplasia than the DMBA only group. CONCLUSION: These results suggest that application of acid and acid/pepsin mixtures may promote experimental carcinogenesis in the hamster cheek pouch.
DESIGN: Retrospective analysis of a case series. SETTING: Referral center, private or institutional practice, hospitalized care. OBJECTIVE: To analyze the level (site) of ipsilateral neck recurrences after supraomohyoid (SOH) dissection in patients with lip, oral, and oropharyngeal cancer treated in a single institution. INTERVENTION: Supraomohyoid neck dissection. PATIENTS AND METHODS: From 1979 to 1997, 154 patients with oral and oropharyngeal carcinoma and no palpable lymph nodes at the neck underwent ipsilateral elective SOH dissection. RESULTS: Tumor sites were the lip, 5 cases (3.3%); oral cavity, 128 cases (83.1%); and oropharynx, 21 cases (13.6%). Tumor stages were T1, 13 cases (8.4%); T2, 77 cases (50.0%); T3, 40 cases (27.0%); and T4, 22 cases (14.3%). There were 7 cases (4.5%) of ipsilateral neck recurrences. Three were beyond the limits of the SOH dissection, and 4 were inside these limits. There was no association of neck recurrences with the pathological status of the lymph nodes. Six of the 7 recurrences were in patients who underwent postoperative radiotherapy. CONCLUSIONS: The incidence of neck recurrence after selective neck dissection was 4.5%, and it occurred either inside (57.1%) or beyond (42.9%) the limits of the selective neck dissection.
BACKGROUND: Supraomohyoid neck dissection (SOHND) is currently used as a staging procedure for patients with clinically negative nodes in the neck who are at increased risk (>20%) for metastatic disease. OBJECTIVE: To assess the potential role of SOHND in patients with clinically positive nodes at levels I, II, or III. We evaluated, in particular, whether selective neck dissection in patients with clinically positive nodes results in decreased regional control and/or diminished survival. PATIENTS AND METHODS: We retrospectively reviewed the charts of all patients who underwent SOHND from January 1, 1971, to December 31, 1997. The oral cavity and oropharynx represented the primary sites in the majority of the patients. Two-year follow-up information was available on all patients. RESULTS: During the study period, 69 patients underwent 84 SOHNDs. Of the 69 patients, there were 30 patients with clinically negative nodes and 39 patients with clinically positive nodes in the neck. The overall regional control rates were 88% vs 71% for pathologically negative vs positive nodes, respectively, with or without adjuvant radiation therapy. Adjuvant radiation therapy significantly improved regional control in patients with pathologically positive nodes but not in patients with NO disease (P = .005). Similar results were noted in patients with both clinically and pathologically positive nodes. CONCLUSIONS: Supraomohyoid neck dissection in patients with pathologically positive nodes in the neck is inadequate therapy for regional control without postoperative radiation therapy. However, in patients with pathologically positive nodes in the neck, SOHND with postoperative radiation therapy can achieve regional control comparable to that of comprehensive neck dissection and postoperative radiation therapy.
OBJECTIVE: To evaluate the possibility, complications, and efficacy of endoscopic neck dissection (END) in a porcine model. DESIGN: Experimental self-controlled study. SUBJECTS: Minipigs. INTERVENTION: Endoscopic neck dissection was performed using general anesthesia with techniques adapted from laparoscopic surgery. The tissue specimens removed were divided according to porcine equivalents of human neck groups. After the completion of END, open-neck dissection was performed using standard surgical techniques, and the remaining tissue within each neck group was retrieved. A pathologist evaluated each specimen without knowing its exact origin in terms of neck group or side and the type of surgical technique used. For each specimen, the number of retrieved lymph nodes and their anatomical integrity were analyzed. RESULTS: Ten neck dissections were performed in 8 minipigs without any major complications. The number of retrieved lymph nodes by END was 18.4 +/- 7.4 (mean +/- SD). Completed open-neck dissection retrieved an additional 3.3 +/- 1.8 lymph nodes. The efficacy rate of END was 88% +/- 10% (+/ -SD). The majority of retrieved lymph nodes were intact, with less than 5% of nodes exhibiting crushing artifacts. CONCLUSIONS: Endoscopic neck dissection in a porcine model seems to be free of major complications and able to retrieve the majority of neck lymph nodes. A larger number of animals and their survival need to be studied before human studies can begin.
OBJECTIVE: To report our experience in treating 4 cases of recurrent siacloceles with botulinum toxin type A after partoid surgery. DESIGN: This is a prospective, nonrandomized, nonblinded pilot study describing a new use for botulinum toxin type A. SETTING: Tertiary academic medical center. PATIENTS:Four patients (2 men and 2 women) with persistent postparotidectomy sialoceles who had undergone various treatment failures were included. The diagnosis was made by fine-needle aspiration of the mass based on well-recognized cytologic features of the entity, as well as an elevated amylase level and no evidence of tumor or infection. INTERVENTIONS: Sialoceles were aspirated before local injection of botulinum toxin type A (30-50 U) subcutaneously. MAIN OUTCOME MEASURES: The patients were followed up 1 week after receiving botulinum toxin type A injection and then at monthly intervals. They were extensively questioned and examined for any evidence of side effects or recurrence. RESULTS: All patients had total resolution of sialocele or external salivary fistula within 1 month of treatment. None of the patients to date have demonstrated recurrences at 7 through 13 months, and there were no complications, particularly facial nerve weakness. CONCLUSION: Our findings suggested that botulinum toxin type A offers a highly effective, safe, and noninvasive method of treatment in postparotidectomy sialocele.
OBJECTIVE: To evaluate sensory changes in the head and neck region associated with selective neck dissection with or without preservation of cervical root branches. DESIGN: Retrospective cohort study. SETTING: University tertiary referral hospital and a Veterans Affairs hospital. PATIENTS: Fifty-seven patients who had undergone 84 neck dissections with or without preservation of the sensory cervical root branches 3 or more months before evaluation. INTERVENTIONS: Questionnaire combined with head and neck sensory examination. MAIN OUTCOME MEASURES: Neck and facial sensory function. RESULTS: Neck dissections with preservation of the cervical rootlets were most likely to be associated with a small area of anesthesia in the upper neck below the body of the mandible and anterior to the mid-body of the mandible (P=.03). Neck dissections without rootlet-preserving technique increased the area of anesthesia to include all other areas of the neck (P= .02). CONCLUSIONS: Preservation of the cervical root branches resulted in a small, limited, and uniform area of the neck rendered permanently anesthetic. Conversely, sacrifice of the nerve branches led to a pattern of anesthesia involving the entire neck.
BACKGROUND: The occurrence of second primary neoplasms in patients with head and neck carcinoma assumes greater importance as our ability to control local disease improves. Both the primary lesions and the therapeutic interventions can predispose patients to pulmonary complications. OBJECTIVE: To explore the incidence of pulmonary complications in patients with head and neck cancer who also undergo lung surgery. DESIGN: Survey; case series. SETTING: A tertiary care university hospital. RESULTS: The clinic and hospital charts of 32 patients with multiple interventions of the head and neck and lung were retrospectively reviewed, and data were recorded on the clinical and pathologic specifics of primary and secondary neoplasms, pulmonary complications, and treatment outcomes. Twenty-eight (88%) of these patients underwent a diagnostic or therapeutic surgical procedure for a head and neck primary neoplasm. All patients (100%) underwent a pulmonary resection for malignant or nonmalignant pulmonary disease. Overall, 31 patients (97%) experienced either major or minor pulmonary complications after surgery, 51 (79%) of which occurred during the immediate postoperative course. Major complications occurred in 11 patients (34%), which were fatal in one. CONCLUSIONS: Our data suggest that patients with head and neck cancer who also experience a second pulmonary disease requiring lung resection are at high risk of developing serious pulmonary complications. These risks should be considered in planning optimal therapy.
BACKGROUND: The sentinel lymph node (SLN) biopsy is revolutionizing the surgical management of primary malignant melanoma. It allows accurate nodal staging, and targets patients who may benefit from regional lymphadenectomy and systemic therapy; however, its use in the management of head and neck melanoma has not been widely accepted. METHODS: A retrospective review of patients treated for clinical stages I and II malignant melanoma of the head and neck with dynamic lymphoscintigraphy and gamma probe-guided SLN biopsy. RESULTS: Fifty-eight patients (47 male and 11 female) were identified. Primary melanoma sites included the scalp (21), ear (8), face (13), neck (15), and eyelid (1). Primary tumor staging was T2 (11), T3 (24), and T4 (23). Dynamic lymphoscintigraphy visualized SLNs in 57 patients (98.3%). In 43 cases (75%) a single draining nodal basin was identified, and in 14 cases there were multiple draining nodal basins. Sentinel lymph nodes were successfully identified in 72 (96%) of 75 nodal basins. Positive SLNs were identified in 10 patients (17.5%). Sentinal lymph node positivity by tumor staging was T3, 16.7% and T4, 27.3%. Completion lymphadenectomy revealed residual disease in 3 patients (30%). Relapse occurred in 10 (21.3%) of the 47 patients with negative SLN biopsy results and 7 (70%) of those with positive results. CONCLUSIONS: Gamma probe-guided SLN localization in the head and neck region was successful in 96% of draining nodal basins. It can target regional lymphadenectomy in patients who may benefit from regional nodal dissection.
Natural aroma compounds are of major interest to the flavor and fragrance industry. Due to the limited sources for natural aromas, there is a growing interest in developing alternative sources for natural aroma compounds, and in particular aromatic aldehydes. In several microbial species aromatic aldehydes are detected as intermediates in the degradation pathway of phenylpropanoids. Thus, bioconversion of phenylpropanoids is one possible route for the production of these aroma compounds. The present work describes the isolation of microbial strains, capable of producing vanillin from isoeugenol. Bacterial strains isolated from soil, were screened for their ability to transform isoeugenol to vanillin. One of these strains, strain B2, was found to produce high amounts of vanillin when grown in the presence of isoeugenol, and was also capable of growing on isoeugenol as the sole carbon source. Based on its fatty acids profile, strain B2 was identified as a Bacillus subtilis sp. The bioconversion capabilities of strain B2 were tested in growing cultures and cell free extracts. In the presence of isoeugenol, a growing cultures of B. subtilis B2 produced 0.61 g l-1 vanillin (molar yield of 12.4%), whereas cell free extracts resulted in 0.9 g l-1 vanillin (molar yield of 14%).
Recently the existence of 'heavy chain' immunoglobulins in Camelidae has been described. However, as yet there is no data on the binding of this type of antibody to haptens. In addition, it was not a priori predictable whether the binding domains (VHH) of these antibodies could be produced and secreted by the lower eukaryotic micro-organism Saccharomyces cerevisiae. In the present study these questions are addressed. Heavy chain immunoglobulins directed against two hapten molecules, the azo-dyes RR6 and RR120 as well as the (proteinaceous) human pregnancy hormone, have been raised in Lama glama. We were able to select specific VHH fragments for all three antigens by direct screening of Escherichia coli or yeast libraries, even without prior enrichment via bio-panning. This is the first example of the isolation of llama anti-hapten VHH domains. Surprisingly, the affinities of the llama VHHs for the RR6 hapten obtained in this way are in the low nM range. Furthermore, some of the antigen specific VHHs were secreted by S. cerevisiae at levels over 100 mg l-1 in shake flask cultures. These two findings extend the possible application areas for the llama VHH fragments significantly.
1H/15N and 13C NMR were used to investigate metabolism in Spodoptera frugiperda (Sf9) cells. Labelled substrates ([2-15N]glutamine, [5-15N]glutamine, [2-15N]glutamate, 15NH4Cl, [2-15N]alanine, and [1-13C]glucose) were added to batch cultures and the concentration of labelled excreted metabolites (alanine, NH4+, glutamine, glycerol, and lactate) were quantified. Cultures with excess glucose and glutamine produce alanine as the main metabolic by-product while no ammonium ions are released. 1H/15N NMR data showed that both the amide and amine-nitrogen of glutamine was incorporated into alanine in these cultures. The amide-nitrogen of glutamine was not transferred to the amine-position in glutamate (for further transamination to alanine) via free NH4+ but directly via an azaserine inhibitable amido-transfer reaction. In glutamine-free media 15NH4+ was consumed and incorporated into alanine. 15NH4+ was also incorporated into the amide-position of glutamine synthesised by the cells. These data suggest that the nitrogen assimilation system, glutamine synthetase/glutamate synthase (NADH-GOGAT), is active in glutamine-deprived cells. In cultures devoid of glucose, ammonium is the main metabolic by-product while no alanine is formed. The ammonium ions stem both from the amide and amine-nitrogen of glutamine, most likely via glutaminase and glutamate dehydrogenase. 13C NMR revealed that the [1-13C] label from glucose appeared in glycerol, alanine, lactate, and in extracellular glutamine. Labelling data also showed that intermediates of the tricarboxylic acid cycle were recycled to glycolysis and that carbon sources, other than glucose-derived acetylCoA, entered the cycle. Furthermore, Sf9 cell cultures excreted significant amounts glycerol (1.9-3.2 mM) and ethanol (6 mM), thus highlighting the importance of sinks for reducing equivalents in maintaining the cytosolic redox balance.
Effects of recycling ECF-bleached softwood kraft pulp on pulp properties were evaluated in the laboratory. The tensile strength, fiber flexibility and WRV lost during drying of the pulp were recovered by refining between the cycles which, however, resulted in deteriorated drainage properties. The recycled pulps were treated with purified Trichoderma reesei cellulases and hemicellulases and the changes in fiber properties due to enzymatic treatments were characterized. The endoglucanases (EG I and EG II) significantly improved pulp drainage already at low dosage levels, and EG II was found to be more effective at a given level of carbohydrate solubilization. Combining hemicellulases with the endoglucanase treatments increased the positive effects of the endoglucanases on pulp drainage. However, as a result of the endoglucanase treatments a slight loss in strength was observed. Combining mannanase with endoglucanase treatments appeared to increase this negative effect, whereas the impact of xylanase was not significant. Although the drainage properties of the pulps could be improved by selected enzymes, the water retention capacity of the dried hornified fibers could not be recovered by any of the enzymes tested.
Proteolytically cleaved human 22 kDa growth hormone (22K hGH) between the amino acid residues 134 and 150 by plasmin or other proteases in vitro has been reported to be most active in growth promoting activity. In this study a deleted mutant hGH lacking amino acid residues from 135 to 146 and having more sensitivity to plasmin digestion was produced using the inverse polymerase chain reaction method and the Escherichia coli expression system. The mutant, hGH delta 135-146, was folded and purified effectively and found to be more sensitive to plasmin cleavage to form the two-chain form in vitro. The biological activities of this plasmin sensitive hGH delta 135-146 were tested by in vitro cell proliferation assays and in vivo growth promoting assay. In Ba/F3-hGHR cells, which express receptors for hGH, hGH delta 135-146 showed 10-20% less growth promoting activity than 22K hGH, but expressed comparable quantities of IGF-I mRNA to that of 22K hGH. In Nb2 rat lymphoma cells, which proliferate in response to hGH via the lactogenic receptors, hGH delta 135-146 showed equivalent activities to those of 22K hGH at lower concentrations. By the body weight gain test using hypophysectomized rats, a lower dose (2.5 nmol kg-1) of hGH delta 135-146 exhibited an equivalent activity to that of wild type 22K hGH, but a higher dose (25 nmol kg-1) of the mutant showed less growth promoting activity than 22K hGH. These results indicated that the plasmin sensitive recombinant hGH delta 135-146 failed to show higher biological activity than the 22K hGH in vivo, suggesting the unsuccessful formation of the active two-chain form in vivo.
Recombinant human interferon-gamma and chloramphenicol acetyltransferase I were isolated from two Escherichia coli strains, E. coli LE329 and E. coli XL1-blue and characterized by electrospray ionization mass spectrometry (ESI-MS). The ESI-MS analysis showed higher masses in comparison with the theoretically calculated for both proteins as well as unexpected molecular heterogeneity. The ESI-MS spectral patterns of the proteins depended on the host strain used and were more heterogenous for the proteins isolated from E. coli LE392. One of the proteins (human interferon-gamma obtained from E. coli XL1-blue) was further subjected to BrCN cleavage. The ESI-MS analysis of the polypeptide mixture revealed shift in the molecular mass for two peptides including the last 26 amino acids of the human interferon-gamma molecule.
A conventional anion exchange column packed with porous particles (BioScale Q2), and a novel continuous-bed column (UNO Q1) were compared for displacement separation of dairy whey proteins with polyacrylic acid as displacer. The steric mass action model was investigated as a means to aid and accelerate this development. Characteristic charges and steric factors were measured for the proteins and the displacer according to the model, and used together with the affinity constant derived from the adsorption isotherms for simulations, as well as for the construction of the affinity and operating regime plots. If possible, the latter two were used to select conditions for the actual experiments. In the case of the particle-based column, experimental results and simulations did not agree. In addition, the operating regime plot could not be constructed. The affinity plot did predict the order in the displacement train correctly, but gave misleading information concerning the possible effect of a change in displacer concentration. This is taken to be a result of the porous nature of the particles, which handicaps, to some extent, the interaction of the proteins and the displacer molecules with the adsorptive surface. Results were considerably better in case of the continuous-bed column, where there is no intraparticulate surface.
The extracellular xylanase from Bacillus stearothermophilus T-6 is a thermostable alkaline tolerant enzyme that was found to bleach pulp optimally at pH 9 and 65 degrees C, and was successfully used in a large-scale bio-bleaching mill trial. In an attempt to obtain a heavy atom derivative suitable for complete X-ray analysis, xylanase T-6 was labeled biosynthetically with seleno-methionine, resulting in a 'built-in' array of atoms with specific X-ray anomalous scattering signal. Optimization of growth conditions resulted in over 0.8 g of homogeneous seleno-methionine xylanase T-6 per liter culture. The seleno-methionine enzyme was shown to be fully active and produced single crystals suitable for complete multiple wavelength anomalous diffraction (MAD) structural analysis.
In situ hybridization techniques have been an important research tool since first introduced 30 years ago, and more recently clinical applications have been expanding greatly. Still, further improvements in the assay sensitivity and protocols that are amenable to routine clinical use are desired. We use a novel photo-cross-linking technology to irreversibly bind short oligonucleotide probes to the target sequence following a hybridization period. The cross-linking agent is incorporated into the backbone of the probe and is activated to react with pyrimidines in the opposite strand by near-UV (300-370 nm) irradiation. By locking the probe to the target, very stringent wash conditions can be used that would otherwise completely remove probes that are hybridized but not cross-linked to the target. Consequently, the probe-specific signal is maximized, while the background signal is minimized to the greatest extent possible with the stringency of the wash. The use of short, photo-cross-linkable probes presents a new strategy for maximizing the sensitivity of probe hybridization or signal amplification-based in situ techniques.
Lipid-mediated transfection was compared to adenoviral-mediated gene transfer in COS-7 cells as well as human monocyte-derived macrophages (HMDM). For this purpose, we monitored enhanced green fluorescent protein (EGFP) expression by fluorescence microscopy and quantified gene transfer by competitive PCR. Transfection of COS-7 cells with a novel lipid formulation for DNA transfer was highly effective in COS-7 cells. On average, 30% of the cells were fluorescent 48 h after transfection. In HMDM, the same formulation resulted in the expression of EGFP in less than 0.5% of cells. We measured plasmid DNA by quantitative PCR in lipid-transfected macrophages and found that each macrophage contained on average 2 fg of plasmid DNA 24 h after transfection, that is, more than 400 molecules of plasmid DNA entered each cell. Despite the high level of reporter DNA in lipid transfected cells, expression of the fluorescent protein was suppressed in more than 99.5% of the macrophages. We also used adenoviral gene transfer to introduce the foreign DNA into both COS-7 cells and HMDM. Even though the multiplicity of infection was less than 30, expression of EGFP was observed in nearly all COS-7 cells and in more than 80% of HMDM 48 h after transfection. Despite major advances in the field of lipid-mediated transfection of HMDM, the lipid formulations that are available commercially cannot compete with the efficiency of adenoviral gene transfer.
Several techniques are available that detect variations in gene expression between cellular populations. These include subtractive hybridization (SH), differential colony hybridization (DCH) and mRNA differential display, all based on the analysis of mRNA. The first two techniques, however, are limited because they require large amounts of mRNA for SH or several rounds of screening for DCH. Differential display overcomes both of these limitations. However, the conventional differential display technique is plagued by false positives and is labor intensive. The identification of genes that are truly differentially expressed, therefore, becomes a formidable task. We describe a modified differential display technique that overcomes the limitations of the conventional technique. This new technique eliminates a source of false positives, decreases the time required to screen a set of primers and reduces the use of radioactivity.
A new method to produce hot-start conditions in PCR is described. Short double-stranded DNA fragments were found to inhibit the activity of DNA polymerases from Thermus aquaticus and Thermus flavus. This inhibition is not sequence specific, but exclusively dependent on the melting temperature of the fragments as shown by its correlation to their melting curves as measured. This property is exploited by adding fragments of the appropriate length to the PCR mixture during the reaction setup and thereby preventing the DNA polymerases from extending primers annealed nonspecifically at lower than the optimal temperature. By amplifying ten copies of phage lambda DNA in the presence of 2 micrograms of nonspecific DNA, it is shown for three different primer pairs how the melting temperatures of the double-stranded DNA fragments have to be adapted to the cycle profiles to obtain predominantly specific products in the 0.5 microgram range.
Present methods for DNA isolation of stool have various limitations such as the amount of stool used, the requirement of lavage fluids or the use of fresh stool. In this paper, a new method is described for the isolation of human nucleic acids from stool, which is independent from the moment of collection. Fecal samples as dry as possible were collected from 75 patients; two grams of stool were mixed with a lysis buffer containing phenol. DNA yields of crude stool were variable and ranged from 9-1686 micrograms/g of feces. With dot blots in 9 of the 75 cases, the human DNA was identified and ranged from 0.06%-46%. In the remaining 66 cases, human genomic DNA was detected by nested PCR, using human K-ras gene amplification as an example. Amplification products were confirmed for human K-ras with the exonuclease-amplification coupled capture technique (EXACCT). In conclusion, the developed DNA isolation method can be used for the study of large numbers of stool samples, is independent of the age or method of stool collection and is suitable for large-scale screening studies.
The isolation of intact, functional RNA from conifer spp. is not easy, especially from those tissues that are heavily lignified and characterized by a low number of living cells. An efficient procedure for isolating RNA from combined wood and bark tissues of conifers was developed based on a protocol optimized for the extraction of RNA from pollen and one for the isolation of RNA from woody stems. This protocol does not involve the use of phenol, and no ultracentrifugation was required. In addition, the protocol overcame the problems of RNA degradation and low yield due to oxidation by polyphenolics and co-precipitation with polysaccharides, both of which are abundant components in conifer bark tissues. The isolated RNA was of high quality and undegraded as gauged by spectrophotometric readings and electrophoresis in denaturing agarose gels. Quality was further assessed through the subsequent use of the RNA in reverse transcription and RT-PCR, indicating that it could be used for a number of downstream purposes including Northern blot hybridization and cDNA library construction. Using this modified protocol, 80-150 micrograms of RNA was routinely obtained from 1 g of fresh material. This protocol was also used for the isolation of RNA from needles of spruce spp., from which 750-950 micrograms RNA per gram of starting material could routinely be obtained.
Two approaches are described for stably conjugating peptides, proteins and oligonucleotides onto plasmid DNA. Both methods use a peptide nucleic acid (PNA) clamp, which binds irreversibly and specifically to a binding site cloned into the plasmid. The first approach uses a biotin-conjugated PNA clamp that can be used to introduce functional biotin groups onto the plasmid to which streptavidin can bind. Atomic force microscopy images of linearized plasmid show streptavidin localized at the predicted PNA binding site on the DNA strand. Peptides and oligonucleotides containing free thiol groups were conjugated to maleimide streptavidin, and these streptavidin conjugates were bound to the biotin-PNA-labeled plasmid. In this way, peptides and oligonucleotides could be brought into stable association with the plasmid. A second approach used a maleimide-conjugated PNA clamp. Methods are described for conjugating thiolated peptides and oligonucleotides directly to the maleimide-PNA-DNA hybrid. This straightforward technology offers an easy approach to introduce functional groups onto plasmid DNA without disturbing its transcriptional activity.
The conjugation of enzymes, fluorescent or radioactive labels, cross-linkers and other moieties to antibodies is a commonly performed procedure in biochemical research. Using reduced disulphides, conjugation can be an inconvenient, multistep, time- and material-consuming process. We have developed a reduction technique based on UV irradiation, which lacks these drawbacks. Antibodies are irradiated in a sealed vial for a few minutes by a common laboratory UV source in the presence of stannous ions, following the depletion of atmospheric oxygen. The preparation may subsequently be conjugated with thiol-reactive probes such as maleimide derivatives, with no need for any prior purification or concentration. This simple, rapid and effective reduction and conjugation process results in a fully functional immunoglobulin conjugate that can be used for a variety of biochemical applications.
While many novel associations predicted by two-hybrid library screens reflect actual biological associations of two proteins in vivo, at times the functional co-relevance of two proteins scored as interacting in the two-hybrid system is unlikely. The reason for this positive score remains obscure, which leads to designating such clones as false positives. After investigating the effect of over-expressing a series of putative false positives in yeast, we determined that expression of some of these clones induces an array of biological effects in yeast, including altered growth rate and cell permeability, that bias perceived activity of LacZ reporters. Based on these observations, we identify four simple strategies that can assist in determining whether a protein is likely to have been selected in a two-hybrid screen because of indirect metabolic effects.
The performance of conventional cell-free gene expression systems based on the Escherichia coli S30 extract can be significantly improved by using expression vectors that encode viral structural elements known to enhance translation in vivo and to protect mRNA from ribonuclease action. The expression vectors reported here are designed to produce a functionally active protein carrying the Strep-tag oligopeptide at its C-terminus. They can be used in translation, transcription-translation or replication-translation reactions. Depending on its type, the reaction yields up to 40 micrograms per mL, or about 1 nmol of a standard protein. The presence of Strep-tag allows the synthesized protein to be easily isolated on a streptavidin-agarose column under mild conditions and the entire procedure to be completed within one working day. The results show that standard low-cost, cell-free systems can serve for rapid preparation of purified proteins in amounts that can satisfy a number of needs of a research laboratory.
We have developed a simple procedure for the preparation of plant genomic DNA using FTA paper. Plant leaves were crushed against FTA paper, and the genomic DNA was purified using simple, nonorganic reagents. The 18S rRNA gene and the gene encoding the ribulose-1, 5-bisphosphate carboxylase/oxygenase large subunit (rbcL) from the chloroplast genome were detected by PCR amplification of DNA on FTA paper. DNA amplification was successful using extracts from 16 dicot and monocot plants. Studies of specific plant extracts revealed that extracts of leaf samples could be collected and stored at room temperature on FTA paper without a decrease in the DNA amplification success rate for more than a month. Both the 18S RNA gene and the rbcL gene were detected in the genomic DNA isolated from various soybean cultivars stored in this manner. Furthermore, by modestly increasing the number of cycles of DNA amplification, we were able to detect the uidA gene in transgenic tobacco and rice leaves as well as a single copy gene linked to the resistance gene of cyst nematode race 3 using genomic DNA isolated on FTA paper. These results demonstrate that genomic DNA isolated using FTA paper can be used for the detection of plant genes, from a wide range of plants with either high or low gene copy number and of either nuclear or cytoplasmic origin.
Cleavase Fragment Length Polymorphism (CFLP) analysis is a convenient, accurate and highly sensitive method for the detection and localization of nucleic acid mutations. The assay is well suited for high-throughput screening and can be used to detect mutations in known and unknown nucleic acid samples. A recent improvement in the CFLP assay termed "temperature ramping" or "ramping" is reported here. This procedural improvement eliminates the need for time and temperature optimizations before the actual sample analysis. In this study, we compare the CFLP ramping procedure to the conventional CFLP optimization procedure and demonstrate equal, and in some cases improved, detection of point mutations. With ramping, CFLP reactions are identical for all DNA fragments analyzed, which allows for increased sample throughput, decreased assay time and lower overall cost.
The dystrophin-glycoprotein complex (DGC) links the cytoskeleton of muscle fibers to their extracellular matrix. Using knockout mice, we show that a cytoplasmic DGC component, alpha-dystrobrevin (alpha-DB), is dispensable for formation of the neuromuscular junction (NMJ) but required for maturation of its postsynaptic apparatus. We also analyzed double and triple mutants lacking other cytoskeletal DGC components (utrophin and dystrophin) and myotubes lacking a alpha-DB or a transmembrane DGC component (dystroglycan). Our results suggest that alpha-DB acts via its linkage to the DGC to enhance the stability of postsynaptic specializations following their DGC-independent formation; dystroglycan may play additional roles in assembling synaptic basal lamina. Together, these results demonstrate involvement of distinct protein complexes in the formation and maintenance of the synapse and implicate the DGC in the latter process.
Motor axons form topographic maps on muscles: rostral motor pools innervate rostral muscles, and rostral portions of motor pools innervate rostral fibers within their targets. Here, we implicate A subfamily ephrins in this topographic mapping. First, developing muscles express all five of the ephrin-A genes. Second, rostrally and caudally derived motor axons differ in sensitivity to outgrowth inhibition by ephrin-A5. Third, the topographic map of motor axons on the gluteus muscle is degraded in transgenic mice that overexpress ephrin-A5 in muscles. Fourth, topographic mapping is impaired in muscles of mutant mice lacking ephrin-A2 plus ephrin-A5. Thus, ephrins mediate or modulate positionally selective synapse formation. In addition, the rostrocaudal position of at least one motor pool is altered in ephrin-A5 mutant mice, indicating that ephrins affect nerve-muscle matching by intraspinal as well as intramuscular mechanisms.
The pleiotropic functions of small GTPase Rho present a challenge to its genetic analysis in multicellular organisms. We report here the use of the MARCM (mosaic analysis with a repressible cell marker) system to analyze the function of RhoA in the developing Drosophila brain. Clones of cells homozygous for null RhoA mutations were specifically labeled in the mushroom body (MB) neurons of mosaic brains. We found that RhoA is required for neuroblast (Nb) proliferation but not for neuronal survival. Surprisingly, RhoA is not required for MB neurons to establish normal axon projections. However, neurons lacking RhoA overextend their dendrites, and expression of activated RhoA causes a reduction of dendritic complexity. Thus, RhoA is an important regulator of dendritic morphogenesis, while distinct mechanisms are used for axonal morphogenesis.
During development, basic helix-loop-helix (bHLH) proteins regulate formation of neurons from multipotent progenitor cells. However, bHLH factors linked to gliogenesis have not been described. We have isolated a pair of oligodendrocyte lineage genes (Olg-1 and Olg-2) that encode bHLH proteins and are tightly associated with development of oligodendrocytes in the vertebrate central nervous system (CNS). Ectopic expression of Olg-1 in rat cortical progenitor cell cultures promotes formation of oligodendrocyte precursors. In developing mouse embryos, Olg gene expression overlaps but precedes the earliest known markers of the oligodendrocyte lineage. Olg genes are expressed at the telencephalon-diencephalon border and adjacent to the floor plate, a source of the secreted signaling molecule Sonic hedgehog (Shh). Gain- and loss-of-function analyses in transgenic mice demonstrate that Shh is both necessary and sufficient for Olg gene expression in vivo.
Basic helix-loop-helix (bHLH) transcription factors have been identified for neurons and their precursors but not for glial cells. We have identified two bHLH factors, Oligo1 and Oligo2, that are specifically expressed in zones of neuroepithelium from which oligodendrocyte precursors emerge, as well as in the precursors themselves. Expression of Oligo2 in the spinal cord precedes that of platelet-derived growth factor receptor alpha (PDGFRalpha), the earliest known marker of oligodendrocyte precursors, by several days. Ectopic expression of Oligo2 in vivo causes ectopic expression of Sox10, an HMG-box transcription factor expressed in oligodendrocyte and other glial precursors. These data identify Oligo genes as the earliest known markers of oligodendrocyte lineage determination and suggest they play a causal role in this process.
The neurotrophin survival dependence of peripheral neurons in vitro is regulated by the proapoptotic BCL-2 homolog BAX. To study peripheral neuron development in the absence of neurotrophin signaling, we have generated mice that are double null for BAX and nerve growth factor (NGF), and BAX and the NGF receptor TrkA. All dorsal root ganglion (DRG) neurons that normally die in the absence of NGF/TrkA signaling survive if BAX is also eliminated. These neurons extend axons through the dorsal roots and collateral branches into the dorsal horn. In contrast, superficial cutaneous innervation is absent. Furthermore, rescued sensory neurons fail to express biochemical markers characteristic of the nociceptive phenotype. These findings establish that NGF/TrkA signaling regulates peripheral target field innervation and is required for the full phenotypic differentiation of sensory neurons.
We have combined genetic and biochemical approaches to analyze the function of the RNA-binding protein Nova-1, the paraneoplastic opsoclonus-myoclonus ataxia (POMA) antigen. Nova-1 null mice die postnatally from a motor deficit associated with apoptotic death of spinal and brainstem neurons. Nova-1 null mice show specific splicing defects in two inhibitory receptor pre-mRNAs, glycine alpha2 exon 3A (GlyRalpha2 E3A) and GABA(A) exon gamma2L. Nova protein in brain extracts specifically bound to a previously identified GlyRalpha2 intronic (UCAUY)3 Nova target sequence, and Nova-1 acted directly on this element to increase E3A splicing in cotransfection assays. We conclude that Nova-1 binds RNA in a sequence-specific manner to regulate neuronal pre-mRNA alternative splicing; the defect in splicing in Nova-1 null mice provides a model for understanding the motor dysfunction in POMA.
A neurotransmitter transporter can potentially mediate uptake or release of substrate, and its stoichiometry is a key factor that controls the driving force and thus the neurotransmitter flux direction. We have used a combination of electrophysiology and radio-tracing techniques to evaluate the stoichiometries of two glycine transporters involved in glycinergic or glutamatergic transmission. We show that GlyT2a, a transporter present in glycinergic boutons, has a stoichiometry of 3 Na+/Cl-/glycine, which predicts effective glycine accumulation in all physiological conditions. GlyT1b, a glial transporter, has a stoichiometry of 2 Na+/Cl-/ glycine, which predicts that glycine can be exported or imported, depending on physiological conditions. GlyT1b may thus modulate glutamatergic synapses by increasing or decreasing the glycine concentration around N-methyl-D-aspartate receptors (NMDARs).
The discrete localization of ion channels is a critical determinant of neuronal excitability. We show here that the dendritic K+ channels Kv2.1 and Kv2.2 were differentially targeted in cultured hippocampal neurons. Kv2.1 was found in high-density clusters on the soma and proximal dendrites, while Kv2.2 was uniformly distributed throughout the soma and dendrites. Chimeras revealed a proximal restriction and clustering domain on the cytoplasmic tail of Kv2.1. Truncations and internal deletions revealed a 26-amino acid targeting signal within which four residues were critical for localization. This signal is not related to other known sequences for neuronal and epithelial membrane protein targeting and represents a novel cytoplasmic signal responsible for proximal restriction and clustering.
Inhibition of "leak" potassium (K+) channels is a widespread CNS mechanism by which transmitters induce slow excitation. We show that TASK-1, a two pore domain K+ channel, provides a prominent leak K+ current and target for neurotransmitter modulation in hypoglossal motoneurons (HMs). TASK-1 mRNA is present at high levels in motoneurons, including HMs, which express a K+ current with pH- and voltage-dependent properties virtually identical to those of the cloned channel. This pH-sensitive K+ channel was fully inhibited by serotonin, norepinephrine, substance P, thyrotropin-releasing hormone, and 3,5-dihydroxyphenylglycine, a group I metabotropic glutamate receptor agonist. The neurotransmitter effect was entirely reconstituted in HEK 293 cells coexpressing TASK-1 and the TRH-R1 receptor. Given its expression patterns and the widespread prevalence of this neuromodulatory mechanism, TASK-1 also likely supports this action in other CNS neurons.
Voltage-gated K+ channels contain a central pore domain and four surrounding voltage-sensing domains. How and where changes in the structure of the voltage-sensing domains couple to the pore domain so as to gate ion conduction is not understood. The crystal structure of KcsA, a bacterial K+ channel homologous to the pore domain of voltage-gated K+ channels, provides a starting point for addressing this question. Guided by this structure, we used tryptophan-scanning mutagenesis on the transmembrane shell of the pore domain in the Shaker voltage-gated K+ channel to localize potential protein-protein and protein-lipid interfaces. Some mutants cause only minor changes in gating and when mapped onto the KcsA structure cluster away from the interface between pore domain subunits. In contrast, mutants producing large changes in gating tend to cluster near this interface. These results imply that voltage-sensing domains interact with localized regions near the interface between adjacent pore domain subunits.
Axonal regeneration in the lesioned mammalian central nervous system is abortive, and this causes permanent disabilities in individuals with spinal cord injuries. In adult rats, olfactory ensheathing glia (OEG) transplants successfully led to functional and structural recovery after complete spinal cord transection. From 3 to 7 months post surgery, all OEG-transplanted animals recovered locomotor functions and sensorimotor reflexes. They presented voluntary hindlimb movements, they supported their body weight, and their hindlimbs responded to light skin contact and proprioceptive stimuli. In addition, relevant motor axons (corticospinal, raphespinal, and coeruleospinal) regenerated for long distances within caudal cord stumps. Therefore, OEG transplantation provides a useful repair strategy in adult mammals with traumatic spinal cord injuries. Our results with these cells could lead to new therapies for the treatment of spinal cord lesions in humans.
The circadian clock in the suprachiasmatic nuclei (SCN) is comprised of a cell-autonomous, autoregulatory transcriptional/translational feedback loop. Its molecular components include three period and two cryptochrome genes. We describe circadian patterns of expression of mPER2 and mPER3 in the mouse SCN that are synchronous to those for mPER1, mCRY1, and mCRY2. Coimmunoprecipitation experiments demonstrate in vivo associations of the SCN mPER proteins with each other and with the mCRY proteins, and of mCRY proteins with mTIM, but no mPER/mTIM interactions. Examination of the effects of weak and strong resetting light pulses on SCN clock proteins highlights a central role for mPER1 in photic entrainment, with no acute light effects on either the mCRY or mTIM proteins. These clock protein interactions and photic responses in mice are divergent from those described in Drosophila.
Synchronous activity among synaptically connected interneurons is thought to organize temporal patterns such as gamma and theta rhythms in cortical circuits. Interactions between distinct interneuron circuits may underlie more complex patterns, such as nested rhythms. Here, we demonstrate such an interaction between two groups of CA1 interneurons, GABA(A,slow) and GABA(A,fast) cells, that may contribute to theta and gamma rhythms, respectively. Stratum lacunosum-moleculare (SL-M) stimuli that activate GABA(A,slow) inhibitory postsynaptic currents (IPSCs) in pyramidal cells simultaneously depress the rate and amplitude of spontaneous GABA(A,fast) IPSCs for several hundred milliseconds. This suppression has a similar pharmacological profile to GABA(A,slow) IPSCs, and SL-M stimuli elicit GABA(A,slow) IPSCs in interneurons. We conclude that GABA(A,slow) cells inhibit both pyramidal cells and GABA(A,fast) interneurons and postulate that this interaction contributes to nested theta/gamma rhythms in hippocampus.
In the primary visual cortex of macaque monkeys, laminar and columnar axonal specificity are correlated with functional differences between locations. We describe evidence that embedded within this anatomical framework is finer specificity of functional connections. Photostimulation-based mapping of functional input to 31 layer 3B neurons revealed that input sources to individual cells were highly diverse. Although some input differences were correlated with neuronal anatomy, no 2 neurons received excitatory input from the same cortical layers. Thus, input diversity reveals far more cell types than does anatomical diversity. This implies relatively little functional redundancy; despite trends related to laminar or columnar position, pools of neurons contributing uniquely to visual processing are likely relatively small. These results also imply that similarities in the anatomy of circuits in different cortical areas or species may not indicate similar functional connectivity.
In humans the hippocampus is required for episodic memory, which extends into the spatial and temporal domains. Work on the rodent hippocampus has shown that NMDA receptor (NMDAR) -mediated plasticity is essential for spatial memory. Here, we have examined whether hippocampal NMDARs are also needed for temporal memory. We applied trace fear conditioning to knockout mice lacking NMDARs only in hippocampal CA1 pyramidal cells. This paradigm requires temporal processing because the conditional and unconditional stimuli are separated by 30 s (trace). We found that knockout mice failed to memorize this association but were indistinguishable from normal animals when the trace was removed. Thus, NMDARs in CA1 are crucial for the formation of memories that associate events across time.
In the high vocal center (HVC) of adult songbirds, increases in spontaneous neuronal replacement correlate with song changes and with cell death. We experimentally induced death of specific HVC neuron types in adult male zebra finches using targeted photolysis. Induced death of a projection neuron type that normally turns over resulted in compensatory replacement of the same type. Induced death of the normally nonreplaced type did not stimulate their replacement. In juveniles, death of the latter type increased recruitment of the replaceable kind. We infer that neuronal death regulates the recruitment of replaceable neurons. Song deteriorated in some birds only after elimination of replaceable neurons. Behavioral deficits were transient and followed by variable degrees of recovery. This raises the possibility that induced neuronal replacement can restore a learned behavior.
Diffusion tensor magnetic resonance imaging (MRI) was used to study the microstructural integrity of white matter in adults with poor or normal reading ability. Subjects with reading difficulty exhibited decreased diffusion anisotropy bilaterally in temporoparietal white matter. Axons in these regions were predominantly anterior-posterior in direction. No differences in T1-weighted MRI signal were found between poor readers and control subjects, demonstrating specificity of the group difference to the microstructural characteristics measured by diffusion tensor imaging (DTI). White matter diffusion anisotropy in the temporo-parietal region of the left hemisphere was significantly correlated with reading scores within the reading-impaired adults and within the control group. The anisotropy reflects microstructure of white matter tracts, which may contribute to reading ability by determining the strength of communication between cortical areas involved in visual, auditory, and language processing.
The M-CSF receptor (M-CSFR) is expressed in monocytes-macrophages and their progenitors, and drives growth and development of this blood cell lineage. The M-CSFR is a member of a small family of growth factor receptors exhibiting related structures but distinct tissue-specific functions. This review discusses the early molecular events in the M-CSF signaling mechanisms, positive signals, negative signals, the possible organization of individual signaling pathways, and the problem of achieving specificity in the signal transduction mechanism.
We have previously shown that insulin-like growth factor-1 (IGF-1) induces the expression of latent transforming growth factor beta 1 (LTGF-beta 1) through activation of c-fos and c-jun oncogenes. In this study we investigated whether IGF-1 induced latent TGF-beta 1 has autocrine effects on dermal fibroblasts and described a possible mechanism. Human dermal fibroblasts were treated with either vehicle, IGF-1 alone, or IGF-1 with either anti-TGF-beta 1 neutralizing antibody or mannose-6-phosphate (M6P) and levels of mRNAs for TGF-beta 1, collagenase and the pro alpha 1(I) chain of type I collagen were then evaluated by Northern analysis. Conditioned medium was also collected from treated and untreated cells and assayed for TGF-beta 1 protein by enzyme-linked immunosorbent assay. The results of the Northern analysis revealed a differential effect on the expression of the pro alpha 1(I) chain of type I collagen and collagenase in dermal fibroblasts: mRNA for the former being significantly increased in response to IGF-1 treatment while that for collagenase was markedly suppressed. These effects of IGF-1 were blocked to a significant extent by TGF-beta 1 neutralizing antibody at a concentration of 0.5-2.0 micrograms/ml. As the TGF-beta 1 induced by IGF-1 is inactive in the traditionally used mink lung epithelial cell growth inhibition assay, we explored the possible role of IGF-II/M6P receptors in facilitating these autocrine effects. The results showed that the greater than two-fold increase (201.9 +/- 38 vs 81.8 +/- 13, p < 0.05) in mRNA for the pro alpha 1(I) chain of type I collagen induced by IGF-1 was at least 60% inhibited by M6P in a time-dependent fashion. A direct correlation between the expression of TGF-beta 1 and the pro alpha 1(I) chain of type I collagen was found in response to either IGF-1 alone or IGF-1 with M6P. Treatment of cell cultures with TGF-beta 1 neutralizing antibody mimicked the effect of M6P. In contrast to the effects on expression of type I collagen, the level of collagenase mRNA was markedly reduced by IGF-1 alone and was restored by the administration of M6P. The levels of TGF-beta 1 in conditioned medium from treated and untreated cells showed a similar pattern to that of the mRNA detected by Northern analysis. These findings suggest that IGF-1 induces latent TGF-beta 1 and that the matrix-modulating autocrine effects of LTGF-beta 1 on dermal fibroblasts are facilitated by M6P/IGF-II receptors on these cells.
Osteogenic protein-1 (OP-1) or bone morphogenetic protein-7 (BMP-7) stimulates cartilage formation in mouse bone rudiments in vitro but arrests terminal differentiation of prehypertrophic chondrocytes into hypertrophic chondrocytes. In this study we report that these effects of OP-1 depend on the developmental stage of the bone rudiment, early stages (E14 and E15 metatarsals) being most responsive. E17 metatarsals that already contained a hypertrophic area that had initiated mineralization were no longer affected by OP-1. We then investigated whether the sensitivity of the early long bone rudiments to OP-1 correlated with high expression of the OP-1 binding type I serine/threonine kinase receptors (activin receptor-like kinase: ALK-2/ActR-I, ALK-3/BMPR-IA or ALK-6/BMPR-IB) at this early stage. We did not find any significant difference in overall mRNA levels of these ALKs between stages E14 through E17 as assessed by RNase protection assays. However, by immunohistochemistry we found that ALK-6 staining was strong in E14 early cartilage primordium and its future perichondrium but dropped sharply to low levels in these cell types until onset of chondrocyte (pre)hypertrophy at E16. By contrast, ALK-2 and ALK-3 immunostainings in E14 were barely detectable. We also examined by immunohistochemistry the local synthesis of OP-1. OP-1 was present in E14 early chondrocytes and forming perichondrium but in low amounts; however, production of OP-1 increased in these cell types with age. All three receptor types as well as OP-1 were present in significant amounts in prehypertrophic chondrocytes and late hypertrophic chondrocytes including those undergoing mineralization. The temporary high immunostaining for ALK-6 in the early proliferating chondrocytes and future perichondrium of E14 bone rudiments, and its absence in older bones correlated with the sensitivity of chondrocytes and perichondrium to (exogenous) OP-1. We therefore propose that the effects of OP-1 on these cells in vitro are mediated by ALK-6/BMPR-IB. We furthermore conclude that locally produced OP-1 is a potential autocrine/paracrine growth factor. Increased local production of OP-1 may be partially responsible for the age-related decrease in responsiveness to exogenous OP-1 with respect to hypertrophy and mineralization of cartilage.
Insulin-like growth factor-1 receptor (IGF-1R) has been shown to be important for melanoma cell growth and survival. In this study we first show, using immunohistochemistry, that progression from benign nevi to malignant melanoma is paralleled by an increased expression of IGF-1R and a down-regulation of the cyclin-dependent kinase inhibitor p27Kip1. Even though the expression of p27Kip1 was drastically reduced compared to benign tumors, detectable amounts of it could be assayed by Western blotting in cultured melanoma cells. To analyze whether there is a causative relationship between the IGF-1 pathway and p27Kip1 expression, melanoma cells were treated with alpha IR-3, an antibody blocking the IGF-1 binding to IGF-1R, or Tunicamycin, which inhibits the translocation of IGF-1R to the cell surface. From these studies we could conclude that the overall expression of p27Kip1 is independent of the IGF-1 pathway. In contrast, the association of p27Kip1 with the different cyclins was drastically affected. Both TM and alpha IR-3 decreased the binding of p27Kip1 to cyclin D1, whose expression was drastically reduced. On the other hand there was an increased binding of p27Kip1 to cyclin E and cyclin A. This redistribution of p27Kip1 may be a mechanism for growth arrest and induction of apoptosis following interruption of the IGF-1 pathway in melanoma cells.
The biological activities of fibroblast growth factors (FGF) are mediated by specific cell membrane receptors (FGFR), which have three immunoglobulin-like IgG domains in the extracellular region. The carboxy-terminal segment of the third IgG domain of FGFR1 could be encoded by different exons, designated IIIa, IIIb, or IIIc. While exons IIIb or IIIc encode receptor forms with both intracellular and extracellular domains, the FGF receptor becomes potentially a secreted form lacking the intracellular domain and the transmembrane region when exon IIIa is expressed. Using reverse transcription polymerase chain reaction, we have found that mRNAs encoding the nucleotide sequences of FGFR1-IIIa and FGFR1-IIIc are expressed in HL-60 cells. FGFR1-IIIa fragment was synthesized by a glutathione S-transferase gene fusion system. The purified 33 kDa FGFR1-IIIa fragment fusion protein could bind [125I]-labelled FGF-2 in Western ligand blot analysis. Three species of proteins with the molecular weights of 82, 60, and 50 kDa were identified in serum-free, conditioned medium from HL-60 cells by Western blot using an antiserum against purified FGFR1-IIIa fragment fusion protein. Exposure to FGF-2 caused an increase in [3H]-thymidine incorporation into DNA of HL-60 cells and increased cell proliferation, but the addition of FGFR1-IIIa fragment fusion protein inhibited FGF-2-stimulated DNA synthesis and caused a dose-dependent inhibition of FGF-2-stimulated cell proliferation. The effects on DNA synthesis were partly reversed by antibody against the FGFR1-IIIa fragment. These results indicate that both cell membrane spanning and secreted FGF receptors are expressed in HL-60 cells, and that the actions of FGFs as paracrine growth factors could be modulated by secreted FGF receptor forms.
This study investigated serum levels of basic fibroblast growth factor (b FGF), a potent angiogenic factor, during distinct periods of the female life and compared them with corresponding levels in age-matched males. Healthy females (n = 59) and males (n = 53) were included in the study, divided into six groups: fetuses (cord blood), neonates, children, adults (females in proliferative and secretory phase), pregnant and "elderly" men and women. Serum b FGF levels were measured by an enzyme immunoassay. No statistically significant difference was found between both genders. Blood levels in fetuses and neonates were significantly increased as compared to adults (p = 0.01, p = 0.02, respectively). Restricting the analysis to females, all age groups, but fetuses (p = 0.05), demonstrated no difference when compared to proliferative phase adults. In conclusion, b FGF serum levels do not differ between males and females and are elevated in fetal and neonatal life, when growth and development are enhanced.
The efficacy of human recombinant osteogenic protein-1 (OP-1; bone morphogenetic protein-7) in regeneration of dog larynx was examined by treating thyroid cartilage defects (1.5 cm2) in dogs with thyroid allografts covered with host perichondrium or fascia. Prior to implantation allografts were frozen, thawed and demineralized. The treatment groups were as follows: I--Allograft control implant (n = 3); II--Implants coated with 500 micrograms OP-1 (n = 4); III--Implants coated with 100 micrograms OP-1 (n = 3); IV--Implants coated with 500 micrograms OP-1 and covered with neck fascia (n = 3); and V--Implants extracted with 1 M NaCl and guanidine hydrochloride, and coated with 500 micrograms OP-1 (n = 4). Dogs were sacrificed four months following surgery. Each larynx was removed, carefully dissected and a three-dimensional reconstruction of the defect area was performed on serial sections. The results revealed that the implants of control dogs remained intact with no apparent reduction in size and new tissue formation. OP-1 enriched thyroid allografts, dose dependently induced bone, cartilage and ligament-like structures comprising up to 80% of the total regenerated defect area. Boundaries of the defects healed by formation of new bone when bone resided within the old thyroid cartilage layers. Old cartilage not containing bone within its layers healed by complete integration with newly formed cartilage. Both new bone and cartilage were embedded into layers of new ligament-like tissue which expressed specific morphologic and molecular markers. The three newly formed tissues were tightly connected into a "bone-cartilage-ligament continuum" of tissues, suggesting that OP-1 served as a multiple tissue morphogen in this specific microenvironment.
M. hominis is commonly found as part of the normal flora in the female genital tract, but several studies have shown that it may be involved in a variety of urogenital infections. The basis for clinical manifestations in some patients has varyingly been attributed to host and M. hominis factors. The host factors involved in the infection process are largely unknown. M. hominis have no cell wall and outer membranes, and at present it seems plausible that M. hominis possesses genetic systems allowing the bacteria in vivo to alter its antigenic structure on the membrane surface and consequently circumvent the host immune system. The studies of M. hominis have shown that the antigenic variation is pronounced between surface exposed membrane proteins from different isolates. The genetic background for this variation has been investigated for three surface exposed membrane proteins: P120, Lmp, and Vaa. P120 and P120' are similar proteins in M. hominis without any homology to other known proteins. A hypervariable region in the otherwise conserved P120 protein seems to be very antigenic in patients with immunologically verified M. hominis infection. The remaining part of P120 as well as the entire P120' protein do not seem to elicit significant antibody formation. Two genes in M. hominis, lmp1 and lmp3, contain numerous highly similar 0.5 kb tandem repeats at their 3'-end. The proteins, Lmp1 and Lmp3, are synthesized from the lmp1 and lmp3 genes, respectively. Lmp1 shows size variation among M. hominis isolates. M. hominis isolates investigated in detail show that the size variation of Lmp1 corresponds to the variation in number of 0.5 kb repeats contained within the lmp1 gene. Lmp3 appears to have a lesser tendency to size variation. M. hominis isolates were found with deletions involving the lmp1 stop codon leading to translation of the downstream gene lmp2 and expression of a chimeric Lmp1-Lmp2 protein. The number of repeated elements in the lmp1 gene of a M. hominis isolate correlates with the extent of anti-Lmp antibody induced agglutination between the bacteria. Vaa is a protein involved in cell adherence. vaa is a single copy gene containing tandem repeated elements like the lmp gene family. The number of repeats in the Vaa protein differs between M. hominis isolates leading to size variation. It has been suggested that the number of repeated elements is of importance in the bacteria-host adhesion process. Beside the size variation Vaa demonstrates phase variation due to frequent frame shift mutation in a specific region near the 5'-end of the structural gene. Based on the investigations of M. hominis and other mycoplasmas several genetic mechanisms seem to be responsible for the antigenic variation of surface exposed membrane proteins in mycoplasmas: 1) variation in protein size due to insertions or deletion of repeated elements in the structural gene, 2) presence of multi-gene families, and 3) phase variation due to mutations in the promotor region or the coding region. The influence of specific antibodies on antigenic variation of membrane proteins has not been studied in greater detail in mycoplasmas. In M. hominis it was investigated whether the presence in the culture medium of monoclonal antibodies directed against the repeated elements in the M. hominis Lmp proteins would affect gene structure and consequently protein expression. The presence of anti-Lmp antibodies resulted in overgrowth of bacteria with specific deletions in the repeated elements of lmp1 leaving the lmp3 gene unchanged. The precise mechanism leading to the dominance of M. hominis isolates with fewer 0. (ABSTRACT TRUNCATED)
BACKGROUND: Approximately 10% of patients presenting with dyspepsia to the general practitioner have peptic ulcers; the large majority of which are related to infection with Helicobactor pylori. Office-based tests for H. pylori detection are generally validated and evaluated in selected patient groups. AIM: To evaluate the clinical effectiveness of a whole-blood serology test for infection with Helicobacter pylori in detecting peptic ulcer disease (PUD) in daily general practice. METHOD: A descriptive study of 171 primary care dyspepsia patients selected for open-access endoscopy in primary care and aged between 18 and 75 years, in 92 general practices in central, southern, and eastern parts of the Netherlands. H. pylori status was assessed using the BM-test Helicobacter pylori, which is identical to the Helisal test. Dyspepsia severity score was measured using a validated symptom score. Symptom characteristics and probability of relevant disease were assessed by the general practitioner. Endoscopy was carried out in local hospitals. Diagnostic outcome of both endoscopy and H. pylori reference test was supplied by local specialists. The BM-test was evaluated against endoscopic results. RESULTS: A high number (61.8%) of false-negative BM-tests resulted in a low sensitivity (95% confidence interval [CI] = 48-75%) for detection of H. pylori infection. Only 12 out of 32 patients with PUD had a positive BM-test, resulting in a positive likelihood ratio (LR) for PUD of 1.41 and a negative LR of 0.85. CONCLUSIONS: This study confirms the relatively poor performance of the BM-test in daily general practice, and shows the limited diagnostic value of H. pylori office-tests for detecting PUD in primary care. The discriminative value of the test result is too small to support either a 'test-and-endoscope' of a 'test-and-treat' strategy in general practice.
BACKGROUND: Health needs assessment can guide the appropriate shift to primary care by identifying the most effective and efficient resource allocation to meet the needs of populations. Assessing health care needs will be a continuing challenge for primary care trusts in Scotland (or equivalent groups in other parts of the United Kingdom); however, lessons must be learned from the experience of needs assessment that followed the 'internal market' reforms of the 1990s. AIM: To examine general practitioners' (GPs') awareness and experience of needs assessment, to identify barriers to needs assessment in primary care, and to ascertain how better progress might be made in the future. METHOD: A postal questionnaire survey of 1777 Scottish GPs (a one-in-two sample) was combined with a semistructured interview survey of 'lead' GPs from a random sample of 64 mainland Scottish practices between May and August 1996. RESULTS: Sixty-five per cent (1154) of GPs responded to the questionnaire, of which 54% (965) were completed. Over 73% (47) of interviews were completed. Most GPs were unfamiliar with the concept of needs assessment and there was no evidence that needs assessment had influenced commissioning decisions. Most GPs argued that it was not a 'core' activity and that they lacked training in the relevant skills. While the attitude of the majority was indifferent, cynical, and sometimes hostile, a minority, comprising mostly younger fundholders, was more enthusiastic about needs assessment. CONCLUSION: The motivation and attitude of the majority of GPs present a barrier to needs assessment in primary care. GPs will require more resources and training if they are to undertake this responsibility. Most GPs believe than incentives (financial or organisational) will be necessary. Primary care trusts and equivalent structures should be aware of these attitudes as they seek to establish plans based on estimates of population needs in defined locations.
BACKGROUND: Eating disorders are becoming more apparent in primary care. Descriptions of character traits related to people with eating disorders are rarely reported in the primary care literature and there is little awareness of the implications of alexithymia--a concept that defines the inability to identify or express emotion. We hypothesised that many individuals with active eating disorders have alexithymic traits and a tendency to somatize their distress. AIM: To analyse the character traits and degree of alexithymia of a selected group of women with active eating disorders and in recovery, and to recommend responses by members of the primary care team that might meet the needs of such individuals. METHOD: Letters were sent to 200 female members of the Eating Disorders Association who had agreed to participate in research. Seventy-nine women volunteered to complete four postal questionnaires. This gave a response rate of 38.5%. Responders were categorised into three groups--anorexic, bulimic, and recovered--using the criteria of the Eating Disorders Inventory (EDI-2). The results of the 16PF5 Personality Inventory (16PF5) and the Toronto Alexithymia Scale (TAS-20) were analysed using one-way analysis of variance (ANOVA) and correlated using Pearson's correlation. A biographical questionnaire was also completed. RESULTS: In all three subgroups, high scores were achieved on the 16PF5 on 'apprehension and social sensitivity', while there were significant differences in the scores for 'privateness': a scale that measures the ability to talk about feelings and confide in others. On the TAS-20, 65% of the anorexic and 83% of the bulimic group scored in the alexithymic range compared with 33% of the recovered group. There was a significant negative correlation between alexithymia and social skills such as 'social and emotional expressivity' on the 16PF5. CONCLUSION: The results of this study emphasise the difference between those with active eating disorders who achieved high scores for privacy, introversion, and alexithymia, and those who have recovered. These character traits give potential helpers an important indication of the areas that can both block and facilitate recovery, and they act as a reminder that the presenting symptoms in eating disorders and other psychosomatic conditions are the outward presentation of internal conflict. It is suggested that effective screening and needs assessment will facilitate a more appropriate and prompt therapeutic response. This may be provided in the primary care setting where appropriate training has occurred.
BACKGROUND: Despite the recommendation of the Department of Health that patients with asthma receive annual vaccination against influenza, uptake remains unsatisfactory with many patients suspicious that vaccination is harmful. AIM: To examine the effect of influenza vaccination on asthmatic patients typical of a general practice setting. METHOD: A multicentre study with 56 patients participating from 14 practices in England and Scotland. Patients completed peak expiratory flow rate (PEFR) and symptom diaries for two weeks before and two weeks after influenza vaccination. RESULTS: A non-significant fall in baseline PEFR of 10.5 l/min, from an average of 431.5 l/min, was observed after influenza vaccination, representing a 2% change from baseline. A significant increase in night time reliever use of 0.17 puffs per night (P < 0.01) was found. Non-significant increases in number of nights per week with sleep disturbed due to asthma, severity of night-time and day-time symptoms, and day-time reliever use were also noted. CONCLUSION: Influenza is an important cause of morbidity and mortality in asthmatics. This study confirms the safety of influenza vaccination in patients with asthma typical of those seen in primary care. General practitioners need not hesitate in recommending this valuable intervention to their asthmatic patients and should consider ways in which uptake can be improved.
BACKGROUND: Acute paediatric admissions have risen steadily over the past 20 years. During the same period, practice-based child health clinics have increased, although provision is less common in areas of deprivation where hospital use is greatest. AIM: To investigate the contribution of practice-based, preventive child health services to rates of hospital utilisation in children under five years of age. METHOD: A cross-sectional retrospective study examining practice variations in paediatric acute admissions, outpatient referrals, and accident and emergency (A&E) department attendances in the East London and the City Health authority, including all 164 practices in the inner-city boroughs of Hackney, Newham, Tower Hamlets, and the City of London. The main outcome measures were practice-based paediatric hospital attendance rates, for discrete age and sex bands, for the year to 31 March 1996. RESULTS: Hospital use varied with age and sex, with the rates being highest for the youngest children and for boys. The median A&E attendance rate (including reattendances) for boys up to one year of age was 897 per thousand children per practice. In east London, 62% of practices are registered for child health surveillance and 71% provide a child health clinic. Practice approval for child health surveillance, and the provision of child health clinics, did not account for differences between practices in hospital use, but proportionally greater health visiting hours were significantly related to lower rates of emergency hospital admission by young children. Multivariate analyses revealed that up to 23% of the variation between practice admission rates could be explained by health visiting hours. CONCLUSIONS: We found significant associations between the amount of health visiting time available to the practice population and rates of acute admission and outpatient referral among children up to five years of age. These findings suggest that increasing health visitor provision could contribute to lower paediatric emergency admission and outpatient referral rates. A small change would have a significant effect, particularly among the youngest children, given that during the study year 10,000 children under two years of age in east London were either admitted or referred to hospital.
BACKGROUND: There is concern about the educational impact and possible stress on registrars of new out-of-hours co-operatives. AIM: To compare the confidence in managing out-of-hours problems of registrars in traditional on-call rotas and co-operatives with that of their trainers. To determine how frequently registrars discussed problems out-of-hours with their trainers, and to compare the referral pattern of registrars with their trainers out-of-hours. METHOD: Analysis of log diaries of out-of-hours experiences kept by registrars and trainers over two, two-month periods in winter and summer. RESULTS: Thirty registrars (out of a possible 51) and 34 (out of a possible 52) trainers took part in the winter, and 18 registrars and 29 trainers in the summer. Registrars were confident in their management, and their confidence increased over the year (59% versus 72% difference = 12%, 95% CI = 6% to 20% for very confident). Registrars varied in their discussion of problems with trainers. When 'a little worried' they discussed their management 30 out of 53 times (57%); if 'very confident', 36 out of 576 times (6%). Registrars during the summer segment of the study referred more frequently to hospital than trainers (20% versus 10% difference = 10%, 95% CI = 3% to 17%. Registrars in traditional rotas recorded a slightly higher but statistically insignificant level of confidence in their management of problems than those registrars in cooperatives. CONCLUSIONS: While many registrars are confident in their work and are using their trainer for information appropriately, some are not. Registrars may be referring to hospital at a much higher rate than their trainers. More research is required to confirm and further explore these findings.
During two years, a rural ambulance helicopter programme saved 41 patients' lives. In 29 of these patients, the decisive medical interventions were carried out by the flight anaesthesiologist before reaching the hospital. We asked an expert panel to assess whether these interventions could have been carried out by a general practitioner (GP). This was the case for 17 (59%) of the 29 patients, while more advances skills, equipment or drugs were needed for 11 (38%). Among these 11, three patients would probably have died without the interventions. We conclude that GPs can manage a majority of life saving missions for a rural ambulance helicopter programme, but the lack of a flight anaesthesiologist may imply substantial health losses for a few patients.
Diet is important in the aetiology and management of many conditions in primary care. Although valid dietary assessment is required for both clinical work and research, no dietary assessment instruments have been validated among patients seen in primary care. A range of simple self-completion dietary assessment questionnaires and established research instruments were compared with an accepted reference standard, a seven-day weighed record, in 111 subjects assessed in a practice nurse-run treatment room. Simple self-completion tools based on food groups and portion sizes perform as well (likelihood ratios for a positive test = 2 to 3) as much more time-consuming instruments. The error in using such instruments is comparable with the error of the standard itself. There is little justification for using time-consuming dietary assessment questionnaires, since simple tools are accurate enough to be clinically useful--to allow practice nurses to target patients for counselling and waste less time on inappropriate counselling--and also useful for research.
A questionnaire was sent to all practice managers in Wessex in June 1997 to assess to what extent practices had stopped relying on paper records in the consultation. Practices that solely used computer records in the consultation ('noteless') did not necessarily consider themselves 'paperless'. Following a recent declaration that all practices should be using an Electronic Health Record by March 2005, the obstacles to this move were investigated and some differences in the way the 'noteless' and 'paperless' groups used computers were identified.
A retrospective analysis was made of the criminal records of 57 patients successfully retained in methadone maintenance at two general practices in Sheffield. Their criminal conviction rates and time spent in prison per year were compared for the periods before and after the start of their methadone programme. Overall, patients retained on methadone programmes in the general practices studied had significantly fewer convictions and cautions, and spent significantly less time in prison than they had before the start of treatment.
Chronic heart failure is a common clinical syndrome that may have different causes. Its incidence and prevalence are predicted to rise substantially over the next 10 years. There are therefore major consequences for resource provision, especially in primary care, where most patients are managed. Chronic heart failure is a serious condition with high morbidity and mortality. There is good evidence to show that treatment with angiotensin-converting enzyme (ACE) inhibitors in patients with left ventricular systolic dysfunction improves symptoms and signs, slows progression of heart failure, reduces hospitalisation rates, and improves survival. Despite this evidence, primary care studies show that patients with heart failure are incorrectly diagnosed and inadequately treated. Most patients present in general practice, and because effective treatment relies on a correct diagnosis, this is a key step in the appropriate management of heart failure. The aim of this paper is to review the evidence about the usefulness of signs, symptoms, and investigations in diagnosing heart failure in primary care. To identify relevant studies for this review, four strategies were used: a MEDLINE search from 1993 to January 1998 using the diagnosis search filter; a MEDLINE search from 1993 to January 1998 using the guideline search filter to locate published heart failure guidelines; a search for review articles in the Cochrane Library; and a check of references in the studies identified. The search terms included MeSH terms and the keywords 'heart failure' and 'diagnosis'. All searches were limited to humans and English language articles. Studies were included in this review on the basis of quality and relevance to primary care. The review shows that symptoms and signs are important because they alert clinicians to the possibility of heart failure as a diagnosis. However, they are not sufficiently specific for confirming left ventricular systolic dysfunction. From the evidence available, a patient with suspected heart failure must have objective tests to confirm the diagnosis. These should include an electrocardiogram and, ideally, an echocardiogram. Further research is also needed on the usefulness of signs and symptoms in primary care, as most studies of heart failure have been conducted in secondary care.
In the course of the consultation in primary care, the general practitioner integrates knowledge of different types that are drawn from different sources. As a consequence of the way practitioners develop expertise, this use of knowledge is often hidden from the conscious mind of the practitioner and often hidden from direct observation. On the other hand, understanding of this use of knowledge is crucial to several necessary developments of the profession of general practice. A method involving collaboration between researcher and practitioner sheds new light on this knowledge-in-use.
BACKGROUND: Symptom and pulmonary function measures of asthma severity are used for severity classification in practice guidelines. However, there is limited methodological evidence in support of their validity and utility. AIM: To validate initial symptom and forced expiratory volume (FEV1) measures of asthma severity with the subsequent risks of exacerbations resulting in emergency room (ER) visits, hospitalisation, and sickness absence from work. In addition, symptom-based measures of change in asthma severity were also evaluated against the concurrent risks of asthma exacerbations. METHOD: A cohort of 361 adult asthmatic patients in general outpatient clinics was studied. At initial interview, frequencies of asthmatic symptoms and nocturnal exacerbations, FEV1, and a severity score combining these measures, were recorded. At re-interview in the third year, the frequencies of asthma exacerbations resulting in ER visits, hospitalisation, and sickness absence, and a self-assessed global measure of change in severity and serially-assessed change in symptom frequencies, were measured. RESULTS: All individual symptom and FEV1 measures were strongly related to the subsequent risks of ER visits, hospitalisation, and sick absence. A severity score of more than 3 (moderate to severe asthma) and self-assessed change in asthma severity were most strongly and significantly associated with greatly increased risks of all outcomes. Individual symptoms and FEV1 measures alone did not show high sensitivities, but the severity score combining these measures gave much more satisfactory validity. Perhaps not surprisingly, self-assessed change in asthma appeared to give the most satisfactory validity. CONCLUSION: These results support the validity and clinical utility of a simple clinical score based on symptom and FEV1 measures, and self-assessed measure of change in severity, for risk classification in contemporary clinical practice guidelines.
Osteosclerosis (oc) is an autosomal recessive lethal mutation that impairs bone resorption by osteoclasts, and induces a general increase of bone density in affected mice. Genetic mapping of the oc mutation was used as a backbone in a positional cloning approach in the pericentromeric region of mouse chromosome 19. Perfect cosegregation of the osteopetrotic phenotype with polymorphic markers enabled the construction of a sequence-ready bacterial artificial chromosome (BAC) contig of this region. Genomic sequencing of a 200-kb area revealed the presence of the mouse homologue to the human gene encoding the osteoclast-specific 116-kDa subunit of the vacuolar proton pump. This gene was located recently on human 11q13, a genomic region conserved with proximal mouse chromosome 19. Sequencing of the 5' end of the gene in oc/oc mice showed a 1.6-kb deletion, including the translation start site, which impairs genuine transcription of this subunit. The inactivation of this osteoclast-specific vacuolar proton ATPase subunit could be responsible for the lack of this enzyme in the apical membranes of osteoclast cells in oc/oc mice, thereby preventing the resorption function of these cells, which leads to the osteopetrotic phenotype.
Alkaline phosphatases are a family of glycoproteins that are able to hydrolize various monophosphate esters at a high pH optimum. Liver/bone/kidney (L/B/K) alkaline phosphatase (ALP) is one of the four major isoenzymes that belong to this family. Apart from its role in normal bone mineralization, other functions of L/B/K ALP remain obscure, both in physiological and in neoplastic conditions, including the bone-forming tumor osteosarcoma. In this study, we transfected the U-2 OS osteosarcoma cell line, which does not show any basal expression of this enzyme, with the full-length gene of L/B/K ALP, and analyzed the in vitro and in vivo features of four transfectants showing different expression of L/B/K ALP. A reduced in vitro ability to invade Matrigel and to grow in a semi-solid medium, together with a lower tumorigenic and metastatic ability in athymic mice, was found to be associated with a high level of cell surface L/B/K ALP activity. Moreover, L/B/K ALP transfectants showed a reduced secretion of matrix metalloproteinase-9 enzyme. These findings indicate a loss of aggressiveness of osteosarcoma cells after the expression of L/B/K ALP on their surface and suggest a new role for this enzyme.
We have recently demonstrated that the receptor for parathyroid hormone (PTH) and PTH-related peptide (PTHrP), PTHR, can be localized to the nucleus of cells within the liver, kidney, uterus, gut, and ovary of the rat. We set out to determine the localization of the PTHR in cultured osteoblast-like cells. MC3T3-E1, ROS 17/2.8, UMR106, and SaOS-2 cells were cultured in alpha-modified eagle medium containing 15% fetal calf serum under standard conditions. Untreated cells were grown on glass coverslips to 75-95% confluence and fixed in 1% paraformaldehyde. For experiments designed to examine cells synchronized by serum starvation, cells were grown on glass coverslips, starved of serum for 46 h, and then fixed at 2-h intervals for a total of 26 h after the addition of serum to the medium. Parallel sets of cells were pulsed with [3H]thymidine to track the DNA duplication interval. The PTHR was localized by immunocytochemistry using a primary antibody raised against a portion of the N-terminal extracellular domain of the PTHR. The results presented herein indicate that the PTHR attains a nuclear localization in each cell line examined. In UMR106 cells, PTHR immunoreactivity was restricted to the nucleolus. After cell synchronization, MC3T3-E1 cells double approximately 24 h after the addition of serum. Immunocytochemistry for the PTHR in these cells showed that the receptor staining is initially diffuse for the first 6 h, then becomes more perinuclear in distribution by 12-16 h. Nuclear localization of the receptor is achieved approximately 16-20 h after the addition of serum and remains there throughout the mitotic phase. Intense staining of mitotic and postmitotic cells was observed. No change in cell proliferation kinetics was observed in MC3T3-E1 cells cultured in the presence of 25 nM PTH(1-34). These data suggest an important role for the PTHR in the nucleus of MC3T3-E1 cells at the time of DNA synthesis and mitosis.
Bone cells undergo changes in cell structure during phenotypic development. Parathyroid hormone (PTH) induces a change in osteoblast shape, a determinant of collagen expression. We hypothesize that alterations in bone cell shape reflect and direct gene expression as governed, in part, by nuclear organization. In this study, we determined whether the expression of nuclear matrix proteins that mediate nuclear architecture, NuMA, topoisomerase II (topo II)-alpha, and -beta, were altered during osteoblast development and response to PTH in vivo. NuMA forms an interphase nuclear scaffold in some cells, the absence of which may accommodate alterations in nuclear organization necessary for specific functions. Topo II enzymes are expressed in bone cells; the alpha-isoform is specific to proliferating cells. We used immunohistochemistry and flow cytometry to determine whether NuMA is expressed in the primary spongiosa of the rat metaphyseal femur and whether expression of NuMA, topo II-alpha, and II-beta changes during osteoblast development or with PTH treatment. NuMA and topo II-beta were expressed in marrow cells, osteoblasts, osteocytes, and chondrocytes. These proteins were not detected in osteoclasts in vivo, but were observed in cultured cells. Bone marrow cells expressed topo II-alpha. All three proteins were expressed in cultures of rat osteoblast-like UMR-106 cells. PTH treatment downregulated the number of topo II-alpha-immunopositive cells, correlated with a decrease in S-phase cells, in both bone tissue and cell culture. We conclude that, in vivo, nuclear matrix composition is altered during bone cell development and that anabolic doses of PTH attenuate the proliferative capacity of osteogenic cells, in part, by targeting topo II-alpha expression.
Activin, a member of the transforming growth factor-beta (TGF-beta) superfamily, is present in the bone matrix and assumed to be involved in the regulation of bone formation. In the present study, we investigated whether the release of activin from bone is coupled with bone resorption. Neonatal mouse calvaria were cultured in the presence of various stimulators of bone resorption (parathyroid hormone [PTH], interleukin-1beta, prostaglandin E2) for up to 72 h, and the activin activity in the medium was measured using a specific bioassay for activin. Activin activity was accumulated in proportion to the time- and dose-dependent increase in calcium release from bone into the medium (bone resorption). An inhibition of PTH-dependent bone resorption by a bisphosphonate, disodium dichlormethane-1,1-bisphosphonic acid (Cl2MBP), completely blocked release of activin activity from bone into the medium. In primary culture of calvarial cells, however, neither PTH nor Cl2MBP affected activin production. These findings indicate that release of activin activity from bone tissue is strongly coupled to bone resorption. Because activin possesses osteogenic activities, activin released locally from bone might be involved in the regulation of bone formation in the physiological process of bone remodeling, as has been suggested for TGF-beta.
An immunoassay for cross-linked N-telopeptides of type I collagen (NTx) in urine or serum has proven to give a sensitive index of osteoclast-mediated bone resorption. We show that recombinant human cathepsin K is highly active in releasing the NTx neoepitope in 100% yield from bone type I collagen. Cathepsins S, L, and B were also active but at 57%, 36%, and 27% of the yield of K, respectively. The matrix metalloproteinases that were tested, stromelysin, collagenase 3, or matrilysin, did not produce any immunoreactivity. Cathepsin K also acted on demineralized bone matrix, releasing NTx epitope and completely dissolving the bone particles in 24-48 h. Proteolytic cleavage of a G-L peptide bond in the alpha2(I)N-telopeptide was shown to be required for recognition by monoclonal antibody 1H11. Peptide analysis identified bonds in the N-telopeptide and helical cross-linking domains adjacent to the cross-linking residues at which cathepsin K cleaved in bone collagen. The sites were consistent with the known substrate specificity of cathepsin K, which prefers a hydrophobic residue or proline in the critical P2 position. The NTx peptides generated by cathepsin K were of low molecular weight, in the range previously found in human urine. Because cathepsin K appears to be essential for the normal resorption of mineralized bone matrix by osteoclasts, these findings help explain the specificity and responsiveness of NTx as a marker of osteoclastic bone resorption in vivo.
Bone remodeling is regulated by local factors and cytokines. Among them, interleukin-6 (IL-6) plays a critical role in bone resorption, and its synthesis is stimulated by osteoresorptive factors. Transforming growth factor-beta (TGF-beta) is present in high amounts in the bone matrix and is a local regulator of bone formation. However, its role in bone resorption remains unclear. In this paper, we report that TGF-beta stimulates IL-6 transcripts in a time- and dose-dependent manner in primary rat osteoblasts isolated from 22-day-old calvariae (Ob cells). The TGF-beta effect on IL-6 mRNA levels does not require de novo protein synthesis because cycloheximide, a protein synthesis inhibitor, does not block the induction. The mechanisms of IL-6 stimulation by TGF-beta is at least partially transcriptional because TGF-beta induces IL-6 heterogenous nuclear RNA, and, to a lesser extent, IL-6 transcription rate as determined by a nuclear run-on assay. Transforming growth factor-beta upregulation of IL-6 may be critical in conditions of increased bone resorption, such as myeloma.
Growth hormone (GH) exerts potent effects on bone metabolism, resulting in an increased bone formation in animals and humans. Acromegaly has been associated with increased bone turnover, whereas the net effect of the increased bone metabolism has been obscured because patients with acromegaly are often associated with hypogonadism. We investigated changes in cortical and cancellous bone in adult rats implanted mammosomatotrophic pituitary tumor cells (GH3) as a model of acromegaly with gonadal dysfunction. Acromegaly model rats were prepared by implanting GH3 cells into female Wistar-Furth rats at 17 weeks of age. At 28 weeks of age, GH3-bearing rats (GH rats) showed very high serum GH levels and a moderate increase in serum prolactin levels, resulting in low circulating estradiol levels. The GH rats showed significant increases in body weight and in length and volume of both the femur and vertebral body. Bone mineral content values of either the midfemur or the whole lumbar body were significantly greater in the GH rats compared with littermate controls, while the areal bone mineral density values of the respective bones were not different between the two groups. The parameters of mechanical strength of the femur were significantly larger in the GH rats than in controls, whereas those of the lumbar vertebral body cylinder specimen were not different between the two groups. Respective normalized mechanical parameters of the femur and the vertebral body were the same in the GH rats as in controls. In the midfemur, the GH rats showed a significant increase in the total cross-sectional area without influencing the bone marrow area, resulting in an increase in the cortical bone area and the moment of inertia compared with controls. The indices of periosteal bone formation in the midfemur were greater in the GH rats compared with controls, but the endocortical bone formation and resorption were not different between the two groups. In the vertebral body cancellous bone, the GH rats had an increase in bone turnover rate, whereas the structural parameters were not different between the two groups. These results from GH3-bearing rats demonstrate that an excess of GH increases cortical bone mass in rats accompanied with estrogen deficiency, while no large effect on vertebral body cancellous bone mass is seen.
Risedronate is a potent pyridinyl bisphosphonate in clinical development for treatment and prevention of osteoporosis, and has been recently approved for treatment of Paget's disease in the United States. An open-label study was conducted to determine the effect of risedronate treatment on pagetic bone lesions in patients with moderate to severe Paget's disease (mean serum alkaline phosphatase levels [ALP] approximately seven times the upper limit of normal). Patients were treated with 30 mg/day oral risedronate for 84 days followed by a 112-day nontreatment period. This 196-day cycle was repeated once in patients whose ALP did not normalize or who experienced relapse, defined as a > or =25% increase in ALP from the lowest value measured. Radiographs of affected anatomical sites in 26 patients were collected at baseline, 6 months, and/or 12 months. Eleven patients received one course and 15 patients received two courses of treatment. Radiographs were examined by a skeletal radiologist who was blinded to their time sequence. Changes in pagetic lesions were categorized as "improved," "deteriorated," or "no change." Between baseline and 6 months, 16 patients improved and 3 deteriorated; at 12 months, 11 patients improved and 2 deteriorated. Most lesions remained unchanged between 6 and 12 months. Improvements were noted in all skeletal sites (tibia, femur, humerus, forearm, pelvis, spine, and skull), but were most pronounced in weight-bearing long bones. In weight-bearing bones, nine lesions had osteolytic fronts. Of these, seven improved and two remained unchanged at 6 months; at 12 months, all but one lesion (which improved) remained unchanged. This radiographic assessment demonstrates that oral risedronate, 30 mg/day in one or two 3-month courses, is highly effective for improving bone lesions in patients with Paget's disease. Risedronate treatment had no deleterious effect on osteolytic lesions in weight-bearing bones; indeed, the majority of lesions with osteolytic fronts were improved after 6 months of risedronate treatment.
The aim of the study was to compare the effects on bone mass and turnover of continuous vs. intermittent clodronate administration on 120 postmenopausal women (average age 61 years) with low bone mass (femoral neck bone mineral density [BMD] of at least -1 SD or more, T-score), with another 30 women as a control group. Participants were given 1800 mg of clodronate every 6 months over 2 years using different treatment patterns: a) two continuous regimens, consisting of a daily oral dose of 400 mg or 100 mg every 10 days by intramuscular injection, the latter being considered continuous because the interval between injections is shorter than the time employed by each bone remodelling unit to complete the resorption phase of a remodelling cycle; and b) two intermittent regimens, consisting of 1800 mg every 6 months administered either as a single 18-h intravenous infusion or by separate infusions of 300 mg over 6 consecutive days. All women, including those in the control group, received calcium and vitamin D supplementation. After 2 years, continuous clodronate regimens caused an increase in BMD both at lumbar spine and proximal femur (L(1-4) BMD = 3.07% and 2.69%; femoral neck = 2.12% and 2.09%, respectively, with intramuscular and oral regimens). Intermittent clodronate administration was associated with a small increase or a stabilization in bone mass (L(1-4) BMD = 0.53% and 1.22%; femoral neck = 0.30% and 0.77%, respectively, with 1- and 6-day intravenous infusion regimens). From the 12th month, changes in spine and femoral neck BMD after continuous regimens were statistically different compared with that obtained with intermittent ones. Twenty-five of the 150 women (16.7%) discontinued the study before the end of the 2-year follow-up, but of these, only 7 dropped out because of adverse events related to the treatment itself. To summarize, intermittent clodronate administration could be a suitable option for the prevention of osteoporosis.
Organ transplantation is now the treatment of choice for many patients with life-threatening chronic diseases. A new set of side effects unique to these groups of patients has become recognized, and bone disease is one of these complications. However, little is known about the effects of myeloablative treatment followed by bone marrow transplantation (BMT) on bone mineral metabolism. We have prospectively investigated 31 patients undergoing BMT for hematologic diseases. Serum concentrations of calcium, phosphorus, creatinine, gonadotropins, sex hormones, and the biochemical markers of bone turnover were measured. The samples were collected before BMT and 1, 2, 3, 4, and 12 weeks, 6 months, and 1 year after BMT. Bone mineral density (BMD) was measured with dual-energy X-ray absorptiometry before BMT and 1 year after BMT. The serum carboxy-terminal cross-linked telopeptide of type I collagen increased progressively until 4 weeks after BMT. Thereafter, it began to decrease and reached basal values after 1 year. Serum osteocalcin decreased progressively until 3 weeks after BMT. After that, it increased and reached basal values after 3 months. No distinct differences were observed in the serum biochemical turnover markers between males and females, or between patients who received total body irradiation and those who did not. One year after BMT, lumbar spine BMD had decreased by 2.2%, and total proximal femoral BMD had decreased by 6.2%. Eighty-six percent of the women (12/14) went into a menopausal state immediately after BMT. This was caused by high gonadotropin levels and low estradiol levels. In contrast, gonadotropin levels and testosterone levels did not change significantly in the male patients after BMT. In conclusion, the rapid impairment of bone formation and the increase in bone resorption, as shown by the biochemical markers in this study, might play a role in post-BMT bone loss.
Microgravity induces significant and progressive bone loss in both humans and animals. This is the consequence of disturbed bone remodeling. We performed a bed rest experiment to simulate microgravity and tried to clarify bone metabolism by measuring biochemical markers of bone turnover. Six healthy volunteers participated in 120 days of bed rest. The parameters of calcium homeostasis, calcitropic hormones, and biochemical markers of bone turnover were examined. After ambulatory control evaluation, all subjects underwent 120 days of bed rest. Metabolic evaluation was performed in a baseline period, and on days 7, 16, 50, 72, 92, and 108 during bed rest, and on days 10 and 25 during a recovery period. Bed rest induced an increase in urinary calcium (Ca) excretion and serum Ca and bone resorption markers. Urine pyridinoline, deoxypyridinoline, and type I collagen cross-linked N-telopeptide increased more rapidly than urinary Ca excretion and serum Ca. Tartrate-resistant acid phosphatase (TRAP) increased even in the recovery period. Carboxy-terminal propeptide of type I collagen, a bone formation marker, significantly decreased on days 50, 92, and 108 of bed rest. These changes of biochemical markers of bone metabolism, except for TRAP, rapidly returned toward control levels in the recovery period. Immunoreactive parathyroid hormone showed a modest decrease during bed rest and a significant increase in the recovery period. Insulin-like growth factor I (IGF-I) and its binding protein, insulin-like growth factor binding protein-3, increased during bed rest, indicating the possibility of resistance to IGF-I in bones under reduced mechanical stress and strain. Bone loss from unloading results from the combination of acceleration of bone resorption and subsequent retardation of bone formation.
Wrist fractures associated with postmenopausal women are only partially explained by osteoporosis. Recent studies have shown that polymorphism of an Spl binding site in the first intron of the collagen I alpha 1 gene (COLIA1) may determine risk for vertebral and nonvertebral fractures in post-menopausal women independent of bone mass. We investigated the relationship between the COLIA1 polymorphism, lumbar spine and femoral neck bone mineral density (BMD), ultrasound stiffness of the heel, anthropometric variables, and risk for wrist fractures in 126 Czech postmenopausal women with low bone mass who suffered one or more wrist fracture in the last 5 years and in 126 postmenopausal women with low bone mass without any fracture. Genotypes for the Spl COLIA1 polymorphism were determined by polymerase chain reaction, digestion with Ball restriction enzyme, and agarose gel electrophoresis. The test discriminates two alleles, S and s, which correspond to the presence of guanine and thymidine, respectively, at the first bases in the Spl-binding site in the first intron of the gene for CO-LIA1. No significant differences were found between the fracture and control group with regard to age, weight, and years since menopause. However, BMD of the lumbar spine and femoral neck and ultrasound stiffness of the heel were significantly lower in patients with prevalent wrist fracture. Femoral neck BMD was the strongest determinant of prevalent fracture of the wrist. COLIA1 genotyping significantly strengthened prediction of prevalent fracture of the wrist. After multivariate adjustment, women in the Ss group had 2.0 times the risk of the women in the SS group (95% confidence interval [CI] = 1.1-3.8), and the women in the ss group had 2.8 times the risk of the women in the SS group (95% CI = 0.5-14.6). The overall gene-dose effect was an odds ratio of 2.1 per copy of the "s" allele (95% CI = 1.2-3.8). In the stepwise logistic regression, COLIA1 acted synergistically with femoral neck BMD and weight in increasing prediction of wrist fracture. The results demonstrate that COLIA1 Sp1 polymorphism is associated with an increased risk of wrist fracture in postmenopausal women independent of BMD and may be helpful in clinical practice by identifying patients with an increased fracture risk.
Structure model type and trabecular thickness are important characteristics in describing cancellous bone architecture. It has been qualitatively observed that a radical change of trabeculae from plate-like to rod-like occurs in aging, bone remodeling, and osteoporosis. Thickness of trabeculae has traditionally been measured using model-based histomorphometric methods on two-dimensional (2-D) sections. However, no quantitative study has been published based on three-dimensional (3-D) methods on the age-related changes in structure model type and trabecular thickness for human peripheral (tibial) cancellous bone. In this study, 160 human proximal tibial cancellous bone specimens from 40 normal donors, aged 16 to 85 years, were collected. These specimens were micro-computed tomography (micro-CT) scanned, then the micro-CT images were segmented using optimal thresholds. From accurate 3-D data sets, structure model type and trabecular thickness were quantified by means of novel 3-D methods. Structure model type was assessed by calculating the structure model index (SMI). The SMI was quantified based on a differential analysis of the triangulated bone surface of a structure. This technique allows quantification of structure model type, such as plate, rod objects, or mixture of plates or rods. Trabecular thickness is calculated directly from 3-D images, which is especially important for an a priori unknown or changing structure. Furthermore, 2-D trabecular thickness was also calculated based on the plate model. Our results showed that structure model type changed towards more rod-like in the elderly, and that trabecular thickness declined significantly with age. These changes become significant after 80 years of age for human tibial cancellous bone, whereas both properties seem to remain relatively unchanged between 20 and 80 years. Although a fairly close relationship was seen between 3-D trabecular thickness and 2-D trabecular thickness, real 3-D trabecular thickness was significantly underestimated using 2-D method.
The capacity of dual x-ray absorptiometry and quantitative ultrasound to discriminate bone loss and to predict the mechanical and microarchitectural properties of cancellous bone in an animal model of osteopenia was evaluated. Thirty-five female Sprague-Dawley rats (10 months old) were randomized into three groups: baseline group, 10 rats killed at the beginning of the study; ovx group, 15 rats ovariectomized; and sham group, 10 rats sham operated. At the beginning and end of the study, all the animals underwent osteosonography to record the proximal tail (C3 vertebra) bone speed of sound. Sixteen weeks after surgery, the animals were euthanized and the L5-6 lumbar vertebrae of each rat were excised for densitometric, biomechanical (compression test), and histomorphometric studies. Significant differences were found among the groups for final speed of sound (p = 0.01). The L5 bone mineral density of the ovx group decreased by 12.1% (p = 0.049) and 12.6% (p = 0.035) compared, respectively, with baseline and sham groups. The biomechanical parameters of the ovx group decreased by 15-47% compared with the other groups, showing significant differences between the ovx and sham groups both for maximal stress (p = 0.026) and elastic modulus (p = 0.013). Histomorphometric parameters of the ovx group showed significant decreases in comparison with other groups. Logistic regression analysis showed that dual X-ray absorptiometry and quantitative ultrasound discriminate ovariectomized and healthy rats with a similar capacity, classifying correctly all rats used in the model in a range of 61-70%. This similar capacity seems to derive from two different capacities to detect bone changes. Dual X-ray absorptiometry, depending on bone mineralization and density, is able to detect modifications in bone stiffness and strength, confirmed also by the correlation with biomechanical data. On the contrary, quantitative ultrasound seems to depend more on cancellous bone microarchitecural changes because it is correlated to histomorphometric parameters.
Intracapsular femoral neck fractures are associated with decreased cortical width and increased proportions of Haversian canals with diameters greater than the normal mean plus 3 SD (i.e., >385 microm). Such canals might be formed if closely associated resorbing osteons merge; a cortical event analogous with the loss of cancellous connectivity. To test this, we investigated the pattern of osteon distribution in the aging femoral neck to determine if remodeling osteons were distributed in anatomical clusters. Femoral neck biopsies from female patients with intracapsular hip fractures (n = 13) were compared with age/gender-matched cadaveric controls (n = 13). Solochrome-stained sections were analyzed for Haversian canal location, canal diameter, and the presence of an osteoid surface. Clustering was investigated using statistical software with a cluster defined as two or more osteoid-bearing osteon centers within 0.75 mm of each other. Clusters occurred more frequently than would be expected by chance (p < 0.001). Fracture cases had more clusters per unit area (3.14 +/- 0.31 clusters/25 mm2 of cortical bone) than controls (1.89 +/- 0.22) (p = 0.002). In fracture cases, the antero-inferior, antero-superior, and infero-anterior regions had more clusters per 25 mm2 than comparable control regions (ant/inf: 4.12 +/- 0.79, 1.70 +/- 0.60,p = 0.025; ant/sup: 5.31 +/- 1.1, 1.80 +/- 0.59,p = 0.013; inf/ant: 3.15 +/- 0.49, 1.27 +/-0.29, p = 0.004). The mean number of clusters per 25 mm2 per region correlated with the mean porosity per region (adjusted r2 = 0.60;p = 0.014), and the total number of giant canals per region correlated with the total number of clusters per region (adjusted r2 = 0.58; p = 0.011). In conclusion, remodeling osteons are clustered or grouped anatomically, and fracture cases have more clusters than controls. Our data suggest that merging of adjacent, clustered osteons during resorption could lead to the rapid development of canals with excessive diameters and focal weakness. Clustering is greatest in those regions that we have previously shown to have the largest relative reductions in bone strength compared with controls and known to be maximally loaded during a sideways fall. This implicates the remodeling process underlying clustering of remodeling osteons in the aetiology of hip fracture.
Over the past eleven years, we have worked together to treat children who are dentally phobic. This has enabled us to develop an understanding of how children come to be dentally fearful. We have constructed a model of child dental fear which helps us in our work. It is important to acknowledge that fear is a normal phenomenon when any of us are exposed to threat. Helping dentally fearful children appraise or evaluate threat, face their fear and build upon their strengths is the task facing dentists and, occasionally, psychologists. The consequences for children of not doing so are extreme difficulty with accepting and ultimately total avoidance of treatment. Both of these can persist into adulthood. First, we propose to discuss the normality of fear in children, placing dental fear within a developmental context. We will then outline a model for assessing and treating dental fear which identifies five discrete but interrelated factors. Each of the factors and its treatment is illustrated with examples.
Implant surgery protocols differ slightly with individual systems. However, basic surgical principles are required to ensure successful osseointegration of the implant in the correct location which allows good aesthetics and loading.
This paper looks at how carefully prescribed special trays can be helpful in everyday dental practice. Guidelines are suggested for the design of custom trays, that will, hopefully, lead to improvements in the quality of working impressions.
OBJECTIVE: To conduct a systematic review of the literature on the longevity of routine dental restorations in permanent posterior teeth, and to identify and examine factors influencing its variability. METHOD: Accepted guidelines were followed. An advisory group oversaw the project. Simple Class I and Class II amalgam, composite resin, glass ionomer and cast gold restorations were covered. Comprehensive searching of electronic databases, hand-searching, and location of 'grey' literature, generated 124 research reports. Those considered relevant were assessed for validity and quality according to agreed criteria. The analysis was descriptive. RESULTS: Eight of 58 relevant research reports were categorised, according to agreed criteria, as being of satisfactory validity and quality. They suggested that 50% of all restorations last 10 to 20 years, although both higher and lower median survival times were reported. The findings were supported by the totality of studies reviewed. However, variability was substantial. Restoration type, materials, the patient, the operator, the practice environment and type of care system appeared to influence longevity. CONCLUSIONS: Many studies were imperfect in design. Those considered to be the most appropriate for analysis were too limited to undertake a formal statistical exploration. Therefore there remains a need for definitive randomised controlled trials of restoration longevity, of sound design and adequate power, employing standardised assessments and appropriate methods of analysis.
AIM: To find out how many patients for whom dental general anaesthesia was requested actually needed it in order to complete treatment. DESIGN: Analysis of clinical outcomes supported by telephone canvassing of parents. METHOD: In summer 1998, eighty two child patients were seen in the Community Dental Service in Rochdale with a request for the provision of dental general anaesthesia (DGA) for the extraction of teeth. Their ages ranged from 3 to 14 years and all were required to attend for a pre-anaesthetic visit. Unless objective indicators of a need for DGA applied, the parents and children were actively discouraged from having DGA, and the alternative of local anaesthetic (LA) was offered. Clinical outcomes and parent satisfaction were recorded after treatment was finished. RESULTS: In 75% of cases it proved possible to complete the extractions without need for DGA; in the 10% of cases where DGA was necessary, it was to deal with the sequelae of dental caries. Fifteen percent of subjects failed to complete treatment. Subjects found to have a need for DGA tended to be younger and with treatment required in more than one sextant. Pain as a presenting symptom, young age and multiple treatment needs were found to be poor predictors of need for DGA and did not automatically preclude successful treatment without DGA. The satisfaction ascertained from users of the service was high and explanation of proposed treatments, especially the comparative risks and benefits of DGA versus LA, was well received. CONCLUSION: There is scope for significant reduction in provision of dental general anaesthesia if current professional guidelines are followed.
The objective of this paper is to propose an evaluation framework for short courses in continuing education for general dental practitioners (GDPs) (so called, Section 63 courses). Existing monitoring and evaluation procedures in the West Midlands deanery were examined and an improved evaluation framework was then devised, piloted and revised. A 5 phase method was used incorporating the examination of existing practice (Phases 1 and 2), development of a new framework (Phase 3), piloting (Phase 4) and revision of the evaluation framework in the light of the pilot. This approach will be implemented in the West Midlands and may be adapted for national use (Phase 5). It was found that existing monitoring and evaluation was inconsistent in prevalence and scope. Those involved in short courses were in favour of a more consistent and visible evaluation, including some assessment of impact-on-practice and cost-effectiveness. In conclusion, meaningful evaluation needs to include four key processes: data gathering; data analysis; dissemination and, action planning (reviewing provision in the light of the data analysis). Thus, this evaluation framework feeds into a quality development cycle designed to ensure high quality and relevant short course provision for general dental practitioners.
A case report is presented which highlights the importance of a good history in arriving at the correct diagnosis in cases where allergy to local anaesthetic is suspected. Management of the patient is discussed and the topic of 'adverse reaction' briefly reviewed.
NHS dentistry has seen many changes recently. The latest has been the introduction of Personal Dental Services. This article describes the experiences of two general dental practitioners in their practice who entered as a first wave pilot. The article explains the steps involved in generating a proposal and preparing a practice to run as a pilot. The authors have highlighted areas of particular concern for others to consider before embarking on a similar journey.
This is the second of two articles looking at dental care for the patient with a cleft lip and palate. Part 1 looked at the needs of the child from birth through to the mixed dentition stage. Part 2 looks at dental care from the mixed dentition stage through to adolescence and young adulthood.
OBJECTIVE: To investigate current GDP oral surgery referral patterns given the anticipated change since the introduction by the General Dental Council of the specialty of surgical dentistry. DESIGN: Postal questionnaire. SETTING: 400 GDPs in Greater Manchester. RESULTS: 84% participation rate. 69% of dentists made a referral because of anticipated difficulty of surgery and 49% because of the complex nature of the patients' medical history. Practitioners who had undergone some oral surgery postgraduate training were more likely to undertake more surgery in their practices (P < 0.01) and to refer more patients for specialist care (P < 0.05). While female practitioners rated their own surgical confidence less highly than male practitioners (P < 0.001), and younger practitioners less than their older colleagues (P < 0.05), there was no significant difference in the number of referrals made. CONCLUSION: The most common reasons for referral were the anticipated difficulty of surgery and patient medical compromise. There was a wide variation between practitioners in the number of patients referred for specialist care. Postgraduate oral surgery training was identified as a factor contributing to this variation. Other practitioner variables, such as sex, experience and type of practice were not found to contribute.
AIMS: To assess the prevalences of caries, of developmental defects of enamel and their interrelationship in Brazilian 9-10-year-olds from areas of contrasting fluoridation histories. METHODS: Systematic random sampling procedures were used to select children from an area where water had been fluoridated in 1963 and from a second area where water had been fluoridated since 1998. Clinical examinations for caries were carried out using the DMFT index and WHO diagnostic criteria. Developmental defects of enamel on upper incisors were diagnosed using the DDE index. RESULTS: A difference of 40% in DMFT was observed, with a lower prevalence of disease in the area fluoridated since 1963. Diffuse opacities affected 14.3% of the children from the area fluoridated since 1963 compared with only 2.4% in the area fluoridated in 1998. Children living in the area fluoridated in 1963 who had diffuse defects had twice the chance of being free from caries compared with those living in the same area who had no defects or who had only demarcated or hypoplastic defects. CONCLUSIONS: This study confirms previous ones in showing the benefits of water fluoridation. Diffuse opacities of upper incisors affected relatively few subjects in either of the two areas.
OBJECTIVES: To analyse the working patterns of all those who qualified from the Liverpool School of Dental Hygiene over a 20-year period. To assess the proportion who give up practice, the degree of part-time work, career breaks, job satisfaction, availability of continuing professional education etc. METHOD: A questionnaire sent to all 226 hygienists who qualified from the School between 1997 and 1998, whether still enrolled as dental hygienists or not. RESULTS: Responses were received from 83% of whom 89% were still working as hygienists, the majority in general practice. 46% had taken an employment break, mostly for maternity reasons but a significant number for other reasons. Around 80% expressed good job satisfaction. Although there is a high level of part-time work, especially after career breaks, few had experienced difficulty in finding employment. One third of respondents considered that the availability of continuing professional education was 'poor' or 'very poor'. CONCLUSIONS: The great majority of hygienists enjoy good job satisfaction and work in more than one general dental practice. Comparisons suggest that they continue to work in their chosen career at least as much as female dentists do in theirs. The reasons for this are thought to be that hygienists tend to be recruited from the ranks of highly motivated, mature dental nurses and that the profession lends itself to part-time work which can be combined with family commitments. There are perceived deficiencies in the availability of continuing professional education, which may be remedied by the development of distance learning packages and Section 63 type courses designed specifically for them.
The aim of this paper is to report the views of academic dentists about careers in academic dentistry assessed by method of a postal questionnaire survey. The subjects of the survey were dentists in academic posts in the United Kingdom. The incentives in pursuing an academic career which respondents rated most highly were the opportunity to teach and the variety of work in an academic career. The greatest disincentives were competing pressures from service work, teaching and research, and the difficulty of getting research grants. Many would like to spend more time on research and less on service work and teaching. The length of time required for training, and the quality of training, was a concern, particularly for junior academics. Most respondents rated the enjoyment of their job highly but scored much lower on satisfaction with the time their job left for domestic and leisure activities. By contrast with academic medicine, in academic dentistry there is typically greater emphasis on teaching and less on research. In conclusion, the balance of activities in academic posts, particularly between service work, teaching and research, needs to be regularly reviewed. The development of a more structured training programme for junior academics, which does not disadvantage academic dentists when compared with their NHS colleagues, may be required.
The Embryo Collection of the Hubrecht Laboratory is a treasure house of comparative embryology. It is the largest and most important collection of its kind in the world, and consists of thousands of vertebrate embryos stored in alcohol, or prepared as histological sections. Many elusive species are included in the collection, some represented by complete developmental series. The accompanying archives offer a remarkable insight into the methods used to collect embryos form wild animals, as well as the motives behind the founders of the collection. Carefully maintained, documented and catalogued, the collection is available for study by all interested scientists. We argue that this collection is one of the greatest biodiversity resources in existence.
Pieter Nieuwkoop, who died September 18, 1996, at age 79 in Utrecht, The Netherlands, is remembered by developmental biologists for his numerous research contributions and integrative hypotheses over the past 50 years, especially in the areas of neural induction, meso-endoderm induction, and germ cell induction in chordates. Most of his experimentation was done on the embryos of amphibia, the preferred vertebrate embryo of the early years of the 20th century. One of his last publications contains a comparison of the experimental advantages and disadvantages of anuran and urodele amphibians (Nieuwkoop, 1996). The significance of his findings and interpretations for developmental biology can be estimated from the fact that researchers of many laboratories worldwide continue to work on the phenomena he first described and to extend the hypotheses he first formulated. The aim of this article is to review Nieuwkoop's main contributions and to cite the recent extensions by others.
This posthumous review of early embryonic inductions concludes: 1) the amphibian egg has only two distinct components, animal and vegetal. Interactions at their mutual boundary forms meso-endoderm. This is "meso-endoderm induction", not just "mesoderm induction". 2) The dorso-ventral polarity of the yolk mass implies a dorsally situated inducing centre. 3) Accumulation of cells into one, two, three or many cell masses [problastopores] along the circumference of the meso-endoderm results in as many axes, implying a self-organizing capacity of meso-endoderm. 4) Induction of the meso-endoderm is slow, spreading cell to cell through the animal moiety from the boundary of the vegetal yolk mass towards the animal pole. 5) Interaction between mesoderm and ectoderm is a separate step leading to cranio-caudal differentiation of the archenteron roof. 6) The initial invaginating endoderm and mesoderm, representing the future pharynx endoderm and prechordal plate mesoderm, first contacts the most posterior presumptive neurectoderm after having passed the still uninvaginated trunk mesoderm. At that moment an antero-posterior level neural induction actually starts. 7) The ectoderm contraction wave coincides spatially and temporally with the induced neural plate. 8) Two successive homoiogenetic waves of inductive activity pass through the presumptive neurectoderm in the anterior direction, the first one, "activation", giving rise to neural differentiation and ultimately forebrain, the second one, "transformation", to more caudal CNS structures. These are separate, successive steps in CNS regional induction. 9) The midbrain represents a secondary formation in the neural plate. 10) The observed changes in morphogenesis may depend upon separate, successive binary decisions via [cell and] nuclear state splitters [involving differentiation waves].
Spatially and temporally restricted expression of the Hox genes along the main and appendicular axes is essential for correct patterning of vertebrate embryos. In this overview we discuss the latest data that shed light on the mechanisms underlying the generation of the expression domains of the Hox genes. The molecular genetic interactions governing initial transcription of the Hox genes in the posterior part of the primitive streak during mouse and chick gastrulation remain enigmatic. But the recent discovery by Kondo and Duboule (Cell, 97, 1999, 407-417) of a "cluster repressive regulation", will undoubtedly lead to a better understanding of the molecular genetic mechanism underlying colinear and sequential initiation of Hox gene transcription. Recently progress has been booked in characterizing the basal processes driving progression of the Hox expression domains during their establishment. Hox expression is still labile while being established. The transcriptional state of Hox genes in anterior tissues can be reprogrammed under the influence of more posterior locations. Posteriorizing activity may involve RA and FGF signaling. It is only when these interactions and, in some cases at least, regulatory interactions with Hox and cdx gene products occur appropriately, that the Hox expression domains would be correctly established. After the Hox expression domains have been established, regulatory processes involving the products of Polycomb and trithorax- Group genes start operating, perpetuating the transcriptional state of the Hox genes within and outside the expression domains. Whether control at the level of chromatin structure, believed to operate during the late maintenance phase of Hox gene expression, is also involved in regulating concerted initial expression of these genes, is a possibility that has been suggested.
Aristaless-related genes, a subset of the Paired-related homeobox genes, have in the past few years emerged as a group of regulators of essential events during vertebrate embryogenesis. One group of aristaless-related genes has been linked to the morphogenesis of the craniofacial and appendicular skeleton by their expression patterns and by the phenotypes of natural and artificial mouse mutants. Expression and function in the nervous system characterise a second group, and a third group, the Pitx genes, have been shown to have many different roles, including functions in the pituitary, left-right determination and limb development.
Understanding why metazoan Hox/HOM-C genes are expressed in spatiotemporal sequences showing colinearity with their genomic sequence is a central challenge in developmental biology. Here, we studied the consequences of ectopically expressing Hox genes to investigate whether Hox-Hox interactions might help to order gene expression during very early vertebrate embryogenesis. Our study revealed conserved autoregulatory loops for the Hox4 and Hox7 paralogue groups, detected following ectopic expression Hoxb-4 or HOXD4, and Hoxa-7, respectively. We also detected specific induction of 5' posterior Hox genes; Hoxb-5 to Hoxb-9, following ectopic expression of Hoxb-4/HOXD4; Hoxb-8 and Hoxb-9 following ectopic expression of Hoxa-7. Additionally, we observed specific repression of 3' anterior genes, following ectopic expression of Hox4 and Hox7 paralogues. We found that induction of Hoxb-4 and Hoxb-5 by Hoxb-4 can be direct, whereas induction of Hoxb-7 is indirect, suggesting the possibility of an activating cascade. Finally, we found that activation of Hoxb-4 itself and of posterior Hox genes by Hoxb-4 can be both non-cell-autonomous, as well as direct. We believe that our findings could be important for understanding how a highly ordered Hox expression sequence is set up in the early vertebrate embryo.
Here, we review the WNT pathway and its regulation at different levels. We focus on the transcriptional regulation of WNT target genes, in light of the recently identified negative regulators, i.e. relatives of groucho and CBP.
Signalling between cells in the developing vertebrate embryo is essential for normal embryonic development. In the mid 1970's, signal transduction research started at the Hubrecht Laboratory with special emphasis on analysis of the signalling mechanisms that direct cell proliferation and differentiation. The introduction of in vitro model systems contributed tremendously to the success of the signal transduction research at the Hubrecht Laboratory. Initially neuroblastoma cell lines, and later embryonal carcinoma and embryonal stem cells played an important role in identification of the molecular key players in developmental signalling. For instance, embryonal carcinoma cells were used to identify and characterise polypeptide growth factors. Growth factor signalling research was extended to analysis of growth factor receptor activation. Moreover, the second messenger systems that are linked to growth factor receptors were studied, as well as the nuclear responses to growth factor receptor activation. Finally, the role of growth factor signalling in differentiation was established using embryonal carcinoma cells. Here, we will review work that was characteristic for the growth factor receptor signalling research that was done at the Hubrecht Laboratory between 1980 and the early 1990's.
Embryonal carcinoma and embryonic stem cells have been very useful models for identifying some of the factors that regulate differentiation in early mammalian development. Here, we present a brief history of their original isolation and characterization and of their later introduction into the Hubrecht Laboratory. We illustrate in a review their contribution to our current understanding of the function of transforming growth factor beta and ligands binding to the receptors of a related factor, activin, in development with some of our own work.
The formation of parietal endoderm (PE) from primitive endoderm (PrE) immediately after implantation of the early mouse embryo can be seen as the earliest example of an epithelio-mesenchyme transition (EMT) in murine development. Since EMT and EMI (epithelium-mesenchyme interactions) are at the very heart of morphogenesis, identifying molecular mechanisms governing these processes is of utmost importance. An excellent in vitro model system to study PE formation, i.e. F9 embryonal carcinoma cells, is available to this end. In the present paper we review our own recent results and those of others using these cells, and present our current view on the molecular mechanisms involved in PE formation.
Receptor Protein-Tyrosine Phosphatases (RPTPs) belong to the superfamily of protein-tyrosine phosphatases and have the intrinsic ability to transduce signals across the cell membrane. We are beginning to understand the role of RPTPs in development of invertebrates, due to elegant genetic studies. In contrast, relatively little is known about the role of RPTPs in vertebrate development. Signalling by RPTPs has predominantly been studied in mammalian cell systems, which has led to important insights into potential ligands, into regulation of RPTP activity and into potential RPTP substrates. Here, we will introduce the RPTPs, and discuss the function of the LAR-subfamily of RPTPs. In addition, we focus on the function and signalling of the haematopoietic RPTP, CD45. Finally, we will discuss the structure and function of RPTPalpha, the RPTP that is the subject of our studies.
Retinoids and estrogens are small lipophilic compounds fulfilling important biological roles in vertebrate development, reproduction and homeostasis. Both types of ligands are regulators of gene transcription by binding to (nuclear) proteins acting as ligand-activatable transcription factors, members of the nuclear receptor gene superfamily. Retinoids and their multiple receptors (RARs/RXRs) are particularly well-known for their role in early development and spermatogenesis, while much less is known about the two estrogen receptors (ERalpha/beta) during development. In this article we describe some of our previous and present work in both areas of research.
Classical work on germ cells in fishes has dealt with three main issues; their embryonic origin, the proliferation, and migration pathway during embryonic and larval development. Until recently, primordial germ cells (PGCs) have been studied in a number of fishes using morphological criteria only. The identification of the Drosophila vasa homolog gene of zebrafish now allows comparison of these morphological data with vasa RNA expression patterns in zebrafish. Teleost PGCs can be distinguished from somatic cells by their distinct morphology, at the earliest during gastrulation, and in most fishes their number varies between 10 and 30 during pregonial development. Mitosis is generally not observed in PGCs at extragonadal locations, whereas they are mitotically active once at the gonadal ridges. During gastrulation, PGCs appear to translocate from the epiblast to the hypoblast and during somitogenesis they are found associated with the most peripheral yolk syncitial layer (YSL). From the peripheral YSL they migrate through the median mesoderm into the dorsal mesoderm and then to the dorsal mesentery, where they establish the gonad primordia with mesenchymal cells. Vasa RNA positive cells, the PGCs of the zebrafish conform to these general observations. Interestingly, classical descriptive and experimental data can now be reevaluated using vasa as a molecular marker of the fish germ line. The power of zebrafish genetics together with possibilities of experimental embryology should accelerate research on aspects of vertebrate germ line development such as PGC migration, division and apoptosis, as well as (in) fertility. The present review summarizes some of the classical data on germ line development in fishes in relation to recent data on vasa expression in zebrafish and compares these findings, where appropriate, with those in other model organisms. Special emphasis is placed on vasa gene expression as a potential universal germ line marker and suggestions are made for novel, zebrafish specific approaches to investigate the vertebrate germ line.
This paper discusses current efforts to produce databases of gene expression for the major model embryos used in developmental biology. The efforts to build these resources were motivated by the need for immediate internet access to all types of research data, and the production of these databases is a major and new challenge for bioinformatics. Thus far bioinformatics has mainly been concerned with textually oriented resources and data, much of it concerned with gene and protein sequences. Because the genetic basis of developmental biology is integrated with developmental anatomy, these databases require the use of images to link molecular data with spatial information. In order to standardise database formats, digital atlases of some model systems are being produced that include integrated anatomical descriptions and these are being linked to appropriate genetic data. Integrating such image-based, searchable data into databases makes new demands on the field of bioinformatics and we consider here the imaging modalities that are used to obtain information and we discuss in particular the production of 3D images from serial sections. Next, we consider how to integrate textual and spatial descriptions of gene expression and the key tool needed to make this possible, i.e. anatomical nomenclature. A short review of internet resources on developmental biology is also given and future prospects for the development of these databases are discussed.
The use of clonal analysis to obtain a fate map of the epiblast of the mouse embryo and to investigate cell distribution during gastrulation and early neurulation is described in a personal reminiscence. A revised fate map of the epiblast at 6.5 days gestation is provided, and the development of 3-dimensional, quantitative image analysis techniques outlined.
A link between atopic dermatitis (AD) and emotional dysfunction is now well established but consideration of this has yet to be widely incorporated into clinical management. A biopsychosocial model of the mind-body interaction is considered in conjunction with the theory of childhood emotional development and the impact of a diseased skin, for its effect on a child growing up with AD. AD inevitably adds to the difficulties of parenting, so that parents and children may become locked into dysfunctional relationships. This adds to the stresses for all concerned, and may contribute to the deterioration of the child's skin condition as part of a psychosomatic vicious cycle. There is a strong indication that the psychological dimensions of AD should be taken into account as part of routine management. Further research will be needed to establish the effectiveness of any psychological intervention.
Sebum is a complex mixture of lipids, which is secreted by mammalian sebaceous glands, and forms a fluid film over the skin surface. After sebum is secreted, it becomes mixed with lipid from the keratinizing epithelium and forms the skin surface lipid film (SSLF). Until now, direct fine structural observation of the SSLF has been lacking. In the present work, we viewed the detailed structures of the human SSLF by ruthenium tetroxide staining. The results showed that the SSLF formed an amorphous sheet of variable thickness on the skin surface instead of forming lipid droplets, as had been the usual assumption. In general, its thickness was < 0.5 &mgr;m or even negligible in sebum-poor extremities. However, in the sebum-rich face, its thickness was > 4 &mgr;m in focal areas. Consistent with the thickness of SSLF, the sebum quantity showed great regional variation. It varied from 1 &mgr;g/cm2 (leg) to 189 +/- 42.7 &mgr;g/cm2 (mean +/- SD: face). The SSLF was composed of numerous fine granules of about 4-5 nm in a random orientation. Within the SSLF, variable amounts of deranged lipid lamellae derived from corneocytes were mixed with sebum. As well as on the skin surface, a similar amount of sebum was also found between the desquamating corneocytes in the uppermost several layers of the stratum corneum (SC). We also observed the presence of intercellular lipid lamellae in the outer layers of the SC: their lipid envelope remained intact even in desquamated corneocytes. Our results provide some new insights concerning the structure of the SSLF and its relationship with the SC.
Langerhans cells (LCs) and dendritic epidermal T cells (DETCs) constitute the skin immune system. To demonstrate the kinetics of in vivo activation of murine LCs and DETCs in the elicitation phase of contact hypersensitivity, we measured the cell area positively stained for I-A and gammadeltaT-cell receptor (or Thy-1.2), respectively, under a fluorescence microscope at various time intervals after topical application of dinitrofluorobenzene. The fluorescence-positive area of LCs increased in parallel with that of DETCs at 1 h and 24 h, indicating the biphasic activation of LCs and DETCs. Early activation was hapten-specific and often exhibited close LC-to-DETC apposition. Experiments with in vivo administration of neutralizing anticytokine antibodies revealed that none of interferon (IFN)-gamma, tumour necrosis factor (TNF)-alpha and interleukin (IL)-1beta were involved in the induction of early activation of LCs and DETCs, while TNF-alpha and IL-1beta mediated late activation of LCs, and IFN-gamma and IL-1beta mediated that of DETCs. Our results indicate that LCs and DETCs are synchronously and biphasically activated in the epidermis during the elicitation phase of contact hypersensitivity and suggest that different mechanisms may control early and late activation.
Wound healing in rat skin was studied in standardized wounds inflicted on both hind legs after unilateral sciatic nerve sectioning and/or capsaicin-induced depletion of sensory nerve (C-fibre) neuropeptide content. Daily visual inspection, histological examination and immunohistochemistry with antibodies against substance P, calcitonin gene-related peptide (CGRP), vasoactive intestinal peptide, neuropeptide Y and a pan-neuronal marker, protein gene product 9.5 (PGP 9.5) were used to assess wound healing and determine the distribution of dermal nerve fibres. In controls, nerve fibre density in the wound tissue was low during the first few days after wound infliction, but started to increase on day 4, reaching a peak on day 7 when 25% of medial wounds and 70% of lateral wounds were healed. All wounds were healed on day 11, a scar appearing on day 14 followed by a decrease in nerve fibre density. Capsaicin treatment and/or sciatic nerve sectioning reduced the density of CGRP-immunoreactive nerve fibres by 70% and that of PGP 9.5-immunoreactive fibres by 50%. The capsaicin-induced reduction in PGP 9.5-immunoreactive nerve fibre density is attributable to partial destruction of peripheral nerve fibres. CGRP-immunoreactive and PGP 9.5-immunoreactive nerve fibre density was restored both in capsaicin-treated and denervated groups, reaching a maximum, corresponding to the original level, by days 4-10. Neither the reduction in nerve fibre density following sciatic nerve sectioning nor the impairment of sensory nerve functional capacity following capsaicin treatment affected the rate of wound healing, all wounds being closed on day 11. The study shows that it is difficult to knock out all cutaneous sensory innervation. Thirty per cent of C-fibre innervation seems enough to ensure a normal wound healing.
Transforming growth factor (TGF)-beta1 is a multipotent growth factor with an important role in tissue homeostasis. This growth factor regulates cell proliferation, adhesion, migration and differentiation, as well as extracellular matrix deposition. The temporal secretion and activation of latent TGF-beta1 is thus of major importance to physiological and pathological processes and in wound healing and tumour formation. Cultured skin substitutes, as used to treat extensive acute or chronic skin wounds, offer an attractive model to investigate cellular interactions in cytokine and growth factor expression and response in vitro. In the present investigation, expression of TGF-beta1 was analysed in keratinocyte, fibroblast and melanocyte monolayer cultures, as well as in the dermal vs. epidermal components of reconstituted human skin. Immunohistology, enzyme-linked immunosorbent assay (ELISA) and Northern blotting were used to demonstrate expression at the RNA and protein level. In the monolayer cultures, levels of TGF-beta1 synthesized by melanocytes were observed to be considerably elevated when compared with keratinocytes. Most TGF-beta1, however, was secreted by fibroblasts. The relative contribution of the epidermal and dermal components of the skin substitutes to overall TGF-beta1 levels was determined by comparing results obtained for either component in the presence and absence of fibroblasts and keratinocytes. From results obtained by ELISA it was apparent that TGF-beta1 levels generated predominantly by fibroblasts within the skin substitutes were greatly reduced over time in the presence of keratinocytes. Suppression of fibroblast TGF-beta1 expression in the presence of keratinocytes was also demonstrable at the RNA level by Northern blotting. Results obtained by immunohistochemistry suggest that most, if not all, of the growth factor was present in the latent form. It is therefore most likely that the observed effect results from a factor secreted by keratinocytes, which is capable of suppressing TGF-beta1 synthesis by fibroblasts. These results suggest that expression of TGF-beta1 by fibroblasts is downregulated by paracrine actions of keratinocytes in healing skin.
Nitric oxide (NO) is produced by a wide variety of human cells and affects physiological and pathophysiological processes. In this study, we demonstrated for the first time that neuronal NO synthase (NOS) is expressed in eosinophils infiltrating into the dermis and follicular epidermis in eosinophilic pustular folliculitis (EPF). EPF is a cutaneous inflammatory follicular disorder first described in Japan. The clinical and histological features of EPF are characterized by erythematous papules, infiltration of numerous eosinophils, and a spongiotic appearance of the follicular epidermis, but the pathophysiology of EPF remains unclear. Our results suggest that NO produced from eosinophils plays an important part in the pathogenesis of EPF. Furthermore, we speculate that NOS inhibitors may be useful in the management of EPF.
Pseudoepitheliomatous hyperplasia has occasionally been reported in cutaneous T-cell lymphoma (CTCL). This association raises the question of the relationship between epidermal hyperplasia and the lymphomatous infiltrate. Because epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alpha) have been demonstrated to be involved in epidermal proliferation through binding to EGF receptor (EGFr), we tested the hypothesis that these cytokines could be secreted by lymphomatous cells, and induce the overlying pseudoepitheliomatous hyperplasia. The purposes of this study were: (i) to describe the clinical and immunohistological features of pseudoepitheliomatous hyperplasia; (ii) to determine its frequency in a large series of CTCLs; and (iii) to evaluate the expression of EGF, TGF-alpha and EGFr in CTCL with or without pseudoepitheliomatous hyperplasia. Eleven cases of CTCL with pseudoepitheliomatous hyperplasia were collected from a series of 353 cases of cutaneous lymphoma registered from 1990 to 1996. They consisted of eight of 28 (28.5%) CD30+ large T-cell lymphomas and three of 148 (2%) cases of mycosis fungoides. Epidermal expression of EGF, EGFr and TGF-alpha was stronger in CTCL than in control normal human skin. Lymphomatous T cells expressed EGF and TGF-alpha whereas no expression of these cytokines could be detected in cutaneous and nodal B-cell lymphomas, nor in a normal lymph node. In addition, epidermal expression of EGFr was stronger in CTCL with pseudoepitheliomatous hyperplasia than in control cases of CTCL without pseudoepitheliomatous hyperplasia, suggesting that these cytokines, in association with other factors, are probably involved in the epidermal hyperplasia observed in some cases of CTCL.
Although interferon-alfa (IFN-alpha) has proved effective in treating epidermotropic cutaneous T-cell lymphoma (ECTL), few studies have considered the follow-up of treated patients and whether complete remission was maintained. We studied 51 patients (one stage Ia, seven stage Ib, one stage IIa, 30 stage IIb, 11 stage III (Sézary syndrome) and one stage IV) who received low-dose IFN-alpha as monotherapy for ECTL (mean daily dose of IFN-alpha 2.7 x 106 units for 14.9 months), giving special consideration to the significance of My7 (CD13) antigen expression by epidermal basal cells in predicting the maintenance of complete remission. For a mean follow-up period of 43.4 months, the results showed 21 complete remissions, 13 partial remissions and 17 patients with stable or progressive disease. Twelve patients died during the follow-up (3-52 months). IFN-alpha led to an improved response in the early stages, with a greater number of complete remissions (P = 0.03) and partial remissions (P = 0.01). The mean time to complete remission was 4 months, regardless of clinical stage (P = 0.1). Of 21 patients in complete remission, 57% had a relapse within a mean period of 7.5 months. For patients maintained in complete remission, the mean period of response was 31 months. The length of complete remission was independent of clinical stage, and My7 antigen expression was not predictive of complete remission.Department of Dermatology, Hôtel-Dieu, Pl. A.Ricordeau, 44093 Nantes cedex 1, France.
Alopecia areata (AA) is widely believed to be an autoimmune disease. Hair loss is associated with a peri- and intrafollicular inflammatory infiltrate of anagen hair follicles primarily composed of CD4 + and CD8 + cells. A previous investigation involved in vivo depletion of CD8 + cells in the DEBR rat model to examine the cells' potential pathogenic activity in AA. The rat model is used here in a comparable study of CD4 + cell pathogenic activity. Eight AA affected DEBR rats were given intraperitoneal injections of a CD4 + cell depleting OX-35/OX-38 monoclonal antibody (MoAb) cocktail over a 15-day therapy course. A further eight AA-affected rats comprised a control group and were injected with equivalent volumes of an irrelevant MoAb, OX-21. Changes in both CD4 + and CD8 + peripheral blood cell populations were analysed by flow cytometry, and macrophotography was used to record any changes in hair growth. Of the eight CD4 + cell-depleted rats six responded with hair growth. The rats revealed significant hair growth within 23 days of treatment initiation. With rapid replacement of the CD4 + cell population the newly generated pelage hair was eventually lost. Two control rats also showed limited hair growth within the 112-day study period. In vivo depletion of CD4 + cells partially restores hair growth in AA affected rats. The response suggests that CD4 + cells may be actively involved in the pathogenesis of AA. Further research may elucidate whether CD4 + cells have a direct effect on hair follicles or exert their influence through their classic T helper cell supporting role for CD8 + cells.
Hairs were sampled from long-haired Caucasian females, and cross-sectional measurements were performed using a rotating profile method at fixed humidity (100%). The effect of the hair cycle on medullation was investigated by examining medulla size and form along the lengths of anagen and telogen terminal hairs and also from a composite model of the entire medulla, as produced from a full cycle's growth, by amalgamating the results from the anagen and telogen hairs. The effect of hair shaft cross-sectional size on medullation has been investigated by controlling any effect of the hair cycle on terminal hairs and by use of the maximal medulla size in short, fine (vellous) hairs from the same subjects. All terminal hairs were medullated for the majority of their lengths. The presence and size of the medulla, in terminal hairs, was profoundly affected by the hair cycle. It was largest early in anagen, where the medulla minor axis, major axis and cross-sectional area represented about 26%, 23% and 7% of the corresponding whole hair shaft parameters (at 100% humidity). The medulla was virtually absent towards the end of anagen. The shape of the medulla cross-section was less elliptical than that of the whole hair shaft and was close to circular. There was no significant change in medulla shape through anagen. The form of the medulla was also affected by the hair cycle; approximately, it was continuous for the first 50% of anagen, discontinuous for the next 25% and virtually absent or absent for the final 25%. The maximal size of the medulla, as occurred in early anagen, was markedly associated with the cross-sectional size of the whole hair shaft, both within terminal hairs and between all scalp hairs. The medulla was large in terminal hairs and small or absent in very small hairs. The proportion of the whole hair shaft occupied by the medulla increased with increase in hair size and reached a maximum in terminal hairs, in which the medulla minor axis represented about 30% of the whole hair shaft minor axis. Furthermore, this proportion was constant in the terminal hairs and was not related to whole hair shaft size. Such maximal proportional medullation might represent a defining feature of terminal hairs. Variation in size of the medulla is not the cause of the previously reported cycle-dependent change in cross-sectional size of the whole hair shaft of terminal hairs.
One-third of patients with chronic idiopathic urticaria (CIU) have circulating functional autoantibodies against the high affinity IgE receptor FcepsilonRI, or IgE. The intradermal injection of autologous serum causes a weal and flare reaction in many patients with CIU, and this reaction forms the basis of the autologous serum skin test (ASST). We have determined the parameters of the ASST which define the optimal sensitivity and specificity for the identification of patients with autoantibodies. Two physicians (R.A. S. and C.E.H.G.) performed ASSTs in a total of 155 patients with CIU, 40 healthy control subjects, 15 patients with dermographism, nine with cholinergic urticaria and 10 with atopic eczema. Patients were classified as having functional autoantibodies by demonstrating in vitro serum-evoked histamine release from the basophils of two healthy donors. There were significant differences (P < 0.001) in the mean weal diameter, weal volume, weal redness and flare area of the intradermal serum-induced cutaneous responses at 30 min between patients with CIU with autoantibodies and either those without autoantibodies or control subjects. The optimum combined sensitivity and specificity of the ASST was obtained if a positive test was defined as a red serum-induced weal with a diameter of >/= 1.5 mm than the saline-induced response at 30 min. For R.A.S. and C.E.H.G., the ASST sensitivity was 65% and 71% and specificity was 81% and 78%, respectively. Using these criteria, the following subjects had positive ASSTs: none of 15 dermographics, none of 10 atopics, one of nine cholinergics and one of 40 controls.
An association between pruritus and eating disorders has been suggested. This study examined changes in pruritus during weight restoration in a homogeneous group of women with severe anorexia nervosa (n = 19), using a structured questionnaire, visual analogue scale, clinical examination and a range of serological markers. We demonstrated that itching is a clinical feature of anorexia nervosa, associated with low weight and resolving on weight restoration. Some 58% of the sample suffered pruritus at low weight in a stable hospital environment. There was a significant association between changes in body mass index and severity of pruritus (P = 0.033), with reduced itching on weight restoration. Pruritus occurred in the absence of abnormalities in thyroid, renal and hepatic function, serum androgens, oedema, dermatoses or compulsive washing. Scratching was manifest as 'scratch prurigo' in five cases. Where itching was present, it was experienced as severe. We discuss a variety of possible explanations, including psychopathology, endocrine factors, regional blood flow variation, eczema and the role of central opioid and serotonergic activity. We argue that anorexia nervosa should be considered in all patients at low weight presenting with pruritus, and pruritus should be considered to be a physical symptom of anorexia nervosa.
A study of melanocytic naevi was carried out in southern Spain to examine the relationship between numbers of naevi at different body sites as predictors of whole-body naevus count and to determine whether the naevus count on the arms is valid for identifying the risk factors for total naevi. Subjects were the control group from a case-control study on risk factors for cutaneous melanoma. They were selected from visitors to the University of Granada Hospital (southern Spain) between 1989 and 1993. Of 200 people invited to participate, 146 accepted (73%). Data were collected by personal interview, and melanocytic naevi were counted over the entire body surface by clinical skin examination performed by one dermatologist. Partial correlation coefficients (R) estimated by multiple linear regression were calculated. Comparisons between whole-body naevi and naevi on the arms, and their relationship with risk factors, were assessed by analysis of variance and covariance. Arms in men (adjusted R = 0.88) and thighs in women (adjusted R = 0.82) were the best predictors of total naevi after adjusting for age and sun exposure. Age, occupational and leisure sun exposure, and sunburns showed significant correlations with the total number of naevi. Similar results were found for the naevus count on the arms. In conclusion, the prediction of whole-body numbers of naevi by a naevus count on specific sites differs between men and women: arms in men and thighs in women are the best predictors. Nevertheless, naevus counts on the arms allowed us to study the risk factors for total naevi as well as whole-body naevus count: age and occupational sun exposure were the strongest determinants.
In summer 1989, a skin cancer campaign was organized in the coastal area of western Netherlands. In order to assess the impact of this and future campaigns on detection rates for melanomas of various thicknesses, a frame of reference for the epidemiology of cutaneous melanoma was established. We performed a retrospective investigation of all melanomas diagnosed in this region in the period 1980-95. A total of 3705 (2967 invasive and 738 in situ melanomas) cases was analysed. During the 1980s the age-adjusted incidence of invasive melanoma steadily increased (from five in 100,000 to 11 in 100,000 for men and from nine in 100,000 to 14 in 100,000 for women). Since 1988/89 the rate has remained stable for men and has increased only slightly among women. Detection rates for in situ melanomas followed the same pattern. Mortality rates remained largely unchanged for both sexes (European standard rates are about 2.6 in 100,000 for men and 2.2 in 100,000 for women). The median Breslow thickness decreased from 1.2 to 1.1 mm for men and from 1.1 to 0.8 mm for women. A marked increase occurred in the proportion of thin melanomas in comparison with intermediate and thick tumours, particularly among women. The female/male ratio declined in the period studied from 2.0 to 1.5. Directly after the campaign in 1989 a temporary increase occurred in the total number of melanomas diagnosed, largely due to more thin melanomas (</= 1.50 mm). These findings are discussed, particularly the possible implications of the most remarkable finding of this study: the stabilization of the incidence rates in recent years. We conclude, given the recent stabilization of rates and the relative favourable thickness, that screening or surveillance should be confined to high-risk groups.
The prevalence of atopic dermatitis (AD) was recorded following examination by dermatologists and dermatology registrars of a random sample of 2491 school students throughout the State of Victoria, Australia. The overall prevalence, based on clinical examination, was 16.3% (95% confidence interval, CI 14.1-18.5), being higher in girls (17.7%; 95% CI 15.0-20.4) than boys (14.8%; 95% CI 11.8-17.8). Using the U.K. Working Party Diagnostic Criteria for AD reduced the prevalence to 10.8% (95% CI 9.3-12.3) with the prevalence in girls 12.3% (95% CI 10.1-14.4) and in boys 9.2% (95% CI 7.1-11.4). The prevalence was highest in 4-6 year olds (18.7% on clinical examination, 11.5% using the U.K. Working Party Criteria), decreasing with increasing age to 11.6% on clinical examination (8. 6% on U.K. Working Party Criteria) among 16-18 year olds. Most of those with AD were classified as having mild disease (54.1%), with 32.1% classified as having minimal and 13.8% as having moderate to severe disease. Over 80% of those who reported on the questionnaire that they had dermatitis that was then confirmed on examination had been using one or more products to treat it. Nearly 90% of these products were classified as efficacious, with medical practitioners being the major source of advice for their use (77%). Pharmacists (8%), family/friends (6%) and others (9%), including beauticians and naturopaths, made up the remainder of the persons from whom those affected had sought advice about their treatment. These data, the first community-based prevalence data on AD published from Australia, confirm that the condition is common among those of school age. There is a need for AD to be included among those conditions that are discussed in health education lessons in schools.
Dermatology in-patient units are frequently threatened with reduction or closure, yet there are few objective data regarding the nature and use of in-patient management with which to assess their value. We have surveyed 300 patients admitted during March 1997 to dermatology units throughout Scotland and Northern England, to establish their clinical and social profile, and the outcome of admission. All departments provided phototherapy and out-patient treatment services, and 84% of those admitted lived within an hour's travel of one of these. Three diagnostic groups (psoriasis, eczema and leg ulcers) accounted for 83% of in-patient days. Patients were admitted principally because of disease severity but many, including half of those with psoriasis, had concurrent medical problems such as alcohol abuse, psychiatric disorder or arthropathy. Many patients with psoriasis and leg ulcers were from socially deprived areas, as defined by low Carstairs index scores, and a similar proportion received income support. Eighteen per cent of patients, mainly those with acute disorders, would have needed admission irrespective of dermatology bed availability. Out-patient management was considered the next best alternative for only 28% of patients, and many patients would have been expected to treat themselves. By contrast, 84% of patients admitted were cleared or substantially improved, or had procedures completed as planned, and another 12% were partially improved. Outcomes were particularly good in psoriasis, eczema and infection groups. We have demonstrated that in-patient management is highly effective in providing remission in chronic skin disease, and our survey also suggests that concomitant disability or social factors mean that for many such patients ambulatory care cannot replace this service.
Thirty-five patients with mycologically proven scalp infections were enrolled in a randomized, double-blind clinical trial with oral terbinafine (dose adjusted according to patient weight) for either 1 or 2 weeks. Patients were observed for 12 weeks; after 4 weeks, non-responders were offered an additional 4 weeks of treatment followed by a second observation period. The causative organisms were Microsporum canis (n = 12), Trichophyton tonsurans (n = 12) and other Trichophyton spp. (n = 11). The Trichophyton infections were treated effectively in five of nine (56%) patients treated for 1 week and 12 of 14 (86%) patients treated for 2 weeks. Three of the non-responders were treated for an additional 4 weeks, and one responded. In the Microsporum group only one of seven patients treated for 1 week and none of five treated for 2 weeks responded. However, treatment was effective in four of six (66%) patients treated for an additional 4 weeks. Mild to moderate adverse events believed to be drug related occurred in four patients in each of the two groups. Terbinafine is well tolerated, and requires 2 weeks of treatment in most patients with Trichophyton scalp infections and 4 weeks or more in Microsporum scalp infections, to achieve a successful clinical and mycological response.
Laser removal of tattoos is not generally or readily available on the National Health Service and removal in privately run clinics is expensive. For those seeking removal of their tattoo this can create significant financial hardship. We investigated the psychological, social and financial impact of tattoos on the lives of those patients requesting laser removal. Sixty-eight patients with a mean age of 36 years participated. Twenty-seven (40%) had one tattoo, whereas eight (12%) possessed 10 or more; 54% had an amateur tattoo. Only 18% had received an explanation of the procedure or side-effects. The mean age at application of the first tattoo was 16 years; 48 (71%) were tattooed below the legal age limit of 18 years. Most tattoos were applied for the sake of fashion. The median duration of regret was 14 years before seeking removal. The main reasons for removal were enhancement of self-esteem and social, domestic and family reasons. In those patients attending for removal, most tattoos are applied impulsively and inexpensively in youth. They are often regretted for decades and create significant psychological, social and financial burdens.
Epidermolytic palmoplantar keratoderma (EPPK) is an autosomal dominant genodermatosis characterized by diffuse keratoderma, typically with an erythematous border. Histologically, palmoplantar epidermis shows suprabasal cytolysis and ultrastructurally, tonofilament aggregation with overlying epidermolytic hyperkeratosis. Mutations in the KRT9 gene, encoding keratin 9 (K9), a cytoskeletal protein expressed exclusively in suprabasal keratinocytes of palmoplantar epidermis, have been reported to cause EPPK. To date, all KRT9 defects reported in EPPK have been missense mutations in exon 1, which encodes the start of the alpha-helical rod domain. However, based on studies of other keratin disorders, it was postulated that mutations at the other end of the rod domain might also produce the EPPK phenotype. Here, we report the first mutation in the 2B domain of KRT9, 1362ins3, leading to an insertion of histidine in the helix termination motif of the K9 polypeptide. Insertional mutations have not been previously described in keratins. The phenotype of this case is similar to EPPK caused by 1A domain mutations, demonstrating that mutations in either of the helix boundary motif sequences of K9 are detrimental to keratin function and keratinocyte structure.
We report a Japanese family with dyschromatosis symmetrica hereditaria (DSH) (MIM 127400 in McKusick's Mendelian Inheritance in Man), a rare autosomal dominant genodermatosis, predominantly occurring among Japanese and Korean individuals. Members of the present family affected with the disease showed a mixture of hyperpigmented and hypopigmented macules distributed on the face and the dorsal aspects of the extremities, which are typical of DSH. As most of the literature on DSH has been written in Japanese, dermatologists outside Japan are not familiar with the condition. In this paper, 185 cases of DSH, most of them reported in Japanese, are reviewed and unique clinical, histological and genetic features of this condition are delineated.
Two elderly women with complex medical histories presented with erythematous patches, in one case involving the face and forearms, and in the other both elbows. Punch biopsies from both patients revealed intravascular proliferations of medium-sized and large cells with luminal occlusion typical of angioendotheliomatosis. Immunostaining did not show either lymphocytic or endothelial cell antigens but was consistent with a histiocytic differentiation of the intravascular cells in both cases, and was further substantiated by ultrastructural examination in one case. One patient received a course of cyclophosphamide therapy over 15 days. Skin lesions faded but did not disappear. The patient died 10 months later from cardiac and renal failure, which was most probably unrelated to the skin lesions. In the other case, lesions diminished but did not entirely resolve with treatment with low doses of oral prednisone. Angioendotheliomatosis can be divided into a malignant variant, which is an angiotropic lymphoma mostly of B-cell phenotype, and a benign, reactive variant, which is characterized by a proliferation of cells expressing endothelial cell markers. Only one case of angioendotheliomatosis with cells of histiocytic differentiation has been published previously under the name of intravascular histiocytosis. Our cases are very similar to the latter. The question arises as to whether intravascular histiocytic cell proliferation is a neoplastic proliferation of histiocytes or an early stage of classic reactive angioendotheliomatosis representing the residual cells associated with organization of microthrombi, which will be later followed by endothelial cell proliferation.
Nasal-type T/natural killer (NK) cell lymphoma, which often shows an angiocentric growth pattern, is a distinct clinicopathological entity highly associated with the Epstein-Barr virus (EBV). This tumour has a characteristic immunophenotype, whereas the cytological spectrum is broad. It is known that a clonal T-cell receptor (TCR) gene rearrangement is not found in this tumour. However, it is still unresolved as to whether the finding of a clonal TCR gene rearrangement excludes the diagnosis of nasal-type T/NK cell lymphoma. We describe a case of nasal-type T/NK cell angiocentric lymphoma, EBV-associated, and showing clonal TCR gamma gene rearrangement. The patient died of sepsis 5 months after diagnosis in spite of aggressive chemotherapy.
A woman with Hailey-Hailey disease, suffering from carcinoma of the vulva, was examined by histology and for the presence of human papillomavirus (HPV) DNA by polymerase chain reaction (PCR) and in situ hybridization. Our diagnosis by histological examination revealed the vulval carcinoma to be a squamous cell carcinoma (SCC), adjacent to lesions of Hailey-Hailey disease and severe dysplasia/carcinoma in situ [vulval intraepithelial neoplasia (VIN) III]. The PCR with consensus primers for the L1 region (L1-PCR) successfully amplified HPV DNA using total DNA extracted from formalin-fixed and paraffin-embedded tissue specimens. Restriction fragment length polymorphism analysis and sequencing of L1-PCR products revealed HPV types 16 and 39. HPV 16-specific primers for the E6 region identified HPV 16 DNA. In situ hybridization analysis with biotinylated HPV 16 and 39 DNA probes revealed the presence of the HPV 39 genome in the nuclei of the tumour cells in the SCC. These results indicate that HPV 16 and 39 are associated with lesions in vulval carcinoma. Regarding the patient's susceptibility to infection in the case of Hailey-Hailey disease, there is a possibility that HPV was inoculated into the lesions of Hailey-Hailey disease and induced those of VIN III and SCC.
Erythroplasia of Queyrat (EQ) is an intraepithelial carcinoma in situ affecting the mucosal surfaces of the penis, with a significant risk of invasion and metastasis. Treatment is often difficult and is associated with significant recurrence rates. Topical 5-aminolaevulinic acid (ALA) photodynamic therapy (PDT) combines a photosensitizer precursor and visible light to produce a photodynamic effect. It has been used successfully to treat benign, premalignant and malignant skin diseases. We present four patients with EQ who have been treated by topical ALA PDT. Of two patients with limited disease one has achieved a long-term complete response (36 months) and the other developed a recurrence at 18 months after a complete response. Two further patients with more extensive disease achieved a significant improvement, allowing easier treatment by laser vaporization. Although topical ALA PDT offers the advantages of tumour specificity, preservation of function and a good cosmetic result, more extensive EQ appears less responsive to this new therapeutic modality using current treatment parameters.
We report the first adult case of staphylococcal scalded skin syndrome (SSSS) due to methicillin-resistant Staphylococcus aureus (MRSA). This case is particularly unusual as the MRSA produced toxic shock syndrome toxin 1 and enterotoxin, but not exfoliatoxin. SSSS was originally described in neonates and is thought to result from exfoliatins which produce subcorneal splitting of the epidermis and are only produced by certain strains of S. aureus. This case reflects the range of toxins that can be associated with SSSS and the clinical manifestations of MRSA infection in adult patients.
Reactive perforating collagenosis (RPC) is characterized by umbilicated papules with a central adherent keratotic plug. Histologically, this condition shows transepidermal elimination of altered dermal collagen bundles into a cup-shaped epidermal depression. The present paper describes eight patients with associated diabetes mellitus who meet the diagnostic criteria for the acquired form of RPC (ARPC). Although half of these patients underwent dialysis, the lesions did not tend to develop after dialysis. Pruritus and the Koebner phenomenon were common, and histologically a microvasculopathy was noted in the dermis of all patients. We speculate that this disease is triggered by a cutaneous response to superficial trauma. Furthermore, this response acts synergistically with vasculopathy in the dermis, primarily in the case of diabetes mellitus. A secondary sign of ARPC may be degenerated collagen fibres as a result of transepidermal elimination.
Annular elastolytic giant cell granuloma is a rare granulomatous skin disease characterized by phagocytosis of elastic fibres by multinucleated giant cells. Lesions are either solitary or grouped in a few annular patches with elevated borders and central atrophy. Sun-exposed areas are more commonly involved than covered skin. The pathogenesis of the disease is still controversial. We report a 72-year-old fair-skinned woman with unusual clinical findings. An irregularly shaped erythematous plaque covered the entire face, and hundreds of lichenoid papules were present on both sun-exposed and covered areas which gradually evolved into annular lesions of about 0.5-1 cm in diameter. Sparing of an old burn scar and a nearly complete lack of elastic fibres in the scar site were noted, illustrating the presumed importance of dermal elastic tissue in the pathogenesis. The course of the disease is chronic. Several treatments have been tried, with variable success. In our patient, improvement was achieved with chloroquine over a period of 16 weeks.
2-Chloracetophenone (CN) is widely used as tear gas by police and civilians for self-defence. It may affect the eyes, respiratory system and skin, sometimes causing serious injuries. Both irritative and allergic contact dermatitis have been described. We report three police officers who experienced accidental escape of CN from their professional tear gas canisters. All of them showed localized dermatitis at the site of contact to CN, while widespread lesions appeared after 4 days in one case. Patch tests with the original involved tear gas dissolved in acetone (at 0.1-0.0001%) indicated an allergic reaction in two patients and an irritative reaction in the third. Occupational contact dermatitis due to CN seems to occur among police officers more often than is generally known. Infrequently, extensive health problems may be caused by CN when lesions spread over the integument. Therefore, an improvement of safety measures in occupational CN gas use is needed, especially aiming at avoidance of accidental leakage of canisters.
Many patients with cutaneous porphyria have curable or controllable disease; untreated porphyria may prove fatal. The genetic defects and mechanisms underlying porphyria are steadily being delineated, treatments have become more appropriate and genetic counselling is now more accurate. A summary of the basic diagnostic features, management and recent advances in the cutaneous porphyrias is presented, based on a workshop held by the British Photodermatology Group.
Keratin 2e (K2e) is expressed in the upper spinous and granular cells of adult epidermis. A highly specific polyclonal antibody was made against a C-terminal peptide of K2e and used to observe K2e expression at different developmental stages. At 12.5 weeks estimated gestational age (EGA) K2e was detected in trunk skin in scattered cells in the intermediate layer. At 13.5 weeks EGA, greater numbers of intermediate cells were stained with variable intensity, and staining in this pattern increased with age. Epidermal sheets from 14 weeks EGA showed that K2e + cells were excluded from developing hair follicles. At 135 days EGA, the following regional patterns were observed: in cheek, trunk, dorsal and ventral knee, elbow and dorsal hand there was moderate to intense staining of upper intermediate keratinocytes excluding cells of the hair canals and sweat ducts. The periumbilical region distinctly lacked K2e staining, while more distal areas showed increasing numbers of K2e + cells. The earliest expression of K2e was at 10 weeks EGA in the presumptive nail bed of developing digits. By 13.5 weeks EGA this pattern had shifted to the proximal nail fold, and K2e was absent in the nail bed. K2e was excluded from developing sweat glands and ducts and from developing hair follicles at the hair germ and early peg stages. By 15 weeks EGA in the fetal hair follicle small groups of cells were K2e + and by 19 weeks K2e + cells were seen at the level of the matrix. Some overlap in staining was detected for K2e with K10, and in palmar skin with K9; however, mostly the filamentous staining patterns for these keratins were distinctive. This study shows that the complex patterns of temporal and regional expression of K2e differ from known patterns for other epidermal keratins and suggest different regulation and function for this epidermal keratin.
Tenascin C is a large extracellular matrix glycoprotein involved in morphogenesis and wound healing. The distribution and expression levels of tenascin were examined in photodamaged skin to investigate the hypothesis that photoaged skin displays characteristics of wound repair. In situ hybridization and semiquantitative immunohistochemistry were performed on paired skin biopsies from patients with varying levels of photodamage, using monoclonal antibodies and cRNA probes for tenascin and its large isoform. In sun-protected skin, tenascin protein was distributed adjacent to the dermoepidermal junction, usually sparsely and discontinuously; tenascin mRNA was detected in dermal fibroblasts and some keratinocytes. In photodamaged skin, tenascin protein was increased in proportion to the clinical level of photodamage (analysis of variance: P < 0.0001, n = 29). With increased photodamage, tenascin protein expression became continuous along the dermoepidermal junction, extending deeper into and sometimes throughout the papillary dermis; tenascin mRNA was detected throughout the epidermis. Large tenascin isoform protein and mRNA distribution mirrored that of pantenascin, suggesting that it may be the predominant species in photodamaged skin. There was no correlation between tenascin expression levels and age or sex, and no seasonal variation was noted. The results indicate that photodamaged skin demonstrates tenascin increases consistent with an early wound healing response. However, tenascin increases in photodamage appear to be permanent and may therefore interfere with effective repair of ultraviolet-induced damage. In conclusion, this study has shown that dermal tenascin expression increases in proportion to the degree of photodamage. In normal skin, the temporal and spatial patterns of tenascin expression during morphogenesis and tissue remodelling are crucial to their correct progression. In photoageing, the 'normal' control of tenascin expression seems to be abrogated.
Hard keratins are expressed in normal hair and nails, and are characterized by a higher cysteine content than cytokeratins. Previous studies have suggested a coexpression of hard keratins and cytokeratins in pilomatrixoma, a benign follicular tumour which could originate from the hair matrix. Human hair keratin basic 1 (hHb1) is a newly characterized hair keratin which is expressed specifically by cortical cells of the normal hair shaft. A preliminary study has suggested that hHb1 could be expressed in pilomatrixoma. In order to confirm this hypothesis, we have studied a series of 128 pilomatrixomas by in situ hybridization, using a 35S-labelled hHb1-specific probe. The anti-sense probe was used as a negative control. Among these pilomatrixomas, six were early cases, 60 were classified into the intermediate stage (either fully developed or early regressive cases) and 62 were late regressive tumours made of shadow cells only. Forty-seven tumours showed hHb1 expression (37%), all being intermediate stage pilomatrixomas. The areas positively stained by the probe were band-like structures made of transitional cells only, which were very close to cells showing tricholemmal keratinization features. Neither the basophilic matrix cells nor the shadow cells expressed hHb1. Our results suggest that pilomatrixomas can differentiate towards cortical cells during their maturation process, as this keratin is specifically expressed in the cortex of the normal hair shaft. These data are consistent with previous studies which showed the expression of a hard keratin group in transitional cells by immunohistochemistry. The histogenesis of basophilic cells of pilomatrixoma is controversial, but it is likely that transitional cells represent an equivalent of the hair cortex.
The importance of a dermal element when providing permanent wound cover for skin loss has become evident as the shortcomings of pure epidermal grafts are recognized. We are developing a skin composite formed from sterilized human de-epidermized acellular dermis, keratinocytes and fibroblasts with the ultimate aim of using this composite to cover full-thickness excised burn wounds. These composites can be prepared with or without basement membrane (BM) antigens initially present on the dermis. This study investigates the importance of retaining BM antigens on the dermis to the production and appearance of these composites in vitro. Skin composites prepared from dermis with BM antigens either present or absent initially were studied throughout 3 weeks. Composites with BM antigens present initially were significantly better than those initially lacking BM antigens in: (i) the degree of epithelial cell attachment to the underlying dermis (hemidesmosomes were seen only in the former); (ii) the morphology of the epithelial layer; (iii) the consistent presence of collagen IV and laminin and the increasing expression of tenascin; and (iv) the amount of soluble collagen IV and fibronectin detected in the conditioned media. We conclude that an initial BM antigen template is vital in this skin composite model for the attachment and differentiation of the epithelial layer and for the subsequent remodelling of the BM in vitro.
Induction of hair growth by skin irritants and its relation to skin protein kinase C (PKC) isoforms were evaluated. Dorsal skin of BALB/c mice was shaved and anthralin (0.1% in corn oil) was applied on one side of the shaved backs in 20 mice daily for 5 days. The corresponding opposite side treated with corn oil served as a control. In another 20 mice, sodium dodecyl sulphate (SDS, 10% in water) was applied on one side of the shaved backs for 5 days by the same procedure as above and the corresponding opposite side treated with water served as control. Visible acceleration of hair growth on anthralin-treated skin was observed as early as 14 days after the application of anthralin and significant hair growth was observed at about 17-20 days. Enhancement of hair growth on SDS-treated skin was observed at about 3 weeks from the beginning of the treatment. None of the mice showed remarkable hair growth on the control side in either group. Mouse skin PKC isoforms levels detected by Western blot showed a similar pattern in both treatment groups. PKC alpha was downregulated initially, and was then elevated from 3 days after anthralin treatment and 14 days after SDS application. PKC beta was unchanged initially, decreased at 8 and 14 days after anthralin and SDS treatment, respectively, and reverted to the control level at 25 days after anthralin treatment, when it was still lower than the control in SDS-treated skin. PKC delta was also unchanged at first, but was elevated from 3 days after anthralin treatment and 14 days after SDS application. These results suggest that involvement of PKC may be a general phenomenon in irritant-induced hair growth in mice. Considering the stimulatory effect of PKC alpha and inhibitory effect of PKC delta on cell growth, we postulate that PKC alpha may be responsible for enhancement of hair growth while PKC delta may inhibit hair growth to keep the hair growth in balance.
The increased number and early activation of cutaneous mast cells is a typical feature of psoriatic inflammation. Interferon-gamma (IFN-gamma) is believed to be one of the important mediators in the cytokine cascade of psoriasis. Human mast cells have been previously reported to release various cytokines upon stimulation including interleukin (IL) -4, IL-5, IL-6, IL-8, IL-13 and tumour necrosis factor-alpha. Here we report that human mast cells synthesize also IFN-gamma at mRNA and protein level and that the number of IFN-gamma producing mast cells is significantly increased in the psoriatic skin. IFN-gamma immunoreactivity in mast cells was demonstrated by staining non-lesional and lesional skin sections from 21 patients with psoriasis. Ten patients with atopic dermatitis (AD) and five healthy persons served as control groups. The percentage (mean +/- SD) of IFN-gamma + mast cells in lesional compared with non-lesional psoriatic skin was 67 +/- 18% vs. 44 +/- 17% (P < 0.0001, paired t-test), respectively, but only 9 +/- 6% vs. 10 +/- 7% in corresponding skin samples of AD. In the skin of healthy controls, only 12 +/- 12% of the mast cells were IFN-gamma +. Using immunoelectron microscopy, we confirmed the ultrastructural localization of IFN-gamma within the granules of mast cells in psoriatic skin. In addition, stimulation of a human mast cell line HMC-1 with phorbol myristate acetate (PMA) (100 nmol/L) for periods of 2-24 h induced expression of IFN-gamma mRNA, which peaked at 24 h. When HMC-1 cells were stimulated with PMA (100 nmol/L) for periods of 0-3 days, the cells released IFN-gamma protein, peaking on day 1. These results provide further evidence for the important role of mast cells in the pathogenesis of psoriasis.
The MELAS syndrome (mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes) belongs to the category of mitochondrial disorders. The most common molecular aetiology of the syndrome is a mutation at base pair (bp) 3243 in the mitochondrial genome (mtDNA). The phenotype is varied and, apart from central nervous system involvement, the patients with this mutation may present with neurosensory hearing loss, diabetes mellitus and cardiomyopathy. These phenotypes suggest that organs dependent on aerobic metabolism suffer most. We investigated the possible clinical and physiological manifestations of impaired energy metabolism in the skin of 28 patients with the bp 3243 mutation in mtDNA. Non-invasive sonography and laser Doppler flowmetry were used to measure skin thickness and the blood flow of the skin. Skin collagen synthesis was assayed from suction blister fluid. Evaporimetry was used to assess the re-epithelialization rate of suction blister wounds. Histochemistry and immunohistochemistry were used to evaluate the melanocytes and pigment in the skin. Vitiligo was found in three of the 28 patients (11%), which was markedly more than in the general population. Histological findings showed an absence of pigment, but an apparently normal distribution of melanocytes in the dermoepidermal junction. Seborrhoeic eczema and atopy were also somewhat more frequent. No features of premature ageing, such as a marked decrease in skin thickness, blood flow, collagen synthesis or re-epithelialization rate, were demonstrated.
One of the goals of antifungal therapy is to combine an anti-inflammatory activity with antimycotic properties, and therefore it is interesting to evaluate the capacity of active antifungal drugs to interfere with different phases of the inflammatory reaction. In order to identify a possible scavenger property of free radical species of the antifungal agent terbinafine, we studied its activity on the reduction of nitrotetrazolium blue chloride (NTB), induced by superoxide anions with the ultraviolet (UV)- and chemical-induced peroxidation of unsaturated lipid targets. NTB reduction was followed by spectrophotometer and the decomposition of squalene or methyl arachidonate by gas chromatography-mass spectrometry. Terbinafine (20 microgram and over) was capable of significantly inhibiting NTB reduction, indicating that the drug scavenges superoxide anion radicals. In these conditions no modifications of the concentration of the drug, as evaluated by high performance liquid chromatography, were observed. The UV- or chemically induced peroxidation of squalene and arachidonic acid was significantly reduced in the presence of 50 microgram of terbinafine, suggesting that the substance interferes with the chemical properties of peroxyl radicals. In all the tests used the degree of inhibition was proportional to the amount of free radicals generated. In conclusion our results indicate that terbinafine, at therapeutic concentrations, can be considered to be a free radical interceptor in vitro and could exert a mild anti-inflammatory activity in vivo.
We have recently shown in a cohort of patients (n = 67) with mucous membrane pemphigoid, 63 of whom had cicatricial pemphigoid (CP), that the presence of both IgG and IgA circulating antibasement membrane zone antibodies was associated with a more severe and persistent disease. In this study we sought to ascertain whether in CP, serial titres of IgG and IgA might provide a reliable marker of disease activity. Serum titres for IgG and IgA antibodies were assayed at 6- to 12-monthly intervals in 56 patients over 32.2 +/- 20.3 (mean +/- SD) months. Antibody titres were correlated with the clinical score recorded at each follow-up visit. Sequential titres for both IgG (P < 0.001) and IgA (P = 0.015) were significantly associated with variations in disease activity. Greatest improvement was seen in patients with the greatest change in either IgG or IgA antibody titre. We suggest that serial antibody titres may therefore be useful in the assessment and management of CP.
Several studies, in particular in adult groups, have evaluated the involvement of mites in the pathogenesis of atopic dermatitis (AD). This still remains controversial. The objective of this study was to determine the level of house dust mites (HDMs) in the beds of a group of children with AD and correlate these levels with their allergometric assessment. Forty-one children with AD underwent allergometric tests (prick test, patch test and radioallergosorbent test, RAST) and the concentration levels of HDMs in their homes were evaluated. Our data show that about half of the children (51%) with AD presented Dermatophagoides pteronyssinus positivity (prick test and/or RAST and/or patch test). Dust was collected in the period October-November from the children's beds, by the same two operators, using a dust-collection device. The dust mite level was tested by an enzyme-linked immunosorbent assay with antibody against Der p 1 allergen. Ten children (24%) presented a Der p 1 concentration > 2 microgram/g of dust (the value assumed to be a risk level for sensitization), 20 (49%) between 0.1 and 2 microgram/g and 11 (27%) < 0.1 microgram/g of dust. In the group with the highest Der p 1 concentration (> 2 microgram/g dust) nine children (90%) presented an allergometric D. pteronyssinus sensitivity, the difference with the other two groups being statistically significant at P < 0.018. The results of the present study show that the highest HDM concentrations were observed in the group with an allergometric D. pteronyssinus positivity (prick test and/or RAST and/or patch test). It is hypothesized that a higher HDM concentration may elicit D. pteronyssinus IgE sensitization and delayed hypersensitivity in children with AD.
Renal transplant recipients have an increased risk of developing skin cancers, which are often multiple and aggressive. Frequently, these tumours develop on a background of widespread epidermal dysplasia. Systemic retinoids are known inhibitors of skin cancer but reports of their use in renal transplant patients are limited. We describe our experience using 0.3 mg/kg daily of acitretin in 16 patients over a 5-year period. Overall, there was a significant reduction in the number of new tumours excised in 12 of 16 patients during treatment compared with the same pretreatment interval. A significant chemoprophylactic effect was shown for up to 4 years of treatment. Patients with five or more tumours prior to acitretin benefited most. Two patients discontinued treatment because of side-effects and two patients developed hyperlipidaemia. Two patients with end-stage graft failure proceeded to haemodialysis. The introduction of low-dose acitretin proved to be a useful strategy in the long-term reduction of skin cancer in renal transplant recipients with multiple skin cancers and extensive epidermal dysplasia.
The ideal psoralen-ultraviolet A (PUVA) regimen for chronic plaque psoriasis has yet to be established. There are four components to a PUVA regimen: the dose of psoralen, the starting dose of UVA, the frequency of treatment and the incremental UVA dose protocol. Recent studies have been directed at trying to optimize the efficacy of PUVA while minimizing acute side-effects and the risk of cutaneous carcinogenesis, believed to be independently related to the cumulative dose of UVA and the total number of treatments. The British Photodermatology Group recommends two twice-weekly PUVA regimens: one starts with 50% of the minimal phototoxic dose (MPD) and uses weekly increments of 40%, 30%, 25%, 20%, 15%, 10% and 5% of the previous dose to a maximum of 14.5 J/cm2; the other starts with a fixed dose based on skin type and uses weekly dose increments of 40%, decreasing to 20% once erythema develops. We undertook a prospective randomized controlled trial comparing these regimens in 85 Irish patients. The clearance rate with the MPD regimen was lower than with the skin type regimen, 67.5% vs. 95% (P < 0.05). The reasons for treatment failure were grade 3 erythema and severe PUVA itch. There was a trend suggesting that patients with skin types I and II, but not skin type III, required a higher cumulative UVA dose and fewer exposures to clear with the MPD regimen than the skin type regimen, although this did not reach statistical significance. Grades 2 or 3 erythema were very common in both treatment groups (52. 5% of the skin type group and 45% of the MPD group). This is the third study to suggest that patients with skin types I and II receive a higher total UVA dose when the starting dose is 50-70% of the MPD (rather than 0.5 J/cm2 for skin type I and 1.0 J/cm2 for skin type II) and when large dose increments are used. We suggest that smaller dose increments should be used in patients with skin types I and II.
Chronic forms of urticaria are common, often adversely impacting on quality of life. No formal studies have assessed the extent and nature of disability in different types of urticaria. The Dermatology Life Quality Index (DLQI) is a simple and validated 10-item questionnaire designed to measure and compare disability in different skin conditions. In this study, we aimed to assess the disability in different urticarial groups using the DLQI, allowing comparison with previously published DLQI scores in common skin diseases. The DLQI was administered to 170 consecutive patients attending a specialist urticaria clinic over a 4-month period. Consistent with previous studies using the DLQI, mean scores were not influenced by gender or age. Patients with chronic idiopathic urticaria without a concurrent physical urticaria (n = 47) suffered moderate quality of life impairment (mean +/- SD DLQI 25 +/- 24%). In comparison, patients with chronic idiopathic urticaria with concurrent delayed pressure urticaria (DPU) (n = 26) suffered significantly higher quality of life impairment (mean +/- SD DLQI 43 +/- 23%, 95% confidence interval for difference 7-29%). Disability in this group was greatest in the dimensions of work/study, symptoms/feelings and leisure. Subjects with another form of physical urticaria, cholinergic urticaria, also endured high levels of disability (n = 9, mean +/- SD DLQI 50 +/- 34%). From our urticaria study group, we have shown that subjects with DPU and cholinergic urticaria endure the most quality of life impairment. The mean DLQI scores demonstrated in these groups are comparable with those previously seen in severe atopic dermatitis out-patients (60%) and higher than those seen in out-patients with psoriasis (29.7%), acne (24.3%) and vitiligo (16.1%).
Skin diseases such as acne are sometimes thought of as unimportant, even trivial, when compared with diseases of other organ systems. To address this point directly, validated generic questionnaires were used to assess morbidity in acne patients and compare it with morbidity in patients with other chronic diseases. For 111 acne patients referred to a dermatologist, quality of life was measured using the Dermatology Life Quality Index, Rosenberg's measure of self-esteem, a version of the General Health Questionnaire (GHQ-28) and the Short Form 36 (SF-36). Clinical severity was measured using the Leeds Acne Grade. Population quality of life data for the SF-36 instrument were available from a random sample of adult local residents (n = 9334) some of whom reported a variety of long-standing disabling diseases. All quality of life instruments showed substantial deficits for acne patients that correlated with each other but not with clinically assessed acne severity. The acne patients (a relatively severely affected group) reported levels of social, psychological and emotional problems that were as great as those reported by patients with chronic disabling asthma, epilepsy, diabetes, back pain or arthritis. Acne is not a trivial disease in comparison with other chronic conditions. This should be recognized in the allocation of health care resources.
Excessive axillary hyperhidrosis is a socially embarrassing condition which severely reduces quality of life and may result in psychological disturbance. Available treatment strategies do not resolve the problem in all cases. Intracutaneous injections of botulinum A toxin have been shown to be effective in patients with local sweating disorders. We performed a randomized, double-blind, within-group comparison in 13 individuals (nine women and four men) with severe axillary hyperhidrosis resistant to conventional treatment, to study the effect of intracutaneous injections of botulinum toxin on sweating. A total dose of 200 mouse units of botulinum A toxin (Dysport(R)) was injected into six different sites in one axilla, whereas the other was injected with sterile saline. Objective quantification of sweat production was performed using digitized ninhydrin-stained sheets. Three weeks after treatment, the mean difference in ninhydrin staining between botulinum-treated and placebo-treated axillae was -34.5% (P < 0.001), after 8 weeks -36.9% (P < 0.001) and after 13 weeks -28.4% (P < 0.001). Subjective rating of sweat production was evaluated on a visual analogue scale (0, no sweating, to 100, most severe sweating). Three weeks after treatment the difference between the botulinum-treated and placebo-treated axillae was -56.5% (P < 0.001), after 8 weeks -67.4% (P < 0.001) and after 13 weeks -62.5% (P < 0.001). No serious side-effects were observed.
A questionnaire to all 463 consultant members of the British Association of Dermatologists regarding nursing developments received 183 responses (40%) from over 127 departments. Sixty-nine per cent of respondents have nurse-run clinics, anticipated to increase to 91% at base hospitals and 79% in community settings. Fifty-seven per cent have protocols for nurses to alter treatments; 20% have nurses who initiate treatments. Twenty-six per cent have nurses who prescribe with medical countersignature, and 5% have nurses who can prescribe without medical countersignature (likely to increase fourfold). Seventy-two per cent anticipate nurses administering cryotherapy within the next few years. Few respondents supported nurses treating malignancies or lesions not seen by a dermatologist, but supervised procedures such as curettage, shave biopsy and punch biopsy were all likely to increase from about 15% to 30-40%. These interventional procedures, and particularly prescribing without medical countersignature, were currently most frequent in units without specialist registrar support. Obstacles to extending nursing roles included opposition from administration or nursing hierarchies (about 40%), funding problems (about 25%) and uncertainties from departmental nurses themselves (17%). There is clearly support from dermatologists for these developments, but the importance of adequate training, protocols and support from all relevant staff is paramount.
Although gamma-linolenic acid (GLA) has been shown to correct deficiencies in skin lipids associated with reduced delta-6-desaturase activity which should result in improvement of dysregulation of inflammation and immunity in atopic eczema, clinical studies with evening primrose oil containing 10% GLA have yielded contradictory results. We have therefore examined the effect of a higher percentage (at least 23%) GLA-containing borage oil in adults with stable atopic eczema of moderate severity in a double-blind, multicentre study. One hundred and sixty patients were randomized to take daily either 500 mg of borage oil-containing capsules or the bland lipid miglyol as a placebo over a 24-week period. Use of topical diflucortolone-21-valerate cream was allowed as rescue medication, with the amount used until response being defined as primary, and clinical improvement as secondary efficacy criteria. Although several clinical symptoms improved compared with placebo, the overall response to borage oil did not reach statistical significance. Significant differences in favour of borage oil were, however, observed in a subgroup excluding patients who failed to show increased erythrocyte dihomo-gamma-linolenic acid levels and in whom adherence to inclusion criteria and the study protocol were questionable. GLA metabolites increased in borage oil-treated patients only, and serum IgE showed a trend to decrease on overall and subgroup analysis. No substance-related adverse effects were observed. This study shows no overall efficacy of GLA-containing borage oil in atopic eczema, with steroid use being the primary response parameter, although it suggests that a subgroup of patients may benefit from this well-tolerated treatment.
We report a large family with ichthyosis bullosa of Siemens (IBS) including eight affected members spanning three generations. The classical features of the disease were consistently observed with blistering, superficial peeling of the skin, and localized lichenified hyperkeratosis mainly confined to the limbs. Phenotypic variation, however, was also observed with some individuals exhibiting unusual clinical features. Specifically, the index patient was erythrodermic at birth; she subsequently developed a widespread pustular eruption. Erythroderma is classically absent in IBS and pustulation is very unusual. She also had hypertrichosis of the limbs, as did an affected female first cousin. This has not previously been reported in IBS. Electron microscopy showed complex aggregates of keratin in the spinous and granular layers associated, in places, with remarkably little cell lysis. Sequencing of genomic DNA revealed a mutation (E493K) in keratin 2e. A review of the literature on IBS indicates that E493K is the most commonly reported mutation to date and might represent a mutational hotspot for this disease.
Although bullous pemphigoid and cicatricial pemphigoid are sometimes associated with malignancy, it remains uncertain whether such an association is pathogenetically related or just a coincidence attributable to the advanced age of the patients. We report a 61-year-old patient with antiepiligrin (laminin 5) cicatricial pemphigoid (AeCP) associated with an advanced gastric carcinoma. The gastric carcinoma cells in this patient were shown to produce laminin 5 by immunofluorescence microscopy, and the patient's serum contained autoantibodies directed against laminin 5 on immunoprecipitation. Furthermore, the blistering symptoms and the titre of antibasement membrane zone antibodies coordinately changed with the resection and subsequent relapse of the gastric cancer. These observations suggest that the gastric carcinoma producing laminin 5 may have induced the production of autoantibodies to this laminin, which were pathogenic to the skin and mucous membranes in this patient. This report demonstrates a link between this autoimmune subepithelial blistering disease and malignancy. It is of interest and potential great importance to examine other cases of AeCP for such a potential association.
Basal cell carcinoma (BCC), the most common malignant neoplasm of the skin, has many different clinical and histological appearances. Four patients with a new distinctive form, polypoid BCC, are described. Polypoid BCC is clinically distinguished from other types of BCC by being pedunculated and having a stalk that connects it to the skin surface. Histologically, it is distinctive in that the tumour aggregations are restricted to the exophytic polypoid zone.
Sézary syndrome (SS) is a rare cutaneous T-cell lymphoma. SS usually develops de novo. We describe a 23-year-old man with a proven history of severe atopic dermatitis since childhood, who developed SS. This case contributes to the discussion about the possibility of a relationship between inflammatory dermatitis, atopy and subsequent SS. We provide criteria that should be fulfilled to define such an association.Department of Dermatology, University Hospital Utrecht, PO Box 85500, 3508 GA Utrecht, The Netherlands. haselen@digd.azu.nl
The term Richter's syndrome is used to describe the transformation of chronic lymphatic leukaemia (CLL) into a high-grade systemic lymphoma and is associated with a poor prognosis. We have undertaken detailed molecular studies in two patients with cutaneous B-cell lymphoma (CBCL) and CLL. Patient 1 exhibited a low-grade CBCL with different immunoglobulin gene rearrangements in blood and skin. By contrast, patient 2 showed identical gene rearrangements, confirmed by gene sequencing, and died within 4 months of presentation. The latter patient fulfilled the criteria for a diagnosis of cutaneous Richter's syndrome, whereas the former patient demonstrated the coincidence of CLL with a primary CBCL. Our results highlight the importance of gene rearrangement studies with sequencing for the accurate diagnosis of cutaneous Richter's syndrome.
Patients with hydroa vacciniforme (HV)-like eruptions and malignant potential have been reported from Asia and Mexico, and those patients frequently had an associated latent Epstein-Barr virus (EBV) infection. In order to elucidate the association of latent EBV infection with HV, we studied six children with typical manifestations of HV by detection of EBV genes and EBV-related RNAs in biopsy specimens from cutaneous lesions. Cutaneous lesions of all six children with typical HV contained EBV-encoded small nuclear RNA (EBER)+ cells in 3-10% of the dermal infiltrates, whereas no Bam HI-H, l-fragment (BHLF) mRNA, or transcripts encoding EA-D antigen, were detected. No EBER + cells were detected in other inflammatory or benign lymphoproliferative skin disorders tested. Polymerase chain reaction amplification confirmed the presence of EBV DNA sequences in five of six biopsy specimens from the patients. Latent EBV infection is associated with the development of cutaneous lesions of HV.
Tinea capitis is rare in adults, although a few cases have been described in HIV-infected patients. We present two cases in black African adults who were HIV positive. In one, the condition led to the diagnosis of HIV infection. It is possible that the rarity of tinea capitis in HIV-positive adults could be related to increased colonization of their scalp by Pityrosporum (Malassezia) spp. In patients well controlled with an antiviral therapy, the treatment of tinea capitis seems no more difficult than in non-immunosuppressed patients. There is possibly a relation between clinical presentation and degree of immunodeficiency.
Of 11 patients with primary or secondary antiphospholipid syndrome (APS), four exhibited papules or nodules on the finger, sole or leg as the initial cutaneous manifestation. Histological examination demonstrated thrombosed vessels or vessels containing organized thrombi in the dermis or in the subcutaneous fat tissue. Cutaneous papules and nodules should be recognized as skin manifestations of APS. Screening tests for antiphospholipid antibodies and lupus anticoagulant are required in patients with cutaneous papules or nodules of unknown aetiology. In cases of positive antiphospholipid antibodies and/or lupus anticoagulant, histological examination is critical in the establishment of the diagnosis of APS.
Longitudinal melanonychia displaying features of keratinized acanthoma is described in two patients. In both cases, a pigmented band consisting of a subungual keratinized epithelial ridge originated in the nail bed. We have been able to show, using the clinical and histological findings, that the origin of the pigment is linked to its synthesis within the acanthoma of the nail bed. These lesions are reminiscent of pigmented seborrhoeic keratosis. The horny cysts are replaced, because of the special physiological longitudinal arrangement of the ridges in the nail bed, by a single, prominent, longitudinal, keratinized and pigmented mass.
We report a 13-year-old girl with extensive striae and an acneiform eruption following surgery for complex congenital heart disease. These findings were associated with elevated serum and urinary cortisol levels with loss of diurnal rhythm. The resolution of the eruption and the fading of her striae coincided in time with normalization of her blood parameters on day 72 postoperatively. We conclude that the cause of steroid excess in our patient was stress induced by the cardiac surgery and a complicated and protracted postoperative course. To our knowledge, this is the first report in the English language literature of skin changes due to endogenous hypercortisolaemia caused by intense physical and emotional stress.
We report a case of extensive phytophotodermatitis in a hiker. This was caused by the application of the juice of a member of the Rutaceae family, Citrus hystrix, as a folk remedy to ward off biting insects. Besides the Rutaceae, plants belonging to the families Umbelliferae, Moraceae and Leguminosae also contain psoralens and can cause phytophotodermatitis. In temperate countries, cases are often seen among children playing outdoors during summer, when psoralens are most abundant in wild and garden plants.
We have used microdialysis in the dermis for assessing penetration kinetics of salicylic acid (SA) in healthy volunteers (n = 18), following application on the volar aspect of the left forearm. Penetration was monitored at four locations: in normal (unmodified) skin and in skin with perturbed barrier function from (i) repeated tape stripping (ii) irritant dermatitis from 1 or 2% sodium lauryl sulphate (SLS) for 24 h and (iii) delipidization by acetone. The order of the treatments was randomized according to a latin square design. Epidermal barrier function and skin irritation were assessed in each location using evaporimetry and colorimetry. Transepidermal water loss (TEWL) values confirmed that both mild (acetone), moderate (1% SLS) and severe barrier damage (tape stripping and 2% SLS) had occurred. Microdialysis sampling with two parallel probes in the dermis was performed in each of the four treatment areas for every subject. SA (5% in ethanol) was applied in a chamber glued to the skin overlying the microdialysis probes and sampling was continued for 4 h. SA was detectable in all samples and measurable in all samples from penetration through perturbed skin. Comparing the SA penetration in barrier-perturbed skin with the penetration in unmodified skin in the same subject, the mean SA penetration increase was 2.2-fold in acetone-treated skin (P = 0.012), 46-fold in mild dermatitis and 146- and 157-fold in severe dermatitis and tape stripped skin, respectively (P < 0.001). The penetration of SA significantly correlated with the measurements of barrier perturbation by TEWL (P = 0.01) and erythema (P = 0.02) for each individual. Microdialysis sampling of SA penetration was more sensitive than non-invasive measuring techniques in detecting significant barrier perturbation in acetone-treated skin. A positive dose-response relationship for the percutaneous penetration of SA in response to increasing SLS pretreatment concentrations and thus the degree of irritant dermatitis was found. When analysing data by location on the forearm, a tendency towards an intraregional variation in the reactivity to barrier damage was found, with the most proximal location displaying higher reactivity scores than the most distal location in response to the same barrier perturbation procedures. The penetration of SA was not significantly different between locations. In conclusion, using microdialysis in the dermis to obtain real-time dermal pharmacokinetics in the target organ, this study demonstrates highly increased and differentiated cutaneous penetration of SA in barrier-perturbed skin. The measured drug penetration was demonstrated to correlate with non-invasive quantification of barrier damage.
Little research is currently available related to pain management by neuroscience nurses. However, due to concerns about the potential for altering neurological status, some neurosurgery patients may not receive optimal pain management. This paper describes findings from a pain related survey which was distributed during the Canadian Association of Neuroscience Nurses June 1998 national conference. The survey was intended to assess Canadian neuroscience nurses pain management knowledge and to explore pain management techniques after intracranial surgery. While 60% of respondents answered four pain assessment and management case study related questions correctly, some respondents rated pain differently when it was expressed by a smiling or grimacing patient. The most common methods for pain control after intracranial surgery included intermittent codeine and/or morphine, often by intramuscular injection. Findings from this study suggest that some neuroscience nurses require further education about pain management and that many patients do not receive optimal pain management after intracranial surgery.
There has been little specific investigation of personality and behaviour changes following spinal cord injury (SCI) and only limited consideration of the possible impact of concurrent traumatic brain injury (TBI). By mail-out questionnaire, we evaluated personality and behaviour changes in a married group (n = 9) with traumatic SCI, who knew their partners prior to injury, and who had not been identified as having concurrent TBI on referral to the Canadian Paraplegic Association. Both the person with SCI (and the partner) completed the revised Adjective Checklist and by their combined report, there were significant personality and behaviour changes. Unexpectedly, five individuals described post-traumatic amnesia (PTA) > or = 3 days. Subsequently, participants' reports were further divided into two groups--"longer PTA" and "shorter PTA". The "longer PTA" group self-reported less change and more positive change than did their partners. The "longer PTA" partners described changes that are consistent with the profile of TBI. The "shorter PTA" group described themselves more negatively than did their partners. Given the size of the groups (n = 5, n = 4), these findings are presented to illustrate trends and to stimulate further research.
On the pediatric neurosciences unit of British Columbia's Children's Hospital we are in the throes of a nursing staff crisis. In the last year alone we hired ten new graduates to work as casuals on our unit. With a two and a half day hospital orientation, five preceptor shifts, and a Competency Based Education Plan in hand, we send them off to the trenches. We know these nurses have little nursing experience and even less Neuroscience nursing experience. Yet, we expect them to care for patients and families whose problems they may not understand. For a preceptor, and senior colleague, this is a disturbing situation. We recognize that in their orientation shifts they have not even begun to experience the challenges of a Neuroscience unit. Have they cared for a child who has had a postoperative laminectomy? Do they recognize subtle seizures? Have they sat with a family who have just learned that their child has a brain tumor? No. We expect them to care for patients and families with minimal support. This is the reality of nursing today. Many of us pride ourselves on being committed pediatric Neuroscience nurses. Our physicians rely upon our assessment skills and they trust our intuition! We believe we have earned that trust. How can we convey our enthusiasm and excitement to our perceptees so they are motivated to stay and become experienced pediatric Neuroscience nurses? In this presentation we outline our paper to remedy this situation.
BACKGROUND: Cytokine receptors are exceptionally valuable targets for immunotherapy. For example, the high affinity IL-2 receptor is expressed by abnormal T cells in patients with certain lymphoid malignancies or autoimmune disorders and in individuals rejecting allografts whereas it is not expressed by normal resting cells. DESIGN: To exploit this difference in receptor expression in normal resting cells and leukemic cells we have introduced different forms of IL-2 receptor directed therapy including an unmodified murine antibody to the alpha subunit of the IL-2 receptor (anti-Tac), humanized anti-Tac as well as this antibody armed with truncated Pseudomonas exotoxin or alpha- and beta-emitting radionuclides (e.g., 211At and 90Y). In particular, unmodified murine anti-Tac was used in the therapy of HTLV-I-associated adult T-cell leukemia (ATL). RESULTS: Six of nineteen patients treated with this antibody underwent a partial (four) or complete (two) remission. In a subsequent clinical trial involving anti-Tac armed with 90Y over 50% of the patients with ATL treated underwent a partial or complete remission. CONCLUSIONS: New agents under development include humanized antibodies directed toward shared cytokine receptors such as IL-2/15R beta used by both IL-2 and IL-15 as well as to a shared signal transduction element Jak3 utilized by the T-cell stimulatory cytokines IL-2, IL-4, IL-7, IL-9 and IL-15. Thus our emerging understanding of cytokine receptors and their signaling pathways taken in conjunction with the ability to produce humanized antibodies armed with radionuclides or toxins are providing novel perspectives for the treatment of leukemia and lymphoma.
BACKGROUND: In view of the successful use of serotherapy in many B-cell malignancies, we and others have sought to identify tumor selective antigens for the serotherapy of plasma cell dyscrasias (PCD) including multiple myeloma (MM), and Waldenstrom's macroglobulinemia (WM). We recently identified Muc-1 core protein as a MM selective antigen. Though Muc-1 core protein is abundantly expressed on most MM plasma cells, expression of this antigen can be absent, or weak on some plasma cells which could potentially result in the selection of Muc-1 core protein negative clones following serotherapy of PCD. In addition to Muc-1 core protein, we have also been examining the use of CD20 directed serotherapy for PCD. DESIGN: As part of these efforts, we recently initiated a phase II clinical trial examining the use of Rituximab (Rituxan, MabThera) as a single agent in MM patients; as well several WM patients have been treated with Rituximab at our Institutions. RESULTS: In previous studies, we have shown that CD20 is abundantly expressed on the plasma cells of most WM patients; in contrast, CD20 is expressed on plasma cells from a minority of MM patients, and in these patients expression of CD20 can be weak or heterogeneous with both CD20+ and CD20- plasma cells present. As such, we have sought out clinically useful inducers of Muc-1 core protein, and of CD20 on malignant plasma cells. CONCLUSIONS: These efforts resulted in the identification of dexamethasone (Dex) as a potent inducer of Muc-1 core protein on MM plasma cells, and interferon-gamma (IFN-gamma) as a potent inducer of CD20 on MM plasma cells and B-cells. Importantly, these agents induced their respective antigens at pharmacologically achievable doses.
Primary cutaneous lymphomas differ significantly from their nodal equivalents in clinical behaviour and prognosis, and often require a different therapeutic approach. Since currently used classification systems for non-Hodgkin lymphomas do not or insufficiently recognize the special character of these lymphomas, primary cutaneous lymphomas are not uncommonly diagnosed incorrectly, and/or treated inappropriately with unnecessarily aggressive therapies. For that reason the Cutaneous Lymphoma Group of the European Organization for Research and Treatment of Cancer (EORTC) has recently proposed a separate classification for the group of primary cutaneous lymphomas. The EORTC Classification is consistently based on a combination of clinical, histological, immunophenotypical and genetic criteria, and includes well-defined and recognizable disease entities. It contains a limited number of cutaneous T-cell lymphomas and cutaneous B-cell lymphomas, which comprise more than 95% of all primary cutaneous lymphomas. The clinical significance of this classification has been validated by long-term follow-up data of more than 800 patients with a primary cutaneous lymphoma. The basic principles of the EORTC Classification will be presented, and current controversies between the EORTC Classification on the one hand, and the R.E.A.L. Classification and the proposed WHO Classification on the other will be discussed.
BACKGROUND: B-lymphoproliferative post-transplant disorder (BLPD) is a severe complication of organ and bone marrow transplantation. The reduction of immuno-suppressive therapy or surgery for localized disease may cure some BLPDs. Other therapeutic approaches such as chemotherapy and antiviral drugs are toxic and of limited efficacy. Adoptive immunotherapy with donor T-cell infusions has yielded promising results but is, at the present time, easily applicable only in bone marrow-transplanted patients. Anti-B-cell Murine monoclonal antibodies (MoAbs) have proven effective but are no longer available for human use. We report the activity of a humanized anti CD 20 Mo Ab (Rituximab-MABTHERA Roche) in 32 episodes of BLPD treated in 14 French centers. PATIENTS AND METHODS: Between November 1997 and September 1998, 32 patients were diagnosed with BLPD. Twenty-six patients had undergone solid organ transplants (liver 8, kidney 8, heart 4, lung 3, heart lung 1, kidney-pancreas 1, liver-kidney 1) and six patients had received bone marrow transplantations. The median age of the patients was 34 years (3-67 years) and the median delay between graft and tumor 5 months (1-156 months). In organ recipients, tumors were classified as polymorphic and monomorphic in 10 and 15 cases, respectively; 4 of 6 bone marrow transplant recipients were treated without pathology documentation because of a rise in EBV load, fever and lymph node enlargement. Tumors were associated with EBV in 22 of 26 tested cases. Rituximab was used as first-line therapy in 30 patients (after reduction of immunosuppressive treatment in 27 patients) and as salvage therapy in 2 patients (after failure of chemotherapy). The median time from diagnosis of BLPD to treatment with Rituximab was 14 days (1-110 days). Two patients received eight infusions, twenty-six patients four infusions, one patient three infusions and three patients two infusions of 375 mg/m2. RESULTS: The tolerance of rituximab was good. The overall response rate was 69%, with 20 complete responses and 2 partial responses. In solid organ transplant the response rate was 65% (15 CR and 2 PR) while it was 83% in bone marrow-transplanted patients (5 CR). With a median follow-up of 8 months (1-16 months) 24 patients are still alive. The one-year projected survival is 73%. Of the 22 patients who achieved response, 15 patients (11 solid organ transplant and 4 bone marrow transplant) are alive with no evidence of disease, 4 patients relapsed a median of 7 months (3-10 months) after treatment and 3 died while in CR of concurrent diseases. Of the 10 patients who did not respond to Rituximab 5 are alive with no evidence of disease after salvage therapy. CONCLUSIONS: The use of rituximab appears to be a safe and relatively efficient therapy in BLPDs. The results need to be confirmed in a prospective multicentric trial.
BACKGROUND: A retrospective analysis was performed to delineate the factors associated with response, and to determine the duration of response, in 87 patients with CD20-positive mantle-cell lymphoma (MCL) treated with Rituximab (chimeric monoclonal anti-CD20 antibody) in two prior studies. PATIENTS AND METHODS: Patients with newly-diagnosed MCL (MCL1, n = 37), and previously-treated MCL (MCL2, n = 50), received single-agent Rituximab, in the context of two multicentre clinical studies using different schedules and doses, conducted in 1996 and 1997. A follow-up analysis was performed at the end of 1998, including all 81 patients who completed therapy. Statistical modeling of factors associated with response was performed using ordered logistic regression. The duration of complete (CR) and partial response (PR), and the time to disease progression (TTP), were also derived. RESULTS: The overall response rate (RR) was 34% (30 of 87) (81 evaluable patients, RR 37%; CR 14%), and was equivalent for MCL1 and MCL2. On univariate analysis, elevated LDH (P = 0.004); prior therapy with alkylating agents (P = 0.01) or fludarabine phosphate (P = 0.04); WHO performance status = 2 (P = 0.02); MCL2 refractory to last prior therapy (P = 0.04); and splenomegaly (P = 0.04), each at the time of treatment with Rituximab, were significantly associated with a lower RR. On multivariate analysis, only LDH (P = 0.007) and prior alkylating agents (P = 0.03) retained statistical significance. At a median follow-up of 1.4 years, the median TTP was 7 months. The median duration of response was one year, and was significantly longer for patients achieving CR vs. PR (P = 0.04). CONCLUSIONS: Rituximab is active in MCL, and can induce complete responses in a minority of patients. Elevated LDH at the time of therapy, and prior therapy with alkylating agents, are associated with a significantly lower RR. The duration of response of one year is similar to that previously reported in follicular lymphoma.
BACKGROUND: Clinical activity of the anti CD-20 monoclonal antibody Rituximab has been reported in patients with follicular lymphoma (FL) and mantle-cell lymphoma (MCL). PATIENTS AND METHODS: 120 patients with bi-dimensionally measurable FL or MCL (R.E.A.L. Classification) were treated with Rituximab 375 mg/m2/week for 4 weeks. A central pathology review confirmed the diagnosis of FL in 76 of 78 and of MCL in 39 of 42 cases. The response was evaluated after 8 weeks and confirmed after 12 weeks from the start of treatment. RESULTS: The toxicity of the treatment was, as expected, grade 1-2 fever and rigors during the first infusion and mild asthenia during the treatment period. Serious adverse events, probably or possibly related to the study treatment, included four deaths (3 of cardiac origin, 1 caused by P. carinii pneumonia) and 10 further nonfatal cases, including a permanent agranulocytosis and one case of heart failure. Response rate at week 12 was 52% for FL and 22% for MCL. After treatment, the BCL-2 rearrangement disappeared in 15 of 29 blood but only in 5 of 23 bone marrow samples; BCL-1 disappeared in 5 of 12 blood and 0 of 7 bone marrow specimens, as determined by PCR. CONCLUSIONS: Rituximab is an active agent for the treatment of FL, while its efficacy is modest in MCL. The effect in reducing minimal residual disease is more pronounced on the blood than it is on the bone marrow.
BACKGROUND: Mantle-cell lymphoma (MCL) is genetically characterized by the translocation t(11;14)(q13;q32) leading to an overexpression of cyclin-D1, but additional chromosomal abnormalities appear to be required for MCL pathogenesis. PATIENTS AND METHODS: Deletions involving chromosome 11q, which were recently found as recurrent aberrations in MCL, were analyzed at the molecular level in a series of 81 MCL by fluorescence in situ hybridization (FISH) with probes from a contiguous set of yeast artificial chromosomes (YACs) spanning bands 11q14-q24. RESULTS AND CONCLUSIONS: Loss of chromosome 11 material was observed in 37 of the 81 MCL cases (46%). The consensus deletion comprised YAC 801e11 containing the ATM gene. The minimal region of loss was further narrowed with P1-derived artificial chromosome (PAC) probes. This allowed the identification of a deletion confined to the genomic region of ATM, which, together with intragenic mutations found in the coding sequence, suggests a role of ATM as a tumor suppressor gene in MCL.
BACKGROUND: Chromosome translocations involving the immunoglobulin heavy chain gene (IgH) on 14q32 are a seminal event in the pathogenesis of many B-cell malignancies. Since myeloma is a post-germinal center tumor of mature, isotype switched plasma cells, we hypothesized that 14q32 translocations would usually involve IgH switch regions. MATERIALS AND METHODS: We analyzed a panel of 21 human myeloma cell lines using a Southern blot assay to detect illegitimate rearrangements involving the switch regions. We then cloned the breakpoints, developed probes for FISH analysis, and characterized the oncogenes dysregulated by the translocations. RESULTS: Only half of the cell lines demonstrated a 14q32 abnormality by conventional karyotypic analysis, but we were able to identify translocations involving IgH switch regions in 15 of 21 lines, including all of the lines in which a 14q32 translocations was not identified by conventional karyotypic analysis. Six cell lines have an Ig translocation involving 11q13 with overexpression of cyclin D1. Six cell lines have an Ig translocation involving 16q23 with overexpression of c-maf. Five lines have an Ig translocations involving 4p16 with overexpression of FGFR3 and a novel gene, MMSET. The 4p16 breakpoints occur within the 5' introns of MMSET, and are associated with IgH-MMSET hybrid mRNA transcripts. The remaining five cell lines have translocations involving other loci, including: 6p25 (MUM1), 8q24 (c-myc), and 21q22 (?AML1). CONCLUSIONS: Recurrent Ig translocations identify at least three distinct molecular subtypes of myeloma. Our long-term goal is to determine if there are phenotypic, prognostic and therapeutic differences associated with these molecular subtypes.
BACKGROUND: We have observed that molecular response, as defined by a PCR-negative status during the first year of therapy, along with beta 2-microglobulin (beta 2M), was the most important variable associated with failure-free survival (FFS) in follicular lymphoma (FL). Herein, we present an update of the previously published MDACC series. PATIENTS AND METHODS: A total of 116 patients (male:female ratio 64:52; median age: 52 years) with indolent FL and BCL-2 rearrangement (at MBR or mcr breakpoints) assessable in peripheral blood (pb) by PCR prior to treatment, and with two or more PCR determinations during the first year, were selected for the present study. RESULTS: Of the 116 patients, 4 who presented with progression and 1 who died of unrelated causes during the first year were excluded from the landmark analysis. One hundred patients (86%) achieved clinical CR and 80 (69%) achieved a negative PCR status within first year. Median FFS was 6.4 years. Five-year FFS was 73% and 28% for molecular responders and nonresponders, respectively (P = 0.001). In spite of this strikingly higher FFS favoring molecular responders, no clearcut plateau was evident in this group. Molecular response assessed in pb (P = 0.001) and serum beta 2M (P < 0.001) were the most important factors to predict FFS in the multivariate analysis. In the subset of patients with normal beta 2M and molecular CR, there was a trend for a plateau in the FFS curve. No significant difference between the groups has been observed so far in terms of survival. CONCLUSIONS: Molecular response assessed in pb using a PCR technique is, along with beta 2M, the most important factor to predict FFS in FL.
BACKGROUND: Lymphoma and leukemia are the commonest malignant diseases in patients with chromosomal breakage syndromes and immunodeficiency (Ataxia teleangiectasia (AT) and Nijmegen breakage syndrome (NBS)). With improved management of infections, malignant disease is more frequently diagnosed and has become one of the commonest causes of death in pediatric AT and NBS. PATIENTS AND METHODS: In three consecutive multicenter therapy trials for pediatric non-Hodgkin's lymphoma (NHL) (NHL-BFM), 1569 patients with newly diagnosed NHL have been registered between 1986 and 1997. Nine patients with AT (n = 5) and NBS (n = 4) were identified and analysed. RESULTS: Median age of patients with AT and NBS at diagnosis of NHL was nine years. NHL-entities differed from non-AT/NBS-patients: diffuse large B-cell lymphomas, n = 7 (78%); ALCL, n = 1; lymphoblastic T-cell lymphoma, n = 1. Cervical nodes, paranasal sinuses and epipharynx were the sites most frequently involved. Stages were: I and II in three patients, III in five and IV in one patient. All patients received polychemotherapy according to tumor-entity and stage, none received radiation. Dose reductions according to individual tolerance concerned mainly ethotrexate, alkylating agents and epipodophyllotoxines. One patient died of toxic complications, two patients relapsed and died, one patient suffered from second malignancy. Five of nine patients are in 1. CCR after a median follow-up of five years. CONCLUSIONS: Patients with AT and NBS suffer from rare entities of pediatric NHL. Curative treatment is possible and should be attempted. Intensity of therapy should be adjusted to individual risk factors and tolerance. Alkylating agents, epipodophyllotoxines should be omitted, dose of MTX should be limited to 1 g/m2. Further cooperative trials using standardized approaches are required.
BACKGROUND: The problem of residual masses on post-treatment CT scans is a continuing dilemma for the oncologist treating malignant lymphomas. These masses may contain active disease or represent only necrotic tumour which continues to shrink without further treatment or post-treatment fibrosis which remains stable on continued follow-up. 18-FDG-PET offers a novel metabolic imaging modality, which can differentiate malignant from benign tissue on the basis of increased glycolytic activity. PATIENTS AND METHODS: Thirty-two patients (15 with Hodgkin's disease (HD) and 17 with aggressive histology non-Hodgkin's lymphoma (NHL)) who had residual masses on their post-treatment CT scans underwent 18-FDG-PET. The post-treatment CT and PET scans were compared and the accuracy of the 18-FDG-PET in assessing residual masses was evaluated using clinical and pathological follow-up data. RESULTS: Nine patients had positive post-treatment 18-FDG-PET, eight (89%) of whom have relapsed. Twenty-three patients had negative post-treatment PET with only two relapses in this group. The 2 patients who relapsed had aggressive NHL while none of the 11 HD patients with negative PET relapsed. The median follow-up of patients in continued complete remission is 38 months. CONCLUSIONS: 18-FDG-PET can differentiate between residual masses containing viable lymphoma where further treatment will be required to achieve cure and those representing ablated disease, where unnecessary treatment and additional morbidity may be avoided.
BACKGROUND: The classification of cutaneous lymphomas has been controversial. The EORTC has proposed that conventional classification schemes are not suitable for cutaneous lymphomas, and that a unique classification system is required. DESIGN: The authors review the suitability of the R.E.A.L. Classification for cutaneous lymphomas, and compare it with the newly proposed EORTC system. The principles of the R.E.A.L. Classification have been adopted by the WHO committees for the classification of hematopoietic and lymphoid neoplasms. Each disease is defined as a distinct entity based on an integration of morphology, immunophenotypic and genetic features, clinical presentation and course, and normal cellular counterpart. If either primary or secondary involvement of the skin is a constant factor, this aspect is considered integral to disease definition. RESULTS: Organ-specific classification schemes may impede the recognition of common features of diseases involving multiple anatomic sites. For example, cutaneous marginal zone B-cell lymphomas (formerly designated cutaneous immunocytomas) mirror the features of MALT lymphomas in other anatomic sites. While the EORTC Classification for cutaneous lymphomas attempts to emphasize certain aspects of these neoplasms of importance to dermatologists, the use of multiple classification systems is a step backward, and may lead to confusion among hematologists/oncologists, and dermatologists. Nevertheless, cutaneous lymphomas often have a more indolent natural history than nodal lymphomas, and may require different therapeutic approaches. Clinical features are an important prognostic factor and should be utilized in guiding therapy. For cutaneous lymphomas the presence or absence of systemic spread is particularly important. Additionally, the site of origin is often important in the definition of disease entities. CONCLUSIONS: Organ-specific classification schemes, such as the EORTC Classification for cutaneous lymphomas, are not required, and indeed may impede the recognition of common features of diseases involving multiple anatomic sites. A common classification system, such as the R.E.A.L./WHO Classification, should be utilized for all lymphomas, regardless of the site of origin.
Follicular lymphomas are characterized by relatively long median survivals and a continuous pattern of relapse. The heterogeneity in these diseases is increasingly appreciated, leading to concerted efforts to define prognostic factors and risk-adapted strategies. The status of multiple options for treatment including interferon, fludarabine, dose intensification with autologous transplantation, therapy targeting the CD20 antigen and novel approaches is reviewed. The long natural history of follicular lymphoma requires mature data for accurate analysis. However, the achievement of molecular remission as a surrogate endpoint is under active investigation. This is an exciting era for the clinical investigation of follicular lymphoma given the large number of candidate therapies and their potential combinations and permutations. Although the goal of primary treatment remains durable remission and cure, the sequential application of effective, non-cross-resistant treatments may also result in a prolongation of median survival time. It is essential that physicians treating patients with follicular lymphoma demonstrate restraint in the application of new treatments and cooperate in the study of new therapies in carefully designed phase II and phase III trials.
BACKGROUND: New treatments are desperately needed to improve the results obtained using CHOP chemotherapy for patients with diffuse large cell lymphoma. In order to develop successful new strategies we need to understand why prior promising therapies have failed and to develop new testable hypotheses based on our current knowledge. PATIENTS AND METHODS: The International Non-Hodgkin's Lymphoma Prognostic Factors Index has provided us with a methodology to compare the expected prognosis of patients on different clinical trials. Many prior, apparent improvements in treatment outcome can now be attributed to the inclusion of patients with better prognoses. RESULTS: Current areas of investigation include: 1) the identification of new active drugs for the treatment of lymphoma, 2) the use of colony stimulating factors to allow dose escalation of the active myelotoxic drugs, 3) the use of strategies which may overcome the problem of resistance to chemotherapy, 4) the combination of monoclonal antibodies with combination chemotherapy and 5) ablative chemotherapy with autologous stem-cell support. CONCLUSIONS: Based on all of the available data, the North American Lymphoma Intergroup has developed the hypothesis that high-intermediate and high risk patients with aggressive lymphoma who receive full course standard induction therapy will benefit form the addition of high dose therapy and has antedated a clinical trial testing that hypothesis.
BACKGROUND: Controversy in lymphoma classification dates back to the first attempts to formulate such classifications. Over the years, much of this controversy arose from the assumption that there had to be a single guiding principle--a 'gold standard'--for classification, and from the existence of multiple different classifications. DESIGN: The International Lymphoma Study Group (I.L.S.G.) developed a consensus list of lymphoid neoplasms, which was published in 1994 as the 'Revised European-American Classification of Lymphoid Neoplasms' (R.E.A.L.). The classification is based on the principle that a classification is a list of 'real' disease entities, which are defined by a combination of morphology, immunophenotype, genetic features, and clinical features. The relative importance of each of these features varies among diseases, and there is no one 'gold standard'. In some tumors morphology is paramount, in others it is immunophenotype, a specific genetic abnormality, or clinical features. An international study of 1300 patients, supported by the San Salvatore Foundation, was conducted to determine whether the R.E.A.L. Classification could be used by expert pathologists and had clinical relevance. Since 1995, the European Association of Pathologists (EAHP) and the Society for Hematopathology (SH) have been developing a new World Health Organization (WHO) Classification of hematologic malignancies, using an updated R.E.A.L. Classification for lymphomas and applying the principles of the R.E.A.L. Classification to myeloid and histiocytic neoplasms. A Clinical Advisory Committee (CAC) was formed to ensure that the WHO Classification will be useful to clinicians. RESULTS: The International Lymphoma Study showed that the R.E.A.L. Classification could be used by pathologists, with inter-observer reproducibility better than for other classifications (> 85%). Immunophenotyping was helpful in some diagnoses, but not required for many others. New entities not specifically recognized in the Working Formulation accounted for 27% of the cases. Diseases that would have been lumped together as 'low grade' or 'intermediate/high grade' in the Working Formulation showed marked differences in survival, confirming that they need to be treated as distinct entities. Clinical features such as the International Prognostic Index were also important in determining patient outcome. The WHO Clinical Advisory Committee concluded that clinical groupings of lymphoid neoplasms was neither necessary nor desirable. Patient treatment is determined by the specific type of lymphoma, with the addition of grade within the tumor type, if applicable, and clinical prognostic factors such as the International Prognostic Index (IPI). CONCLUSIONS: The experience of developing the WHO Classification has produced a new and existing degree of cooperation and communication between oncologists and pathologists from around the world, which should facilitate progress in the understanding and treatment of hematologic malignancies.
INTRODUCTION: Burkitt-like lymphoma (BLL) is a provisional category of B-cell lymphoma which is morphologically intermediate between Burkitt lymphoma (BL) and large B-cell lymphoma (LBCL). The clinical significance of this morphology is controversial. PATIENTS AND METHODS: We examined 41 cases of pediatric B-cell lymphoma by immunohistochemistry for proteins associated with proto-oncogenes c-myc, BCL-2 and BCL-6 and a subset of cases (with adequate slides) for a proliferation-associated marker (Ki-67) and for apoptosis (Apop-Tag). Sixteen cases of BLL, thirteen cases of BL and twelve cases of LBCL were examined. RESULTS: Our results showed BCL-6 expression in 16 of 16 BLL, 4 of 13 BL, and 9 of 12 LBCL; c-myc expression in 14 of 15 BLL, 9 of 13 BL, and 12 of 12 LBCL; and BCL-2 expression in 2 of 16 BLL, 9 of 13 BL, and 6 of 12 LBCL. Mean apoptotic index for BLL was 10.3% (n = 6); for BL was 17.1% (n = 5); and for LBCL was 10.9% (n = 6). Ki-67 was diffusely reactive in all cases tested. There was a significantly higher proportion of BLL than BL which expressed BCL-6 (P = 0.0001). CONCLUSIONS: Labeling for BCL-6 distinguishes BLL from BL. It is likely that in children in North America, BLL is biologically distinct from BL and more closely resembles a subset of LBCL.
BACKGROUND: Primary cerebral non-Hodgkin's lymphomas (NHL) in immunocompetent patients (PCL) are located exclusively in the central nervous system, the eye, or meninges. Clinical management of these patients remains controversial. PATIENTS AND METHODS: Clinical characteristics of the patients and parameters influencing their outcome as of December 1998 were investigated and registered in a database of 226 patients treated in the French Federation of Cancer Centers between 1980 and 1995. RESULTS: Most PCL are diffuse large-cell NHL with a B phenotype. The incidence of PCL has been steadily increasing over the past 20 years in some but not all countries. The overall survival of primary cerebral lymphoma (PCL) patients in the published series, a median of 12-16 months and a five-year survival of 5%-20%, is poor. Several series have now reported long-term survivals of more than 10 years and PCL may therefore be a curable tumor in some patients. The optimal treatment of PCL is not known. Complete resection of the tumor does not improve outcome and multidisciplinary approaches combining chemotherapy and radiotherapy are now commonly used, although the superiority of combination over radiotherapy- or chemotherapy-alone has never been demonstrated in a phase III trial. The optimal chemotherapy regimen, the dose and even the usefulness of brain radiotherapy after chemotherapy are therefore still matters of debate. Recently, several authors have reported a relatively high incidence of late neurological sequelae after PCL treatment. CONCLUSIONS: The optimal treatment of PCL patients remains to be defined. Large cooperative international phase III trials are now required to define and improve the optimal treatment of PCL and reduce its sequelae.
Immunodeficiency-related B-cell disorders are seen after organ transplantation and in congenital and acquired immunodeficiency states. Post-transplant lymphoproliferative disorders (PTLD) comprise a histologic spectrum ranging from hyper-plastic appearing lesions to frank non-Hodgkin's lymphoma or multiple myeloma histology. Multiple clones may co-exist, representing a uniquely different mechanism for lymphomagenesis. The incidence varies from 1% in renal recipients to 8% in lung recipients, but can be markedly increased by the use of anti-T-cell therapies, or by T-cell depletion in bone marrow transplantation. Pre-transplant EBV seronegativity increases risk to as high as 30%-50%. More than 90% of tumors are EBV-associated. Mechanisms for viral lymphomagenesis remain incompletely defined; LMP-1 may function as an oncogene and coprecipitates with TRAF, BCL-2 overexpression has also been identified. A possible direct tumorigenic effect has recently been suggested for cyclosporine. PTLD has a highly variable clinical picture, certain patterns are however seen. Reversibility of PTLD with reduction in immunosuppressives has long been recognized. Predicting reversibility has been difficult. The presence or absence of BCL-6 mutations has recently been identified as being of predictive value. Surgical resection can be curative. Cytotoxics, although problematic, can also be curative. Long term remission has been achieved with anti CD21 and CD24 antibodies; efficacy has been reported anecdotally for interferon alpha and for rituximab. In vitro expanded EBV-specific T cells have been effective as treatment and as prophylaxis in the setting of bone marrow transplantation. EBV viral load measured in blood appears to correlate with the emergence of PTLD and may facilitate prophylactic studies. PTLD is a model of immunodeficiency related EBV lymphomagenesis. Pathogenetic, therapeutic, and prophylactic insights gained from the study of PTLD are likely to be applicable to other immunodeficiency states and to EBV-related lymphoid neoplasia in general.
BACKGROUND: The use of myeloablative intensive therapy followed by autologous or allogeneic stem-cell transplantation (SCT) for treatment of chronic lymphocytic leukemia (CLL) has largely increased over the last years. DESIGN: The present overview updates the available clinical results and discusses important aspects of SCT in patients with CLL including the type of SCT (autologous vs. allogeneic), myeloablative regimens, purging, the predictive value of molecular monitoring of residual disease, and prognostic factors for the outcome of transplant approaches. RESULTS: With appropriate supportive care, autologous SCT is safe and can induce long-lasting clinical and molecular remissions, which may improve the prognosis of patients with CLL. Feasibiliy and efficacy of autologous SCT appears to be best early during the course of the disease, but it is still unclear if autotransplantation can cure the disease even in this favorable subgroup. The role of purging is still unclear. The better disease control observed after allografting appears to be due to graft-versus-leukemia activity and may allow cure in at least a subset of poor-risk patients. Due to the extraordinarily high treatment-related mortality, however, the outcome after allogeneic SCT is still inferior to that after autologous SCT. CONCLUSIONS: Autologous transplantation is a valuable treatment option for younger patients with early or sensitive poor-risk CLL. Selected patients with advanced poor-risk disease and low probability of successful auto-SCT should be considered for allografting. However, it must be kept in mind that both autologous and allogeneic stem-cell transplantation are still experimental procedures and clinical trials further elucidating their value in the treatment of patients with CLL are warranted.
BACKGROUND: Minimal criteria for the diagnosis of multiple myeloma are provided. Monoclonal gammopathy of undetermined significance, smoldering multiple myeloma, primary systemic amyloidosis and metastatic carcinoma must be included in the differential diagnosis. Patients with multiple myeloma should not be treated unless they have an increasing M-protein in the serum or urine, development of anemia, hypercalcemia, renal insufficiency, lytic lesions, fractures or extra-medullary plasmacytomas. PATIENTS AND METHODS: This is a review of patients treated with chemotherapy, autologous stem-cell transplantation and allogeneic transplantation. RESULTS: Comparisons of melphalan and prednisone with a variety of combinations of therapeutic agents produces a higher response rate than with melphalan and prednisone but no significant difference in overall survival. Autologous stem-cell transplantation produces a higher response rate and some prolongation of survival but is not curative. Allogeneic transplantation is associated with a mortality of 40% and is not curative. CONCLUSIONS: If the patient is younger than 70 years, the physician should consider the possibility of an autologous peripheral blood stem-cell transplant. Ideally, this should be done as part of a prospective study. Hematopoietic stem cells are damaged by alkylating agents so they must be collected before these agents are given. Autologous stem-cell transplantation does not produce a cure and most patients will relapse. The appropriate timing of an autologous stem-cell transplant has not been ascertained. Hopefully, better preparative regimens and the removal of contaminated tumor cells from the peripheral blood will make an autologous transplant more effective. Another major question is whether double (tandem) transplants are superior to a single autologous stem-cell transplant. A current French Myeloma Group Study randomized study should answer this question. Allogeneic transplantation for multiple myeloma must be made safer because the transplant-related mortality is 40%. The relapse of multiple myeloma following allogeneic transplant is a major problem and consequently the preparative regimens must be improved. The infusion of donor lymphocytes following relapse after an allogeneic transplant is useful. New approaches with immunologic aspects including the use of dendritic cells and vaccines are of potential importance for the future.
BACKGROUND: Allogeneic bone marrow transplantation (BMT) has been used in patients with low-grade lymphoma (LGL) and chronic lymphocytic leukemia (CLL) with the goal of achieving long-term disease-free survival. PATIENTS AND METHODS: Twenty-nine patients with these diagnoses (LGL = 19, CLL = 10) received allogeneic BMT between September 1995 and January 1999. Median age was 42 (range 20-52) years. Twenty-three of twenty-nine patients (79%) were Ann Arbor or Rai stage IV at the time of transplant; twenty-four (83%) had never achieved complete remission (CR). Donor source was HLA-matched sibling (20), unrelated (8) and syngeneic (1). RESULTS: Seventeen patients are currently alive, a median of 29 months (range 1-85) post-BMT with a median KPS of 90%. Twenty-three of twenty-seven evaluable patients (85%) achieved CR post-BMT. Six patients had refractory/recurrent disease. Death occurred related to transplant complications in eight patients and underlying disease in four. Overall and event-free survival for the whole group is 51% and 44%, respectively. CONCLUSIONS: Allogeneic BMT for young patients with advanced stage LGL or CLL is a feasible strategy that can result in achievement of long-term disease-free survival.
INTRODUCTION: The distribution of the major subtypes of non-Hodgkin's lymphoma (NHL) differs across geographic regions. This is the first study from India that has incorporated immunophenotypic findings while investigating the distribution of NHL subtypes. PATIENTS AND METHODS: All cases diagnosed as NHL between January 1995 and June 1998 in the Department of Pathology and in the Lymphoma Registry, Tata Memorial Hospital, Bombay, were selected for the study. The cases were reviewed by three pathologists and diagnostic problems were discussed by a panel of pathologists with a special interest in lymph node pathology. Of a total of 2831 cases, the diagnosis of NHL was accepted in 2773 cases. RESULTS: B-cell lymphomas formed 79.1% of the NHLs, whereas T-cell lymphomas formed 16.2% of the total. Diffuse large B-cell lymphoma was the most common subtype (34% of all NHLs). Follicular centre-cell lymphomas, B-cell small lymphocytic lymphoma, mantle-cell lymphoma, and marginal zone B-cell lymphomas (including MALT lymphomas) amounted to 12.6%, 5.7%, 3.4%, and 8.2%, respectively. Among the T-cell lymphomas, T-cell lymphoblastic lymphoma, anaplastic large-cell lymphomas of T/null-cell type, and other nodal peripheral T-cell lymphomas accounted for 6%, 4.3%, and 2.9% of all cases, respectively. CONCLUSIONS: The distribution of NHL subtypes in India shows important differences with those from the rest of the world. Follicular lymphoma and mantle-cell lymphoma are less common in India compared to Europe and the USA. Peripheral T-cell lymphomas and T/NK-cell lymphomas of nasal and nasal types, which are common in many other Asian countries, are also less prevalent. T-cell lymphoblastic lymphoma and anaplastic large T/null cell lymphoma are more prevalent in India.
BACKGROUND: The incidence of non-Hodgkin's lymphoma (NHL) has been increasing in Canada. This study assessed the effect of occupational exposure to specific chemicals on the risk of NHL. PATIENTS AND METHODS: Mailed questionnaires were used to obtain data on 1469 newly diagnosed, histologically confirmed NHL cases and 5073 population controls between 1994 and 1997 in eight Canadian provinces. Data was collected on socioeconomic status, life-style, diet, occupation, and years of exposure to any of 17 chemicals. Odds ratios (OR) and 95% confidence intervals (95% CI) were derived by logistic regression. RESULTS: The study found an increased risk of NHL among males exposed to benzidine, mineral, cutting, or lubricating oil, pesticides, and herbicides. Compared with non-exposure to each specific chemical, the adjusted ORs were 1.9 (95% CI: 1.1-3.4) for benzidine, 1.3 (95% CI: 1.0-1.5) for mineral, cutting, or lubricating oil, 1.3 (95% CI: 1.0-1.6) for herbicides, and 1.3 (95% CI: 1.0-1.6) for pesticides. Excess risk of NHL among females was associated with exposure to pesticides and wood dust. ORs increased with increasing exposure in years to benzidine and herbicides for males and with increasing exposure years to wood dust for females. These trends were statistically significant (P < 0.05). CONCLUSIONS: The findings in this study suggest that occupational exposure to specific chemicals plays an important role in the development of NHL in Canada.
BACKGROUND: The t(14;18) translocation, present in 90% of follicular non-Hodgkin's lymphomas (NHL), has been found to exist in low levels in healthy persons. Its clinical/prognostic significance in healthy populations is unknown, and risk factors for its development have not been determined. Our objectives were to assess the prevalence of t(14;18) in individuals without NHL, comparing residents of agricultural settlements (kibbutzim) with city dwellers, as well as first degree relatives of NHL cases. PATIENTS AND METHODS: Residents of kibbutzim and members of two control groups: 1) Jerusalem residents--randomly selected hospital administrative workers and 2) first degree family members of lymphoma patients were interviewed extensively regarding exposures and had blood drawn for t(14;18) determination. The translocation was detected after B-cell purification of blood samples with CD-19 microbeads (Mini-Macs) using nested PCR. The method detects the translocation in a BCL2 positive cell line after dilutions of up to 1:10(5) with normal peripheral blood lymphocytes. RESULTS: Nineteen of two hundred thirty healthy individuals (8.3%) tested were found to be positive for t(14;18). No statistically significant differences in the prevalence of t(14;18) were detected among the rural and urban populations. Five of thirty-four (11.9%) family members tested positive for t(14;18). No age or sex differences between t(14;18) positive and negative individuals were found. No significant association with exposure to specific agricultural or other chemicals was found. CONCLUSIONS: The presence of the t(14;18) translocation in healthy individuals was not associated with agricultural residence in this preliminary study. Whether relatives of patients with NHL are at increased risk will require further study in larger populations. Specific exposures affecting the onset of this translocation have not been ruled out. The significance of this translocation in healthy individuals remains unknown.
BACKGROUND: Treatment strategies in Hodgkin's disease (HD) are changing fundamentally over the last decades. Both radiotherapy and combination chemotherapy are effective treatment modalities. However, the optimal choice of treatment or combinations of treatment is still debated for different prognostic groups. PATIENTS AND METHODS: The German Hodgkin's Lymphoma Study Group (GHSG) initiated randomized clinical trials since 1978. Over the past 20 years, more than 6000 patients with HD in all stages were randomized, treated and followed by the GHSG. Patients are now being recruited from more than 300 clinical centers. RESULTS: As a consequence of different clinical trials, it is now the policy of the GHSG to tailor treatment to the individual risk of patients, giving favorable patients less intensive and less toxic therapy than unfavorable patients. The treatment for early and intermediate stage HD becomes quite similar with few cycles of polychemotherapy followed by involved field irradiation. In advanced stage HD, the introduction of dose intensified chemotherapy (BEACOPP), has improved treatment results and thus will substitute the MOPP or ABVD regimens. CONCLUSIONS: Although most of the patients with HD will be cured by modern treatment strategies, several questions are still subjects of ongoing clinical trials: 1) which chemotherapy regimen in which quantity will be the best with respect to efficacy and toxicity and 2) which dose and field size of radiotherapy is adequate within the combined modality.
BACKGROUND: Between 1987 and 1991, the British National Lymphoma Investigation randomized 459 patients with non-Hodgkin's lymphoma with a large-cell component to either CHOP or the PACEBOM regimen. PATIENTS AND METHODS: Four hundred fifty-nine eligible patients were included in this trial, four hundred one with diffuse large-cell lymphoma and fifty-eight with diffuse mixed-cell lymphoma according to the Working Formulation. Two hundred twenty-six patients were randomized to receive CHOP and two hundred thirty-three to receive PACEBOM. The two arms of the trial were well balanced for all potential prognostic factors. RESULTS: The complete remission rate with PACEBOM was 64% and with CHOP 57% (NS). At eight years, the actuarial cause specific survival (CSS) in the PACEBOM arm is 59% compared to 49% in the CHOP arm (P = 0.09). Patients < 50 years of age fared significantly better in the PACEBOM arm both for CSS and overall survival (P = 0.002) and the CSS was also significantly improved in stage IV disease (P = 0.02). CONCLUSIONS: The mature data from this trial suggest that an etoposide-containing multi-agent weekly regimen can be superior to CHOP.
BACKGROUND: The CUP trial was initiated to analyze the value of high-dose therapy and stem-cell transplantation and purging in patients with relapsed chemosensitive follicular NHL. PATIENTS AND METHODS: After three cycles of chemotherapy responsive patients were randomized to either three more cycles of the same chemotherapy (C), high-dose therapy followed by autologous unpurged (U) or purged (P) stem-cell transplantation. Purging was performed using a cocktail of monoclonals. Pretransplant conditioning consisted of cyclophosphamide (60 mg/kg x 2) and total body irradiation. RESULTS: Of the 140 patients registered from 26 centers in Europe, 89 fulfilled the criteria for randomization (C: 24, U: 33 and P: 32). Reasons for failure to randomize were: no response (28), persistent marrow infiltration (4), patient refusal (7), other (7), no data (5). With the current follow up (median 26 months from randomization) 16 (66%) in C are known to have progressed or relapsed, in contrast to 13 (39%) of U and 12 (37%) of the P patients (P-value 0.002). Overall survival is premature with the current available data. CONCLUSIONS: Patients in U and P arms had higher progression/relapse-free survival rate. There are some suggestions of some improvement in overall survival rate.
BACKGROUND: Mycosis fungoides is the most frequent T-cell lymphoma of the skin. Despite numerous attempts, no tumour antigens have yet been identified. Only in one case has an idiotype-derived peptide been found to trigger CTL of the respective patient. The identification of natural antigens requires the cultivation of large amounts of tumour cells in vitro, which has been possible in two exceptional cases. The identification of synthetic epitopes for tumour-specific CTL with random peptide libraries can overcome this limitation and is a powerful tool for application in the development of immune therapies for a wide range of patients. MATERIALS AND METHODS: The critical amino acids for the construction of epitopes for the CTCL-specific CTL clone My-La CTL were determined with synthetic peptide libraries in positional scanning OX8 format in a standard 61chromium release assay. Sixteen different peptides could be synthesized from the combinatoric of these amino acids with the canonical anchor amino acids for MHC binding. These peptides were tested for their capacity to stimulate My-La CTL and PBMC of an HLA-matched CTCL patient. RESULTS: A synthetic epitope could be identified for My-La CTL, which was recognized in a HLA-restricted manner. The response towards this epitope was comparable to the response towards their natural target My-La. Using these synthetic epitopes, T cells of a HLA-matched patient could be induced in vitro and led to the establishment of different cell lines and clones. Some of these lines recognized the peptides as well as the allogenic but HLA-matched tumour cell line My-La, indicating that they are specific for a naturally expressed tumour antigen. CONCLUSIONS: The identification of synthetic epitopes for tumour-specific CTL clones can be used for the development of vaccines for immune therapies of cancer; such peptides can be applied inter-individually. Synthetic epitopes must not correspond to the natural ones, but they can be even more potent as stimulation of specific T cells and can be fine-tuned to increase the success of the therapy.
Acquired immune deficiency syndrome (AIDS) is an incurable disease at present and so many efforts to conquer this disease are being made around the world. In studies of human immunodeficiency virus (HIV) infection and the disease progression, it has been reported that T cells expressing CD26 are preferentially infected and depleted in HIV-infected individuals. CD26 is a widely distributed 110 kDa cell-surface glycoprotein with known dipeptidyl peptidase IV (DPPIV) activity in its extracellular domain. This ectoenzyme is capable of cleaving N-terminal dipeptides from polypeptides with either proline or alanine residues in the penultimate position. On human T cells, CD26 exhibits the co-stimulatory function and plays an important role in immune response via its ability to bind adenosine deaminase (ADA) and association with CD45. Recent studies have been stripping the veil from over the relationship between CD26 and HIV infection. Susceptibility of cells to HIV infection is correlated with CD26 expression, and HIV transactivator Tat and envelope protein gp120 are reported to interact with CD26. These observations indicate that CD26 is closely involved in HIV cell entry and that CD26-mediated T cell immune response is suppressed. In addition, it has been demonstrated that the anti-HIV and chemotactic activities of RANTES (regulated on activation, normal T cell expressed and secreted) and stromal cell-derived factor-1 (SDF-1) are controlled with the DPPIV activity of CD26. Thus, the regulation of the function of chemokines by CD26/DPPIV appears to be essential for lymphocyte trafficking and infectivity of HIV strains.
The mechanism involved in the development of persistent hepatitis C virus (HCV) infection and the pathogenesis remain unclear. The present review is an accumulation of evidence gathered to date. In addition, it discusses the system developed to characterize HCV.
There are a considerable amount of empirical and theoretic medical literature regarding the possible role of viruses in the development of drug rashes and autoimmune diseases: under these conditions, interactions of viruses with the immune system would serve as an accelerating factor of disease pathogenesis. Recent reports have provided evidence to indicate that immune responses against infections with Epstein Barr (EB) virus and human herpesvirus 6 (HHV-6), which are lymphotropic members of the herpes virus group, not only aid the direct elimination of the virus but also contribute to a favorable milieu for the initiation or acceleration of drug rashes. Viruses that can persist for the lifetime of the host despite strong immune responses against them, such as EB virus and hepatitis C virus (HCV), would be also relevant to the pathogenesis of autoimmune diseases. HCV has been reportedly associated with a wide variety of dermatoses that, in common, show histologically the lichenoid tissue reaction. Although porokeratosis that manifests lichenoid histopathological features had long been regarded as being associated with immunosuppression, we found that HCV could act as trigger for the development of porokeratosis during states of immunosuppression. Thus, the main purpose of this review is to describe recent work on the etiology of drug rashes and autoimmune disease with special reference to viral infections.
Epstein Barr virus (EBV) infection is implicated in various kinds of neoplasms including certain types of cutaneous T or natural killer (NK) cell proliferative disorders. Although a pathogenic role of EBV infection is not clear, some EBV gene products expressed during a latency phase were found to have biological properties leading to cellular gene expression and immortalization. Furthermore, EBV can use an array of strategies to evade host immune responses, and maintain the latent infection. EBV-associated cutaneous lymphoproliferative disorders are prevalent in Asia, and less frequent in western countries where infectious mononucleosis is common in adolescents and young adults. This review introduces recent advances on the mechanism of EBV infection, highlighting unique clinicopathologic manifestations of EBV-associated cutaneous lymphoproliferative disorders.
Human cytomegalovirus (CMV) is a member of the herpes family of viruses. After primary infection, it undergoes latency/persistence. Significant progress has been made in the last few years in detecting CMV. The most available approach to the diagnosis of CMV infection is the direct detection of CMV antigen in nuclei of peripheral blood leukocytes, an assay known as pp65 direct antigenemia test. CMV infection is well controlled in the immunocompetent hosts; however, there are various immunological changes in immune function during and after recovery from CMV infections. Characteristic changes in lymphocyte subsets occur during CMV infection, mainly involving expansion and activation of CD8+ T lymphocytes and NK cells. On the other hand, CMV has an array of immune escape strategies for establishing a life long latent state: CMV inhibits major histocompatibility complex (MHC) class I expression within infected cells and impairs IFN-gamma-induced MHC class II-dependent antigen presentation by macrophages; it can also encode proteins that can interfere with the presentation of viral peptide antigens to T cells. While cutaneous manifestations of CMV seen in immunocompromised patients have been extensively reported, those in adult immunocompetent individuals have received relatively little attention: in this setting the primary CMV infection appears as CMV mononucleosis. At the time of occurrence of the mononucleosis syndrome, a variety of extracutaneous and cutaneous manifestations occur. These clinical symptoms are not the direct consequence of proliferation of CMV in given tissues but indicative of the immunological response toward CMV. The incidence of the appearance of eruptions in CMV mononucleosis is variable. Certain drugs given in the early stage of this disease play an important role in the development of eruption, just as with the ampicillin rashes in the Epstein-Barr virus mononucleosis. Although the mechanism by which drugs trigger the development of rashes in patients with CMV mononucleosis is unknown, it is assumed that CMV is likely to be a potential amplifier of drug rashes induced by activation of drug-specific T cells. By improving methods for detection of CMV, we can recognize that many types of eruptions other than CMV mononucleosis could be induced by primary infection or reactivation of CMV.
Human herpesvirus 6 (HHV-6) and human herpesvirus 7 (HHV-7) are relatively recently discovered beta-herpesvirus. They are prevalent in the human population. Primary infection of HHV-6 has been associated with exanthem subitum and febrile illness. Little information is known about the clinical characteristics of primary infection with HHV-7, although some cases of exanthem subitum have been linked to it. HHV-6 has been recently recognized as an opportunistic pathogen in patients with HIV infection and in transplant recipients. The techniques now available to detect these two viruses remain limited, though putative roles for HHV-6 and HHV-7 in several diseases linked to viral infection have been reported. This report reviews the current knowledge of HHV-6 and HHV-7 biology and their pathogenesis.
AIM: To determine if a non-ophthalmologist can accurately screen for retinopathy of prematurity (ROP) by evaluating the posterior pole blood vessels of the retina. ROP is a common ocular disorder of premature infants and may require multiple screening examinations by an ophthalmologist to allow for timely intervention. Since there is a strong correlation between posterior pole vascular abnormalities and vision threatening ROP, screening examinations performed by non-ophthalmologist may yield useful clinical information in high risk infants. METHODS: Infants born at the Medical University of South Carolina who met screening criteria (n = 142) were examined by a single non-ophthalmologist using a direct ophthalmoscope to evaluate the posterior pole blood vessels for abnormalities of the venules and/or arterioles. To determine the accuracy of the non-ophthalmologist's clinical observations, infants were also examined by an ophthalmologist, using an indirect ophthalmoscope, who graded the posterior pole vessels as normal, dilated venules, or dilated and tortuous venules and arterioles (including "plus disease"). RESULTS: There was significant correlation (p <0.001) between the non-ophthalmologist's and ophthalmologist's diagnoses of posterior pole vascular abnormalities. 47 infants had normal posterior pole blood vessels by the non-ophthalmologist examination. Of these, 31 (66%) were considered to have normal vessels and 16 (34%) to have dilated venules by the ophthalmologist. The non-ophthalmologist correctly identified abnormal posterior pole vessels in all 21 infants diagnosed with abnormal arterioles and venules by the ophthalmologist. No infants with clinically important ROP ("prethreshold" or worse) would have failed detection by this screening method. CONCLUSION: Using a direct ophthalmoscope, a non-ophthalmologist can screen premature infants at risk for ROP by evaluating the posterior pole blood vessels of the retina. While not necessarily recommended for routine clinical practice, this technique may nevertheless be of value to those situations where ophthalmological consultation is unavailable or difficult to obtain.
AIMS: To evaluate the analgesic effect of topical sodium diclofenac 0.1% during retinal laser photocoagulation. METHODS: 87 patients, 45 with proliferative diabetic retinopathy treated with two sessions of panretinal photocoagulation (group A), and 42 patients with non-proliferative diabetic retinopathy who underwent grid treatment of the posterior pole (19 bilaterally) (group B). Sodium diclofenac 0.1% or sodium chloride 0.9% drops were topically applied 30-135 minutes before laser treatment in a masked fashion. Patients who had two sessions were given the alternate drug in the second one. Pain level was evaluated immediately after laser treatment with the visual analogue scale (VAS). The results were statistically analysed. RESULTS: Patients in group A reported pain in 85/90 sessions (94%). The average pain level was 44.2% with sodium diclofenac 0.1% drops and 53.1% with sodium chloride 0.9% drops (p = 0.011 by paired t test). Patients in group B reported pain in only 16/60 sessions (26. 7%), and the pain level ranged from 10% to 60% regardless of the kind of drops used. There was no correlation in either group between level of pain and time interval from application of the drops to laser treatment (30-135 minutes) or average energy level used (100-500 mW). CONCLUSION: Sodium diclofenac 0.1% is useful for pain reduction and should be applied before panretinal photocoagulation.
BACKGROUND/AIMS: A longitudinal study of premature infants was conducted to examine changes in refractive status and their relation with age and factors influencing the occurrence and degree of myopia. Identification of which of the various refractive factors play important parts in relation to myopia in premature infants was attempted. METHODS: Under observation were 125 eyes in 65 patients who were found to demonstrate no signs of retinopathy of prematurity (ROP) or who had grade I or II ROP without or after cryotherapy. Cycloplegic refractions were conducted at 6 months, 3 years, and 6 years of age; at 6 years of age keratometric values, lens thicknesses, and axial lengths were recorded, and anterior chamber depths also were measured. RESULTS: Myopia begins to appear at 6 months of age and its severity increases between the ages of 6 months and 3 years. The condition showed no further progress in subjects older than 3 years. Of the 104 eyes with ROP, those eyes with cicatricial retinopathy tended towards myopia and high myopia while there was no difference in the degree of myopia related to whether or not cryotherapy was conducted. At 6 years of age, the premature infants exhibited shallower anterior chambers, thicker lenses, and higher axial lengths when the degree of the myopia was higher. The keratometric values, however, appeared to bear no relation to the degree of the myopia. CONCLUSION: These results suggest that the occurrence of myopia is related more strongly to whether or not there is cicatricial retinopathy than whether or not there is cryotherapy. Also, the degree of the myopia was found to be related to the depth of the anterior chamber, the thickness of the lens, and the change in axial length but not to keratometric value.
AIMS: To conduct a systematic review of drug induced adverse ocular effects in diabetes to determine if this approach identified any previously unrecognised adverse drug effects; to make a preliminary assessment of the feasibility of this approach in identifying adverse drug reactions; and to assess the current accessibility of this information to prescribing physicians. METHODS: Literature search of online biomedical databases. The search strategy linked eye disorders with adverse drug reactions and diabetes. Source journals were classified as medical, pharmaceutical, diabetes related, or ophthalmological. It was determined whether the reactions identified were recorded in drug datasheets and the British National Formulary. RESULTS: 63 references fulfilled the selection criteria, of which 45 were considered to be relevant to the study. The majority of these were case reports but cross sectional surveys, case-control and cohort studies, and review articles were also identified. 61% of the reactions were not recorded in the British National Formulary and 41% were not recorded in the datasheets. 55% appeared in specialist ophthalmology journals. CONCLUSIONS: This is a feasible approach to the identification of adverse drug reactions. Adverse reactions not listed in the most commonly used reference sources were found. The majority were published in specialist ophthalmology journals which might not be seen by prescribing physicians.
AIMS: To determine the visual and anatomical outcome of surgery for long standing idiopathic macular holes. METHODS: A retrospective review of 24 eyes of all 22 patients who underwent surgery for idiopathic full thickness macular holes (FTMH) symptomatic for between 1 and 3 years. Postoperative follow up was for 6 months. Preoperative and postoperative visual acuities were recorded as well as the presence of anatomical closure of the hole. RESULTS: The mean duration of symptoms was 18.21 (SD 5.42) months). Anatomical closure of the FTMH was achieved in 17 (70.8%) of the eyes at 6 months. The logMAR acuity of the group where closure was achieved improved by a mean of 0.31, equivalent to a change of Snellen acuity from 6/60 to 6/29. Where the hole remained open the acuity deteriorated by a mean logMAR of 0.11 lines, equivalent to a change of Snellen acuity from 6/60 to 5/60. Anatomical closure of the hole was associated with a significantly improved acuity over non-closure (p<0.001). The degree of visual improvement was independent of the preoperative visual acuity (Spearman correlation coefficient 0.03, p=0.888), though preoperative acuity was related to the final acuity (Spearman correlation coefficient 0.701, p<0.001). Over the study period, six patients required cataract surgery, one patient developed secondary glaucoma, and one a retinal detachment. CONCLUSIONS: Vitrectomy with intraocular gas tamponade and postoperative posturing is a well tolerated and effective intervention for long standing macular holes. Anatomical closure of the macular hole is associated with a significant improvement in visual acuity.
BACKGROUND/AIMS: A moderately elevated plasma concentration of the sulphur amino acid homocysteine is an independent risk factor for atherosclerotic vascular disease. Many of the risk factors associated with coronary, cerebral, and peripheral atherosclerotic vascular disease are common to retinal vascular occlusive disease but it is unclear whether elevated plasma concentrations of homocysteine are also associated with such disease. This study assessed the relation between retinal vascular occlusive disease and elevated levels of plasma total homocysteine (tHcy). METHODS: A retrospective case-control study involving hospital based controls and cases with retinal artery, central retinal vein (including hemiretinal vein), and branch retinal vein occlusions was performed. The relation between elevated tHcy, defined as a level greater than or equal to 12 micromol/l and risk of retinal vascular occlusive disease was examined. RESULTS: 87 cases of retinal vascular occlusive disease including 26 cases of retinal artery occlusion, 40 cases with central retinal vein occlusion, and 21 cases of branch retinal vein occlusion were compared with 87 age matched controls. Mean tHcy levels were higher in all disease groups and this difference was significant in patients with retinal artery occlusions (p= 0.032) and patients with central retinal vein occlusion (p=0.0001). When adjusted for known cardiovascular risk factors, tHcy was an independent risk factor for retinal vascular occlusive disease (OR 2.85 (95% CI 1.43-5.68)). CONCLUSIONS: Elevated tHcy is an independent risk factor for retinal vascular occlusive disease. Assessment of tHcy may be important in the investigation and management of patients with retinal vascular occlusive disease.
AIMS: To report the clinical features of five patients with non-progressive central ring scotomas of acute onset associated with excellent retained visual acuity. METHODS: Complete neuro-ophthalmological examinations were performed. Visual fields were performed by tangent screen, Goldmann, or Humphrey perimetry. In some cases further testing was carried out including fundus photography, fluorescein angiography, ERG, VEP, and neuroimaging. RESULTS: The patients were three women and two men whose ages ranged from 25 to 57 years. Four patients were heavy caffeine consumers while the fifth patient experienced an episode of hypotension. Vision loss was acute in all cases. The onset of vision loss was bilateral/simultaneous in three cases, bilateral/sequential in one case, and unilateral in one case. All affected eyes retained visual acuities of 20/25 or better. Colour vision was subnormal in three of four cases. Visual field defects were characterised by a central ring scotoma having an outer diameter less than 10 degrees. Fundus examination demonstrated temporal optic nerve pallor in three patients (five of 10 affected eyes) and reddish, petaloid macular lesions in one patient. Good visual acuity was maintained for the duration of follow up in all five patients. CONCLUSION: Central ring scotomas with excellent retained visual acuity may present as an acute, bilateral syndrome in patients who are heavy caffeine consumers. The configuration of visual field loss and its location, combined with the presence of temporal pallor in five eyes, suggest that the defect localises to the inner layers of the macula. While these cases could be considered an expansion of the clinical spectrum of acute macular neuroretinopathy, some may represent a distinct entity.
BACKGROUND: Despite the fact that visual function has an important role in the quality of life in later years, very few studies have measured visual acuity in population based nationwide samples of British elderly people. Such measurements were carried out in the context of the national diet and nutrition survey of people aged 65 years or over (NDNS). METHODS: NDNS participants, who were living in 80 different randomly selected postcode areas of mainland Britain, were visited at their home by a nurse who measured visual acuity at 3 metres, using the Glasgow acuity card (GAC) method. In addition, a brief questionnaire related to ocular health was administered. RESULTS: Visual acuity was measured in 1362 NDNS participants who were not classified as mentally impaired. Visual impairment (using the WHO low vision criteria) was measured in 195 (14.3%) subjects. Prevalence of visual impairment increased significantly with age (65-74 years 3.1%; 75-84 years 11.6%; 85+ years 35.5%, p<0.001 for trend). Impaired vision was more common in subjects living in a nursing home (odds ratio adjusted for age 2.59 (95% CI 2.23 to 2. 96)) and in women (odds ratio adjusted for age 1.55 (95% CI 1.21 to 1.89)). 132 (9.7%) subjects had previously undergone cataract surgery and another 157 (11.5%) had been told that they currently had cataract. Vision improved 0.2 log units or more (at least one Snellen line) with the aid of a pinhole occluder in 289 subjects (21. 2%). CONCLUSION: Results of this nationwide, community based study confirm that problems with poor distance visual acuity exist in a substantial part of the elderly community, particularly in women and people living in nursing homes.
AIM: To assess the efficacy and safety of an intraoperative intracameral injection of mepivacaine, administered when patients experienced pain during the course of cataract surgery under topical anaesthesia. METHODS: This is a prospective placebo controlled double masked randomised clinical trial. 50 eyes were included; 25 receiving the active compound and 25 receiving placebo. Mepivacaine (2%, 0.4 ml) or placebo was administered intraoperatively under the iris of the patients who experienced pain during the course of phacoemulsification in spite of previous topical anaesthesia. Efficacy was evaluated by the patients themselves using a five point subjective pain rating scale, the Keele verbal pain chart. Safety was measured by assessing intraocular inflammation (clinical evaluation and laser flare meter), intraocular pressure, and endothelial cell count. RESULTS: The pain rating score significantly diminished after intracameral injection in the mepivacaine group (mean 3.0 (95% CI 2.6-3.4) v 0.8 (0.3-1.3), p<10(-4))) while remaining unchanged in the placebo group (2.9 (2.6-3.2) v 2.9 (2.5-3. 3)), the mean reduction in pain score being significantly different between the two groups (p<10(-4)). There was no indication of increased postoperative ocular inflammation, intraocular pressure change, or endothelial cell loss. CONCLUSIONS: These results suggest that it may not be necessary to systematically add intracameral anaesthesia with topical anaesthesia for cataract surgery. An intraoperative intracameral injection, performed only in patients who happen to suffer during surgery, is safe and effective.
AIM: To indicate that congenital idiopathic nystagmus (CIN) and sensory defect nystagmus (SDN) can be vertical or asymmetric in some children. METHODS: Of 276 children presenting with nystagmus for electrophysiological testing, 14 were identified as having CIN or SDN, yet had a nystagmus which was either vertical (n=11) or horizontal asymmetric (n=3). Flash electroretinograms and flash and pattern visual evoked potentials (VEPs) were recorded in all patients. Eye movement assessment, including horizontal optokinetic nystagmus (OKN) testing, was carried out in 11/14 patients. RESULTS: Eight patients (seven with vertical, one with asymmetric horizontal nystagmus) had congenital cone dysfunction. One patient with vertical and another with asymmetric nystagmus had cone-rod dystrophy. One patient with vertical upbeat had congenital stationary night blindness. Two patients (one downbeat, one upbeat nystagmus) had normal electrophysiological, clinical, and brain magnetic resonance imaging findings and were classified as having CIN. One patient with asymmetric nystagmus showed electrophysiological and clinical findings associated with albinism. Horizontal OKN was present in 80% of patients tested, including the three cases with horizontal asymmetric nystagmus. This is atypical in both CIN and SDN, where the OKN is usually absent. CONCLUSIONS: Vertical and asymmetric nystagmus are most commonly associated with serious intracranial pathology and its presence is an indication for neuroimaging studies. However, such nystagmus can occur in children with retinal disease, albinism, and in cases with CIN. These findings stress the importance of non-invasive VEP/ERG testing in all cases of typical and also atypical nystagmus.
AIMS: To compare the effect on intraocular pressure (IOP) of latanoprost monotherapy and timolol-pilocarpine in patients with glaucoma or ocular hypertension with inadequately controlled IOP on topical beta adrenergic antagonists. METHODS: This was a multicentre, randomised, observer masked, 6 week study performed in France and Sweden. 23 centres enrolled 237 patients with glaucoma or ocular hypertension and an IOP of at least 22 mm Hg on treatment with topical beta adrenergic antagonists, alone or in combination. After a 21 day run in period on timolol 0.5% twice daily, patients were randomised either to latanoprost 0.005% once daily or to a fixed combination of timolol-pilocarpine twice daily. Changes in mean diurnal IOP from the baseline to the 6 week visit were determined with an analysis of covariance. RESULTS: Mean diurnal IOP was statistically significantly decreased from baseline in both groups (p<0.001). Switching to latanoprost treatment reduced mean diurnal IOP by 5.4 (SEM 0.3) mm Hg (ANCOVA -22%) and switching to timolol-pilocarpine treatment reduced mean diurnal IOP by 4.9 (0.4) mm Hg (-20%). Blurred vision, decreased visual acuity, decreased twilight vision, and headache were statistically significantly more frequent in the timolol-pilocarpine group. CONCLUSIONS: Latanoprost monotherapy was at least as effective as fixed combination timolol-pilocarpine twice daily treatment in reducing mean diurnal IOP in patients not adequately controlled on topical beta adrenergic antagonists. Latanoprost was better tolerated than timolol-pilocarpine regarding side effects. These results indicate that a switch to latanoprost monotherapy can be attempted before combination therapy is initiated.
AIM: To evaluate the performance of limbal chamber depth estimation as a means of detecting occludable drainage angles and primary angle closure, with or without glaucoma, in an east Asian population, and determine whether an augmented grading scheme would enhance test performance. METHOD: A two phase, cross sectional, community based study was conducted on rural and urban areas of Hövsgöl and Omnögobi provinces, Mongolia. 1800 subjects aged 40 to 93 years were selected and 1717 (95%) of these were examined. Depth of the anterior chamber at the temporal limbus was graded as a percentage fraction of peripheral corneal thickness. An "occludable" angle was one in which the trabecular meshwork was seen in less than 90 degrees of the angle circumference by gonioscopy. Primary angle closure (PAC) was diagnosed in subjects with an occludable angle and either raised pressure or peripheral anterior synechiae. PAC with glaucoma (PACG) was diagnosed in cases with an occludable angle combined with glaucomatous optic neuropathy and consistent visual morbidity. RESULTS: Occludable angles were identified in 140 subjects, 28 of these had PACG. The 15% grade (equivalent to the traditional "grade 1") yielded sensitivity and specificity of 84% and 86% respectively for the detection of occludable angles. The 5% grade gave sensitivity of 91% and specificity of 93% for the detection of PACG. The interobserver agreement for this augmented grading scheme was good (weighted kappa 0.76). CONCLUSIONS: The traditional limbal chamber depth grading scheme offers good performance for detecting occludable drainage angles in this population. The augmented scheme gives enhanced performance in detection of established PACG. The augmented scheme has potential for good interobserver agreement.Department of Preventive Ophthalmology, Institute of Ophthalmology, London.
BACKGROUND/AIMS: Varicella zoster virus (VZV) is a causative agent in acute retinal necrosis (ARN) syndrome. However, in spite of aggressive antiviral therapy, clinical characteristics among patients have varied. Different viral strains were examined to determine their respective role in producing clinical characteristics. The viral strains were also compared with those of previously reported ones. METHODS: To differentiate VZV strains R1 and R5, variable regions of VZV were amplified by nested polymerase chain reaction (PCR) in 11 eyes of 10 patients. Sequence analysis was also performed. RESULTS: Four cases had strains diverted only at the tip of the 3' end of the R1 variable region, similar to that of the H-N3 strain, which was previously reported. Conversely, other cases were diverted to other regions. Interestingly, some of the latter cases showed multiple PCR products in the R1 region that were generated by the truncation of either the 5' or 3' R1 region. Final visual acuities of these patients were less than 0.2. The former cases showed final visual acuities more than 0.4. Only two variants were from the R5 region. No patient had the same viral strain as the European Dumas type. CONCLUSION: These results showed that variable VZV strains participated in ARN. Using PCR of the R1 variable region, it was estimated that patients with a more fulminant type of ARN may have diverse viruses with extensive replication in the affected eyes.
BACKGROUND/AIMS: Fluid transport across the in vitro corneal epithelium is short lived, hence difficult to detect and characterise. Since stable rates of fluid transport across several cultured epithelial cell layers have been demonstrated, the behaviour of confluent SV40 transformed rabbit corneal epithelial cells (tRCEC) grown on permeable supports was examined. METHODS: Fluid transport was determined with a nanoinjector volume clamp; the specific electrical resistance of the layers was 184 (SEM 9) Omega cm(2). tRCEC layers transported fluid (from basal to apical) against a pressure head of 3 cm H(2)O for 2-3 hours. RESULTS: In the first hour, the rate of fluid transport was 5.2 (0.5) microl/h/cm(-2) (n=23), which is comparable with that found in other epithelia. Fluid transport was completely inhibited in 15-30 minutes by either 100 microM ouabain (n=6), 50 microM bumetanide (n=6), or 1 microM endothelin-1 (ET-1; n=6). Preincubation with 10 microM BQ123 (an ET(A) receptor antagonist) obviated inhibition by ET-1 (n=6). ET-1 also caused a 22% decrease in specific resistance. CONCLUSIONS: Fluid transport appears to depend on transepithelial Cl(- )transport since (1) their directions are the same (stroma-->tear), and (2) both bumetanide and ouabain inhibit it with similar time course. tRCEC appear useful to investigate aspects of the physiology and pharmacology of fluid transport across this layer, including receptor mediated control of this process.
AIMS: To examine the hypothesis that apoptosis of infiltrating cells contributes to spontaneous resolution of uveitis in clinically relevant rodent models. METHODS: Experimental melanin induced uveitis (EMIU) was induced in Fischer 344 rats by immunisation with 250 microg bovine ocular melanin. Endotoxin induced uveitis (EIU) was induced by injection of 200 microg Escherichia coli lipopolysaccharide. Formalin fixed, paraffin embedded ocular cross sections were stained by terminal deoxynucleotidyl transferase mediated deoxyuridine triphosphate biotin nick end labelling (TUNEL) to identify apoptotic cells. Indirect immunoperoxidase staining of paraformaldehyde lysine periodate fixed tissue cross sections was used to demonstrate expression of inducible nitric oxide synthase (iNOS). RESULTS: TUNEL positive mononuclear cells were observed in the anterior uvea during both EMIU and EIU at all selected time points. However, whereas the majority of mononuclear cells appeared apoptotic from the outset of disease, neutrophils were notably TUNEL negative at all time points examined. Many infiltrating neutrophils expressed iNOS. CONCLUSION: Apoptosis occurs early in the course of rat EMIU and EIU, and may contribute to resolution of these diseases. In general, infiltrating mononuclear cells die rapidly, while neutrophils survive, producing inducible nitric oxide synthase which may contribute to disease pathogenesis.
BACKGROUND: Pterygium is a relatively common eye disease in the tropics whose aetiology and pathogenesis remain uncertain. As such, interest has focused on understanding the underlying mechanism of pterygia development. METHODS: 15 specimens of pterygia from 15 eyes were examined, together with normal conjunctival tissue from the same eyes for the pattern of gene expression of genes associated with the induction or repression of apoptosis (p53, bcl-2, and bax). In addition, the samples directly for apoptotic cells were examined by the terminal deoxynucleotide transferase (TdT) mediated nick end labelling (TUNEL) methodology. RESULTS: In pterygia specimens apoptotic cells were found mainly confined to the basal layer of cells of the epithelial layer, situated immediately adjacent to the fibrovascular support layer. These cells were shown to express significant levels of p53 and bax, as well as the apoptosis inhibiting protein bcl-2. In contrast, normal conjunctival specimens displayed no bcl-2 expression and apoptotic cells were seen throughout the entire width of the epithelial layer, coupled with high levels of bax expression. CONCLUSION: These results support a model whereby pterygia development is a result of disruption of the normal process of apoptosis occurring in the conjunctiva.
BACKGROUND/AIMS: Accumulating evidence indicates that telomerase activity is repressed in normal human somatic cells but reactivated in cancers and immortal cells, suggesting that activation of telomerase activity has a role in carcinogenesis and immortalisation. To date, telomerase in uveal melanoma and, whether, it may have a role in the development or progression of these tumours has not been described. The expression patterns and the activity of telomerase were investigated in 14 uveal melanoma and these results were correlated with histological and immunohistological features of these tumours. METHODS: A modified PCR based telomeric repeat amplification protocol (TRAP) assay was used to demonstrate telomerase activity in 14 uveal melanomas. In addition, in situ hybridisation was used to demonstrate the expression pattern of the telomerase RNA component (hTR) at the single cell level in eight of these globes. RESULTS: The TRAP assay revealed moderate telomerase activity in all uveal melanomas examined. In situ hybridisation visualised a moderate to high upregulation of hTR in the melanoma cells but not in the admixed reactive cells. There was no correlation among tumour location, cell type, or growth fraction and the amount of telomerase activity. In addition, the cells of the germinative zone of the lens demonstrated a strong hTR expression. CONCLUSION: Telomerase activity is upregulated in uveal melanomas. The expression of hTR was located to the tumour cells and not the reactive tumour infiltrating cells. Strong telomerase expression was also demonstrated in cells of the germinative zone of the lens.
AIMS: To analyse the histopathology of classic and occult choroidal neovascular membrane surgical specimens in age related macular degeneration. METHODS: 35 membranes, from a consecutive series of surgically removed choroidal neovascular membranes in age related macular degeneration, were classified as classic or occult following the guidelines of the Macular Photocoagulation Study. Membranes with classic as well as occult components were considered as mixed membranes. The membranes were serially sectioned and stained with haematoxylin and eosin, Masson trichrome, periodic acid-Schiff, and phosphotungstic acid haematoxylin stain. The correlation has been made in a masked fashion. RESULTS: 31 membranes (19 classic, 10 occult, and two mixed membranes) could be analysed histologically. 18 classic choroidal neovascular membranes had a major subretinal fibrovascular component and 10 of these had an additional, minor fibrovascular component under the retinal pigment epithelium. The 10 occult membranes contained a fibrovascular component under the retinal pigment epithelium and the two mixed membranes contained fibrovascular tissue on both sides of the retinal pigment epithelium. Fibrin and remains of outer segments tended to occur at the lateral edges of classic membranes and to cover the inner surface of occult membranes. CONCLUSION: Classic choroidal neovascularisation in age related macular degeneration is predominantly composed of subretinal fibrovascular tissue while occult choroidal neovascularisation is composed of fibrovascular tissue at the choroidal side of the retinal pigment epithelium.
AIMS: To examine the relation between measures of vision and ability to perform daily living tasks in those visually impaired with macular degeneration. METHODS: A visual functioning index (daily living tasks dependent on vision: DLTV) was used to evaluate patients' perception of their ability to perform vision dependent tasks. Distance visual acuity, near visual acuity, reading speed, and contrast sensitivity were measured in all patients. In addition, a new measure of reading ability was derived, designated the reading index. This takes into account both the size of the text read and the time to read it and is equivalent to the reading speed in words per minute divided by text size in M. RESULTS: The reading index was found to show best associations with the majority of items within the DLTV. Stepwise regression identified the combination of reading index and distance visual acuity as having the best associations with DLTV items. The present study also demonstrated that specific levels of vision as measured by acuity, reading index, and contrast sensitivity corresponded with different perceived amounts of difficulty in the performance of daily living tasks. CONCLUSIONS: This study showed that reading index is valuable in predicting the ability to perform daily living tasks and therefore may be useful in the visual assessment of the visually impaired individual. In addition, this study identified specific levels of vision at which individuals reported different degrees of difficulty in performing daily living tasks.
BACKGROUND: Orbital xanthogranuloma, a diagnosis confirmed histologically, occurs rarely in adults and children. With its characteristic macroscopic appearance the adult form may be associated with a spectrum of biochemical and haematological abnormalities including lymphoproliferative malignancies. METHOD: The clinicopathological features and imaging appearances on computed tomography and magnetic resonance imaging of this condition are described in eight adults and a child. RESULTS: Radiological evidence of proptosis was present in seven patients. In all nine patients an abnormal infiltrative soft tissue mass was seen, with increased fat in six cases. All patients had associated enlargement of extraocular muscles suggestive of infiltration and five had lacrimal gland involvement. Encasement of the optic nerve, bone destruction, and intracranial extension was present only in the child with juvenile xanthogranuloma. Haematological and/or biochemical abnormalities were detected in seven patients and seven patients had other systemic diseases which were considered to have an immune basis. One patient subsequently developed non-Hodgkin's lymphoma. CONCLUSION: The investigation and management of orbital xanthogranulomas requires a multidisciplinary approach even though the diagnosis may be suspected clinically. Imaging delineates the extent of disease and involvement of local structures and may influence the differential diagnosis. The juvenile form may be more locally aggressive, causing bone destruction with consequent intracranial extension.
AIMS: To establish current epidemiological data, risks, and interventional outcomes of newly diagnosed sympathetic ophthalmia (SO). METHODS: Prospective surveillance took place of all permanently employed ophthalmologists in the UK and Republic of Ireland by a monthly reporting card through the British Ophthalmological Surveillance Unit. Case ascertainment was made of newly diagnosed SO from July 1997 and questionnaire data were returned at baseline, 6 months, and 1 year after diagnosis. RESULTS: 23 patients with newly diagnosed SO were recruited over 15 months, corresponding to a minimum estimated incidence of 0.03/100 000. Baseline data were available on 18 patients, in whom SO occurred after surgery in 11 patients, after retinal surgery alone in six patients, and after accidental trauma in seven patients. 12 of the 16 patients with 1 year follow up had a visual acuity of 6/12 or better. Good visual outcome was related to prompt and adequate systemic immunosuppressive therapy. CONCLUSIONS: The incidence of sympathetic ophthalmia is very low. The main current risk is surgery, particularly retinal surgery, but visual prognosis is good if early diagnosis is made and rapid, adequate immunotherapy is commenced.
AIMS: To examine the changes in the retinal nerve fibre layer (NFL) thickness with age and myopia in normal population. METHODS: Retinal nerve fibre layer thickness was measured with a scanning laser polarimeter (NFA-I) in 180 normal subjects of varying age (range 7-83 years) and in 110 eyes of 85 patients with myopia of varying degrees (range -1.00 to -15.00D). They were all voluntary Anatolian people. Superior to nasal (S/N), inferior to nasal (I/N), and the superior to inferior (S/I) ratios were used for the assessment of retinal NFL thickness. RESULTS: The mean superior NFL ratio was 2.96 and the mean inferior NFL ratio was 2.93 in normal subjects. There was a gradual decrease in NFL ratio with increasing age (simple regression analysis, p<0.05). The mean S/I ratio was 1.01 with a large variation. In patients with myopia, the mean superior NFL ratio was 2.60 and the mean inferior NFL ratio was 2.72. Superior and inferior NFL retardations, and S/I ratio in myopic patients were significantly (15.5%, 10.8%, and 4.9% respectively) lower than that of age matched normals (t test, p<0.05). There was also a gradual decrease in NFL thickness with increasing degree of myopia (simple regression analysis, p<0.05). CONCLUSIONS: Nomograms we obtained for retinal NFL thickness may serve as reference points for the assessment of normal Anatolian people and myopic patients in future studies. NFL thicknesses gradually decreased with increasing age. Patients with myopia had significantly lower NFL thicknesses than normal subjects and, although weakened by wide age range of myopic group, there is a linear relation between severity of myopia and NFL thickness in myopic patients.
AIM: To evaluate the efficacy of topical 5-fluorouracil (5-FU) alone, without concurrent surgery or radiotherapy, for the treatment of conjunctival squamous cell carcinoma. METHODS: Eight patients affected by conjunctival squamous cell carcinoma (three recurrent cases, three incompletely excised, and two untreated cases) were treated with 1% 5-FU eye drops. Topical 1% 5-FU was administered four times daily for 4 weeks (one course). Clinical examination (biomicroscopy and photography) and morphological evaluation of conjunctival cytological specimens were used to monitor the efficacy of local chemotherapy, side effects, and recurrences. RESULTS: All patients showed clinical regression of conjunctival carcinoma after topical 1% 5-FU treatment. Neoplastic conjunctiva was completely replaced by normal epithelium within 3 months. Mean follow up was 27 months. One patient needed two courses of local chemotherapy for recurrent disease. An acute transient toxic keratoconjunctivitis was observed in all treated cases; it was easily controlled with topical therapy. No long term side effects were found. CONCLUSIONS: Topical 1% 5-FU is effective in the treatment of recurrent, incompletely excised, and selected untreated conjunctival squamous cell carcinomas. Topical 1% 5-FU has no major complications. This study suggests that topical conjunctival chemotherapy with 1% 5-FU may be useful, at least as adjunctive therapy, in the treatment of conjunctival squamous cell carcinoma.
AIM: To describe a surgical technique for autologous limbal stem cell transplantation and the outcome of a series of patients with unilateral stem cell deficiency. METHODS: A report of six consecutive patients who underwent autologous limbal stem cell transplantation is presented. The primary diagnosis included alkali burn (n=3), conjunctival intraepithelial neoplasia (CIN) (n=1), recurrent pterygium (n=1), and contact lens induced keratopathy (n=1). The autologous transplanted tissue consisted of peripheral cornea, limbus, and conjunctiva obtained from the contralateral eye. Three of the above patients underwent penetrating keratoplasty in association with auto-limbal transplantation. A significant modification to established techniques was the close monitoring of conjunctival epithelial migration in the immediate postoperative period. If conjunctival epithelium threatened to migrate on to the corneal surface, it was mechanically removed at the slit lamp and prevented from crossing the limbus. This was required in three patients. RESULTS: The mean follow up was 18.8 months. The outcome was satisfactory in all cases: a stable corneal surface was restored and there was a substantial improvement in vision and symptoms. One patient had a primary failure of the corneal allograft associated with glaucoma, and 6 months later developed a retinal detachment. No complications were noted in the donor eye with the exception of one patient who developed filamentary keratitis along the edge of the donor site. CONCLUSION: Autologous limbal transplantation with corneal, limbal, and conjunctival carriers was found to be useful for ocular surface reconstruction, over a mid-term follow up, in patients with unilateral stem cell deficiency. Close monitoring of the migration of conjunctival epithelium in the immediate postoperative period, and preventing it from crossing the limbus, ensured that the corneal surface was re-epithelialised exclusively from epithelial cells derived from the transplanted limbal tissue. This approach should improve the success of this procedure.
AIM: Severe mucus deficiency syndromes may require substitution of mucous membrane for re-establishment of the ocular surfaces. The long term results after autologous nasal mucosal transplantation were investigated. METHODS: 55 eyes of 50 patients with severe mucus deficiency syndromes were followed retrospectively after free autologous nasal mucosal transplantation-group A: patients after severe lye, acid, heat burns, or radiation (n=38 eyes), group B: patients with systemic mucosal disease (n=17 eyes). The results of routine clinical examination were recorded and patients were followed for a median of 37 months. 17 biopsies of transplanted nasal mucosa were studied by light microscopy and 22 patients by impression cytology before and at several intervals after mucosal transplantation. RESULTS: All nasal mucosal grafts healed well and no intraoperative complications occurred. During follow up 107 additional surgical procedures were performed including 16 lamellar and 21 penetrating keratoplasties. Subjective complaints improved in 44/47 patients with preoperative symptoms. Best corrected visual acuity at the end of follow up was increased in 23 eyes, 10 eyes (18. 2%) reached a final visual acuity equal to or greater than 20/200. Histopathologically, all (n=17) biopsies showed vital intraepithelial mucin producing goblet cells in the nasal mucosal graft (median 25 cells/field (400x magnification)). The mean density of goblet cells before transplantation was 48/mm(2) and after nasal mucosal grafting 432/mm(2) measured by impression cytology (p<0. 0001). CONCLUSIONS: Functional goblet cells persist in autologous nasal mucosa for up to 10 years after transplantation. In patients with severe mucus deficiency syndromes of different origin nasal mucosal transplantation can re-establish the ocular surface, substitute the mucus components of the tear film, improve symptoms of the patients, and facilitate a moderate increase in visual acuity.
BACKGROUND/AIMS: "Hurricane keratopathy" is the name given to the whorl pattern, highlighted with fluorescein, seen in situations where corneal epithelial cell turnover is exaggerated. Although the condition is well described, follow up data on patients with this condition and its sequelae have only been reported in corneal graft patients. The aim was to study the clinical course of hurricane keratopathy in corneal graft patients and contact lens wearers, and to document any sequelae of this condition. METHODS: Hurricane keratopathy, occurring in 20 eyes with corneal grafts and 16 eyes (six bilateral) wearing rigid gas permeable contact lenses, was studied and followed. The occurrence, pattern, progress, resolution, and residual effects of the whorls were noted. RESULTS: Hurricane keratopathy was noted to occur in grafts as previously reported and also in contact lens wearers, which has hitherto not been reported. The whorls usually appeared within the first 3 weeks postoperatively and persisted up to 4 months. A small epithelial defect (11.1%), heaped epithelial cells (5.6%), and a nebular grade opacity (2.8%), were the only significant sequelae noted at the epicentre of the whorls. Resolution occurred from the periphery towards the centre of the cornea. CONCLUSIONS: The whorl pattern is sustained as long as the stimulus for increased cell turnover is maintained. Once this stimulus is eliminated, the pattern tends to resolve spontaneously.
AIM: To describe the prevalence of and risk factors for pterygium in a population based sample of residents of the Australian state of Victoria who were aged 40 years and older. METHODS: The strata comprised nine randomly selected clusters from the Melbourne statistical division, 14 nursing homes randomly selected from the nursing homes within a 5 kilometre radius of the nine Melbourne clusters, and four randomly selected clusters from rural Victoria. Pterygium was measured in millimetres from the tip to the middle of the base. During an interview, people were queried about previous ocular surgery, including surgical removal of pterygium, and their lifetime exposure to sunlight. RESULTS: 5147 people participated. They ranged in age from 40 to 101 years and 2850 (55.4%) were female. Only one person in the Melbourne cohort reported previous pterygium surgery, and seven rural residents reported previous surgery; this information was unavailable for the nursing home residents. Pterygium was present upon clinical examination in 39 (1.2%) of the 3229 Melbourne residents who had the clinical examination, six (1. 7%) of the nursing home residents, and 96 (6.7%) of the rural residents. The overall weighted population rate in the population was 2.83% (95% CL 2.35, 3.31). The independent risk factors for pterygium were found to be age (OR=1.23, 95% CL=1.06, 1.44), male sex (OR=2.02, 95% CL=1.35, 3.03), rural residence (OR=5.28, 95% CL=3. 56, 7.84), and lifetime ocular sun exposure (OR=1.63, 95% CL=1.18, 2. 25). The attributable risk of sunlight and pterygium was 43.6% (95% CL=42.7, 44.6). The result was the same when ocular UV-B exposure was substituted in the model for broad band sun exposure. CONCLUSION: Pterygium is a significant public health problem in rural areas, primarily as a result of ocular sun exposure.
AIMS: To study the effects of topical nipradilol, a non-selective beta blocker with alpha blocking and nitroglycerin-like activities, on intraocular pressure (IOP) and aqueous humour dynamics in normal humans and in patients with ocular hypertension. METHODS: Nipradilol (0.06%, 0.125%, 0.25%, 0.5%) was applied to normal volunteers (n = 12) to test for IOP lowering effects. In a second group of normal volunteers (n = 11), nipradilol (0.125% and 0.25%) and timolol (0. 5%) were compared for IOP lowering effects. After a single administration of 0.25% nipradilol, IOP, flare intensity in the anterior chamber, aqueous flow, uveoscleral outflow, tonographic outflow facility, and episcleral venous pressure were either directly measured or mathematically calculated. Topical nipradilol (0.25%) was administered to 24 patients with ocular hypertension twice daily for 8 weeks. RESULTS: Administration of 0.25% nipradilol decreased IOP with a maximum reduction of 4.2 mm Hg lasting 12 hours. A single instillation of both 0.25% nipradilol and 0.5% timolol reduced the IOP in normotensive human subjects to the same degree. A single instillation of 0.25% nipradilol decreased the aqueous flow rate in the treated eye by 20%. Nipradilol produced no significant effect in tonographic outflow facility or episcleral venous pressure, but uveoscleral outflow was increased. In patients with ocular hypertension, twice daily instillation of 0.25% nipradilol decreased IOP without tachyphylaxis for the 8 week test period. CONCLUSION: Topical nipradilol (0.25%) reduced IOP by decreasing the aqueous flow rate and probably also by increasing uveoscleral outflow. Nipradilol should be further investigated as a new antiglaucoma drug.
AIMS: To investigate the intraocular penetration of vancomycin eye drops and to compare the conventional method of drop instillation to the lower cul de sac with applying drops to the medial canthus with closed lids. METHODS: This prospective randomised trial evaluated 53 eyes of 53 patients who had undergone extracapsular cataract extraction (ECCE) with intraocular lens implantation. Vancomycin (50 mg/ml) eye drops were applied to either the lower cul de sac with open lids (conventional method), or to the medial canthus with the patient in a supine position and with closed lids. After paracentesis performed during ECCE, an aqueous humour sample was taken and vancomycin concentration was measured using the TDX vancomycin assay (fluorescence polarisation immunoassay). RESULTS: Vancomycin concentration in the anterior chamber were above the minimal inhibitory concentration for Gram positive bacteria in the two methods of drop instillation examined (2.04 (SD 1.9) microg/ml and 1.49 (1.1) microg/ml in the open and closed methods, respectively (p =0.202)). CONCLUSIONS: Vancomycin (50 mg/ml) reaches therapeutic concentration in the anterior chamber after topical drop application. Comparable concentrations were reached when drops were applied in either the lower cul de sac or to the medial canthus with closed lids. The latter method is proposed as likely to improve patient compliance.
AIMS: To determine whether parasol retinal ganglion cells (magnocellular pathway) are selectively lost in the primate model of glaucoma. METHODS: Ocular hypertension was induced in one eye of six Macaca fascicularis monkeys for 6-14 weeks. The retinal ganglion cells in these eyes were labelled retrogradely with the tracer horseradish peroxidase (HRP) implanted into the optic nerve and subsequently examined in retinal whole mount preparations. The degree of retinal ganglion cell loss was estimated from Nissl stained tissue by comparison with the contralateral untreated control eye. RESULTS: In the three glaucomatous retinas with the best labelling 1282 cells could be classified, of which 182 were parasol cells and 1100 were midget cells. Linear regression analysis did not demonstrate a significant reduction in the proportion of parasol to midget cells with increasing cell loss (regression slope 0.023, 95% CI -0.7 to 0.11). Compared with the control eye the cell soma of the remaining retinal ganglion cells in glaucomatous eyes were reduced in size by 20% for parasol cells (p=0.003) and by 16% for midget cells (p <0.001). CONCLUSION: The results of this study do not support the hypothesis that selective loss of parasol retinal ganglion cells occurs in experimental glaucoma. In addition, the change in cell soma size distributions following ocular hypertension suggests that both parasol and midget retinal ganglion cells undergo shrinkage before cell death.
AIMS: To investigate age related alterations in the non-collagenous components of the human lamina cribrosa. METHODS: Fibronectin, elastin, and glial fibrillary acidic protein (GFAP) staining were assessed in young and old laminae cribrosae. An age range (7 days to 96 years) of human laminae cribrosae were analysed for lipid content (n=9), cellularity (n=28), total sulphated glycosaminoglycans (n=28), elastin content (n=9), and water content (n=56), using chloroform-methanol extraction, fluorimetry, the dimethylmethylene blue assay, and ion exchange chromatography, respectively. RESULTS: Qualitatively, an increase in elastin and a decrease in fibronectin and GFAP were demonstrated when young tissue was compared with the elderly. Biochemical analysis of the ageing human lamina cribrosa demonstrated that elastin content increased from 8% to 28% dry tissue weight, total sulphated glycosaminoglycans decreased, and lipid content decreased from 45% to 25%. There were no significant changes in total cellularity or water content. CONCLUSION: These alterations in composition may be indicative of the metabolic state of the lamina cribrosa as it ages, and may contribute to changes in mechanical integrity. Such changes may be implicated in the susceptibility of the elderly lamina cribrosa and also its response to glaucomatous optic neuropathy.
AIMS: To investigate changes in the mechanical compliance of ex vivo human lamina cribrosa with age. METHODS: A laser scanning confocal microscope was used to image the surface of the fluorescently labelled lamina cribrosa in cadaver eyes. A method was developed to determine changes in the volume and strain of the lamina cribrosa created by increases in pressure. The ability of the lamina cribrosa to reverse its deformation on removal of pressure was also measured. RESULTS: Volume and strain measurements both demonstrated that the lamina cribrosa increased in stiffness with age and the level of pressure applied. The ability of the lamina cribrosa to regain its original shape and size on removal of pressure appeared to decrease with age, demonstrating an age related decrease in resilience of the lamina cribrosa. CONCLUSIONS: The mechanical compliance of the human lamina cribrosa decreased with age. Misalignment of compliant cribriform plates in a young eye may exert a lesser stress on nerve axons, than that exerted by the rigid plates of an elderly lamina cribrosa. The resilience of the lamina cribrosa also decreased with age, suggesting an increased susceptibility to plastic flow and permanent deformation. Such changes may be of importance in the explanation of age related optic neuropathy in primary open angle glaucoma.
AIM: To explore why emmetropisation fails in children who have strabismus. METHODS: 289 hypermetropic infants were randomly allocated spectacles and followed. Changes in spherical hypermetropia were compared in those who had strabismus and those who did not. The effect of wearing glasses on these changes was assessed using t tests and regression analysis. RESULTS: Mean spherical hypermetropia decreased in both eyes of "normal" children (p<0.001). The consistent wearing of glasses impeded this process in both eyes (p<0.007). In the children with strabismus, there were no significant changes in either eye, irrespective of treatment (p>0. 05). CONCLUSIONS: In contrast with normal infants, neither eye of those who had strabismus emmetropised, irrespective of whether the incoming vision was clear or blurred. It is suggested that these eyes did not "recognise" the signal of blurred vision, and that they remained long sighted because they were destined to squint. Hence, the children did not squint because they were long sighted, and glasses did not prevent them squinting.
AIMS: To report the clinical findings and visual outcome of patients with extended wear contact lens (EWCL) related bacterial keratitis. METHODS: 11 cases with EWCL related bacterial keratitis were included. Corneal scrapings were obtained for cytology and cultures. RESULTS: Nine patients had unilateral bacterial keratitis and two patients showed bilateral involvement. Corneal scrapings revealed Pseudomonas aeruginosa in seven patients, Staphylococcus aureus coagulase positive in one patient, and Staphylococcus epidermidis in three patients. CONCLUSION: EWCLs may be associated with bacterial keratitis and may result in visual loss. Dispensing contact lenses by optometrists should be performed in consultation with ophthalmologists.
AIM: To examine the results of open lacrimal surgery in patients with Wegener's granulomatosis. METHODS: A retrospective review of patients with Wegener's granulomatosis who underwent lacrimal surgery over a 17 year period. RESULTS: 11 patients were identified and a total of 14 primary dacryocystorhinostomies (DCR) and one revisional DCR were performed; symptomatic relief was achieved in 13/14 operations and one patient required revisional surgery for persistent symptoms. There were no intraoperative and few postoperative complications. CONCLUSIONS: In contrast with some previous reports, open DCR appears to be a safe procedure and it is recommended as a treatment for lacrimal obstruction in patients with Wegener's granulomatosis, but an increase of perioperative immunosuppression is recommended in certain cases.
Replicating adenoviral vectors are a promising new modality for cancer treatment and clinical trials with such vectors are ongoing. Targeting these vectors to cancer cells has been the focus of research. However, even if perfect targeting were to be achieved, a vector still must effectively kill cancer cells and spread throughout the bulk of the tumor. The adenoviral E1b-19kD protein is a potent inhibitor of apoptosis and may therefore compromise the therapeutic efficacy of an adenoviral vector. In this study we have investigated if an E1b-19kD gene deletion could improve the ability of a replicating adenoviral vector to spread through and kill cancer cells. In several lung cancer cell lines an E1b-19kD-deleted virus (Ad337) induced substantially more apoptosis than did a wild-type virus (Ad309), and tumor cell survival was significantly reduced in three of four cell lines. In addition, the apoptotic effects of cisplatin or paclitaxel were augmented by Ad337, but inhibited by wild-type virus. The number of infectious virus particles in the supernatant of infected cells was increased with Ad337 compared with wild-type virus, indicating enhanced early viral release. Ad337, in contrast to Ad309, induced significantly larger plaques after infection of A549 cells. This well-described large plaque phenotype of an E1b-19kD mutant virus is likely the result of early viral release and enhanced cell-to-cell viral spread. Loss of E1b-19kD function caused only minor cell line-specific increase or decrease in viral yield. We conclude that deletion of the E1b-19kD gene may enhance the tumoricidal effects of a replicating adenoviral vector.
We have addressed the possibility that intracellularly expressed miniantibodies directed against the viral capsid protein can be used as antiretroviral agents in gene transfer experiments. R187 is a rat monoclonal antibody that has been reported to recognize the MuLV p30gag capsid polypeptide. We report here that it also binds to the Pr65gag precursor polyprotein. R187 has been cloned and expressed in the form of a single-chain variable fragment (scFv) that shows the same binding specificity as the parental antibody. When expressed intracellularly, the R187 scFv favors the production of viral particles showing reduced infectivity. It, however, exerts no detectable protective effect against infection. This was observed both when using replication-incompetent MuLV-derived vector and replication-competent wild-type MuLV. Although the intimate mechanism of the inhibition is not clear, this work raises the possibility that gene engineering of anti-capsid protein scFvs may offer an additional lead for gene therapy of severe retrovirus-linked diseases.
Glucose responsiveness in the millimolar concentration range is a crucial requirement of a surrogate pancreatic beta cell for insulin replacement therapy of insulin-dependent diabetes. Novel insulin-secreting GK cell clones with millimolar glucose responsiveness were generated from an early-passage glucose-unresponsive RINm5F cell line. This line expressed constitutively both the K(ATP) channel and the GLUT2 glucose transporter; but it had a relative lack of glucokinase. Through overexpression of glucokinase, however, it was possible to generate glucose-responsive clones with a glucokinase-to-hexokinase ratio comparable to that of a normal pancreatic beta cell. This aim, on the other hand, was not achieved through overexpression of the GLUT2 glucose transporter. Raising the expression level of this glucose transporter into the range of rat liver, without correcting the glucokinase-to-hexokinase enzyme ratio, did not render the cells glucose responsive. These glucokinase-overexpressing RINm5F cells also stably maintained their molecular and insulin secretory characteristics in vivo. After implantation into streptozotocin diabetic immunodeficient rats, glucokinase-overexpressing cells retained their insulin responsiveness to physiological glucose stimulation under in vivo conditions. These cells represent a notable step toward the future bioengineering of a surrogate beta cell for insulin replacement therapy in insulin-dependent diabetes mellitus.
Liver toxicity and inflammation were assessed in C57BL/6, CBA, and BALB/c mice injected intravenously with a series of recombinant adenoviruses deleted simultaneously in E1/E3, in E1/E3/E2A, or in E1/E3/E4. All vectors were either devoid of transgenes or carried in E1 the human CFTR cDNA under the control of the CMV promoter. Injection of the E1/E3-deleted vector induced a significant liver dystrophy and inflammatory responses that were accompanied by an increased serum transaminase concentration. The vector toxicity remained elevated on additional deletion of the E2A gene and was further enhanced when hCFTR was expressed. In contrast, additional deletion of E4 led to a reduction in hepatotoxicity, suggesting an active role of E4 gene products in liver injury. However, deletion of E4 also led to a loss of transgene expression. To identify the individual E4 product(s) involved in liver toxicity and in the regulation of transgene expression, a series of isogenic E1/E3-deleted vectors, with or without the hCFTR transgene, and containing various combinations of functional E4 open reading frames (ORFs), were evaluated in vitro and in vivo. We demonstrate that liver injury was markedly reduced with vectors containing either ORF3 alone or ORF3,4 while vectors containing ORF4, ORF6,7 or ORF3,6,7 still displayed elevated hepatotoxicity and inflammatory responses. Moreover, transgene expression was restored when ORF3,4 or ORF3,6,7 was retained in the vector. These results highlight the importance of the E4 gene products in the design of improved in vivo gene transfer vectors.
It has been demonstrated that gene transfer by in vivo electroporation of mouse muscle increases the level of gene expression by more than 100-fold over simple plasmid DNA injection. We tested continuous rat erythropoietin (Epo) delivery by this method in normal rats, using plasmid DNA expressing rat Epo (pCAGGS-Epo) as the vector. A pair of electrodes was inserted into the thigh muscles of rat hind limbs and 100 microg of pCAGGS-Epo was injected between the electrodes. Eight 100-V, 50-msec electric pulses were delivered through the electrodes. Each rat was injected with a total of 400 microg of pCAGGS-Epo, which was delivered to the medial and lateral sides of each thigh. The presence of vector-derived Epo mRNA at the DNA injection site was confirmed by RT-PCR. The serum Epo levels peaked at 122.2 +/- 33.0 mU/ml on day 7 and gradually decreased to 35.9 +/- 18.2 mU/ml on day 32. The hematocrit levels increased continuously, from the preinjection level of 49.5 +/- 1.1 to 67.8 +/- 2.2% on day 32 (p < 0.001). In pCAGGS-Epo treated rats, endogenous Epo secretion was downregulated on day 32. In a control experiment, intramuscular injection of pCAGGS-Epo without subsequent electroporation did not significantly enhance the serum Epo levels. These results demonstrate that muscle-targeted pCAGGS-Epo transfer by in vivo electroporation is a useful procedure for the continuous delivery of Epo.
In contrast to oncoviruses, lentiviruses do not require target cell division for integration into the host genome. Lentiviral vectors can therefore expand the spectrum of target cells susceptible to retroviral gene transfer. To analyze whether vectors based on simian immunodeficiency viruses (SIVs) could be used for gene transfer, a three-plasmid vector-packaging system was developed, in which Gag-Pol and the vector itself are of SIV origin, while Env is derived either from SIV, amphotropic murine leukemia virus (MuLV), or the G glycoprotein of vesicular stomatitis virus (VSV-G). To increase the safety of the SIV vector system, a self-inactivating SIV vector was constructed. After optimization of the SIV gag-pol expression plasmid, a minimal SIV vector, which contained only SIV sequences present on the multiply spliced nef transcript, could still be produced at titers of 2 x 10(5) infectious units/ml. Growth-arrested cells could be transduced with this vector even if vif, vpr, vpx, and nef had been deleted from the packaging construct and the vector.
Subretinal delivery of recombinant adeno-associated virus (rAAV) results in a systemic humoral response in the adult immunocompetent mouse. We characterized this response and determined whether it is possible to readminister rAAV to the subretinal space despite the presence of antibodies to the vector. A systemic humoral response to rAAV capsid proteins was induced by either unilateral subretinal injection or by intradermal administration of 1 x 10(9) infectious units of rAAV carrying the cDNA encoding green fluorescent protein (GFP), rAAV-GFP. Experiments were performed in cohorts of adult C57BL/6 mice. Assessment of systemic humoral response to viral capsid protein was performed through enzyme-linked immunoabsorbent assay (ELISA) and infection inhibition studies of serum samples 3 weeks after virus delivery. The rAAV-GFP virus was readministered by subretinal injection. GFP expression after subretinal administration was evaluated ophthalmoscopically throughout the course of the experiment and histologically at the termination of the experiment. We observed significant systemic humoral responses to viral capsid protein after subretinal delivery of rAAV. Intradermal injection resulted in a larger humoral response (with a higher percentage of neutralizing antibodies) than subretinal injection. Additional transduction events were observed after readministration of rAAV despite the presence of strong humoral response to the vector.
Immunologically sensitized recipients present one of the most critical problems in clinical organ transplantation today, since preformed antibodies rapidly destroy donor tissue expressing specific MHC class I antigens (Ag). Therefore, sensitized patients are either unable to receive a compatible organ, or experience a prolonged waiting period. In this study we examined the effectiveness of donor MHC class I gene therapy in preventing hyperacute rejection (HR) of rat heart allografts in passively sensitized recipients. Our gene therapy strategy to address this problem is based on the phenomenon that liver transplants, which resist antibody-mediated HR, produce soluble MHC class I Ag capable of neutralizing preformed antibodies and suppressing the immune response. To mimic this "liver effect," we used liposomes to transfect cultured recipient (Lewis-RT1.Al) hepatocytes with plasmid DNA encoding the soluble donor MHC class I Ag, RT1.Aa. Control or RT1.Aa-transfected hepatocytes were implanted intrasplenically into Lewis recipients 1 day prior to heterotopic ACI (RT1.Aa) heart transplantation and injection of 6 ml of anti-ACI hyperimmune serum (HIS). Results showed that nearly all recipients receiving ACI-specific HIS and control hepatocytes experienced HR, while none of the recipients receiving HIS and hepatocytes expressing soluble RT1.Aa developed HR. Furthermore, active immunosuppression by soluble RT1.Aa was evidenced by prolongation of allograft survival, compared with controls not receiving HIS. In summary, soluble donor-MHC class I Ag gene therapy can prevent antibody-mediated destruction associated with HR. Future development of a similar strategy in humans may significantly improve the results of clinical organ transplantation in immunologically sensitized recipients.
We have attempted to develop a system for specific enhancement of transgene expression, which has been one of the most important issues in human gene therapy. When an Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA-1) expression vector, pCMV-trEBNA-1, was cotransfected with an origin of latent viral DNA replication (oriP)-harboring plasmid, poriP-CMV-luciferase, luciferase gene expression was up to 20 times greater than in the absence of EBNA-1. This enhancement was regulated mainly at the transcriptional level and was dependent on the oriP sequence and the amount of EBNA-1. However, cointroduction of poriP-CMV-luciferase with purified recombinant EBNA-1 inhibited luciferase gene expression whereas no inhibition was observed when pCMV-luciferase was cointroduced with recombinant EBNA-1. We also introduced poriP-CMV-luciferase into mouse liver via the use of HVJ (hemagglutinating virus of Japan)-liposomes. By 10 days after transfer, luciferase gene expression was decreased to low levels. We then introduced pCMV-trEBNA-1 to mouse liver via HVJ-liposomes on day 10. Luciferase gene expression was reactivated, whereas no reactivation was detected by the injection of EBNA-1 expression plasmid into liver injected with pCMV-luciferase lacking the oriP sequence. Thus, cotransfection of oriP-harboring expression vector with EBNA-1 expression plasmid should be promising for human gene therapy, although the safety of the system must be investigated thoroughly.
Adenoviral vectors are known to transduce hepatocytes in normal liver tissue with high efficiency. The aim of this study was to investigate whether sinusoidal endothelial cells, which separate hepatocytes from the bloodstream in the sinusoidal lumen, are permissive for infection by adenoviruses. We show here that microvascular liver sinusoidal endothelial cells are not infected by adenovirus type 5 in vivo or in vitro unless high MOIs are used. In contrast, macrovascular endothelial cells from aorta are efficiently infected by adenovirus type 5. In addition, Kupffer cells, similar to sinusoidal endothelial cells, are not infected by adenovirus type 5. Liver sinusoidal endothelial cells do not express the integrin receptor alpha(v)beta3, which is required for efficient infection by adenoviruses. Our results demonstrate that hepatocytes are the main cell population of the liver that is infected by adenovirus type 5.
Recent developments in the design and bioanalytical applications of polyion-sensitive electrodes (PSEs) are reviewed. The general electrochemical principles governing the potentiometric response of such polymer membrane-based devices are summarized and new directions for the use of these novel sensors are detailed. These new directions include basic fundamental studies aimed at determining the thermodynamics of polyion extraction into ion exchanger-doped polymeric membranes, new methods to quantitate the anticoagulant drug heparin in whole blood via titrations with polycationic protamine, selective assays of protease activities (and inhibitors of such activities) using natural and synthetic polyionic peptides as substrates, and novel homogeneous immunoassay schemes based on potentiometric polyion detection.
This work presents an amperometric biosensor incorporated into a flow configuration comprising salicylate hydroxylase that catalyses the irreversible hydroxylation of salicylate to catechol in the presence of NADH and molecular oxygen, and tyrosinase that further oxidises catechol giving o-quinone which is electrochemically reduced at -100 mV vs. Ag/AgCl yielding catechol and entering the catalytic oxidation and electrochemical reduction cycling which results in signal amplification and, consequently, low limits of detection. Additional incorporation of glucose dehydrogenase in the enzymatic sequence results in regeneration of NADH provided that glucose is present in the carrier stream and incorporation of a dialysis membrane provides operational stability to the biosensor. The analytical characteristics of this catalytic and electrochemical transduction sequence in a FIA system are: a limit of detection of 3.5 10(-6) M (S/N = 3), a sensitivity of 22.6 nA microM(-1) cm(-2), no loss of response at least after 5 h of continuous operation, and a sample frequency of 15 h(-1). Monitoring of salicylate after ingestion of 500 mg of acetylsalicylic acid has been followed in non-pretreated urine samples and the amount of salicylate in several drugs has been also successfully quantified.
Using the voltammetric method of square-wave voltammetry, a direct electrochemical examination was made of the wild type and Tyr67Phe mutant of both rat cytochrome c and yeast iso-1-cytochrome c. In addition to determining the equilibrium reduction potential (E0') for each cytochrome, the entropy of reaction, deltaS0'(Rxn)(deltaS0'(Rxn) = S0'(Red) - S0'(Ox)), for the reduction process was determined via the non-isothermal method. Having determined deltaS0'(Rxn) and E0', deltaH0' was calculated. For rat cytochrome c, it was found that deltaS0'(Rxn) = -43 J mol(-1) K(-1) for the wild type and -53 J mol(-1) K(-1) for the Tyr67Phe variant, with the deltaH0' for both the wild type and variant nearly identical, indicating that the changes in reduction potential and probably stability are due to changes in deltaS0'(Rxn). In contrast the measured deltaS0'(Rxn) for yeast iso-1-cytochrome c demonstrated significant changes in both entropic and enthalpic contributions in going from wild type to mutant cytochrome c. The entropy of reaction provides information regarding the relative degree of solvation, and very likely the degree of compactness, of the oxidized state versus the reduced state of the redox protein. A thermodynamic scheme and stability derivation are presented that show how the entropies of reaction of wild type versus variant cytochromes contribute to and predict changes in stability in going from oxidized to reduced protein. For yeast iso-1-cytochrome c, the thermodynamically predicted change in stability was very close to the experimentally observed value, based on previous differential scanning calorimetric stability measurements. While such data is not available for rat cytochrome c, consideration of the enormously increased local stability of the rat oxidized cytochrome c variant predicts that the reduced rat variant will be even more stable than the already stabilized oxidized variant.
The structural changes in cytochrome c with temperature have been been followed using a recently developed electrically-heated microelectrode sensor. Differential pulse voltammetry was used to perform electrochemical measurements of cytochrome c oxidation at different temperatures at heated bare gold electrodes contained in phosphate-buffered cytochrome c solution at room temperature. The voltammetric response shows the onset of unfolding and a marked dependence of the signal on electrode temperature. This augurs well for applications of heated electrodes as local probes in the study of the temperature dependence of electron transfer processes of other redox proteins, avoiding problems of bulk deterioration.
The endothelium plays a critical role in maintaining vascular tone by releasing vasoconstrictor and vasodilator substances. Endothelium-derived nitric oxide (NO) is a vasodilator rapidly inactivated by superoxide and by Fe(II) and Fe(III), all found in significant quantities in biological systems. Thus due to the short life of NO in tissue (t1/2 = 3-6 s), in situ quantification of NO is a challenging problem. We designed the present study to perform direct measurements of nitric oxide using the electrochemical porphyrinic sensor. The most significant advantages of this sensor is small size (0.5-8 microm), rapid response time (0.1-1 ms), and low detection limit (10(-9) mol l(-1)). The porphyrinic sensor was used for in vitro and in vivo measurements of NO in an isolated single cell or tissue. Effects of hypertension, endotoxemia, and ischemia/reperfusion on the release of NO and/or its interaction with superoxide (O2-) were delineated. In the single endothelial cell (rabbit endocardium), NO concentration was highest at the cell membrane (950 +/- 50 nmol l(-1)), decreasing exponentially with distance from cell, and becoming undetectable at distances beyond 50 microm. The endothelium of spontaneously hypertensive rats (SHR) released 35% less NO (580 +/- 30 nmol l(-1)) than that of normotensive rats (920 +/- 50 nmol l(-1)), due to the higher production of O2- in SHR rats. Endothelial NO synthase (eNOS) generated NO (140 +/- 20 nmol l(-1)) in lung during the acute phase (first 10-15 min) of endotoxemia, followed by production of NO by inducible NOS. High production of O2- was observed during the entire period of endotoxemia. Ischemia (lower limb of rabbit) caused a significant increase of NO peaking at 15 min and decreasing thereafter, also due to O2- production.
A competitive heterogeneous capillary enzyme immunoassay with electrochemical detection has been developed for phenobarbital in serum. The oxidized primary antibody was attached covalently to the modified interior surface of a microcapillary (22 microl). The competition between analyte phenobarbital and alkaline phosphatase labeled phenobarbital for a limited number of antibody binding sites was complete in 1.5 h. The enzymatic product (p-aminophenol) from the catalytic conversion of the substrate (p-aminophenyl phosphate) was detected by amperometric flow injection analysis. The calibration curve for phenobarbital had a detection limit of 30 microg l(-1) (2.8 pmoles or 0.65 ng) and a range of 30-3000 microg l(-1). The assay could be used to determine the phenobarbital serum concentration in a 4 microl clinical serum sample without pretreatment.
The identified dopamine cell of Planorbis corneus is described as a model system to study neurotransmitter storage and dynamics. Techniques developed with this model system include capillary electrophoresis with electrochemical detection and microelectrochemistry at single cells. These techniques provide a powerful combination to examine single cell neurochemistry. Whole cell and cytoplasmic dopamine concentrations have been quantified with capillary electrophoresis. Additionally, this technique has been used to profile amino acids and to quantify two compartments of neurotransmitter in a single cell. Individual exocytosis events have been monitored at the cell body of the dopamine cell of P. corneus with microelectrodes. In this case, two different types of vesicles have been identified based on the amount of transmitter released. The psychostimulant, amphetamine, has been shown to selectively affect the amount of dopamine in these vesicles with lower to higher doses affecting the larger to smaller vesicle types, respectively. Microelectrochemistry at single nerve cells has also been used to demonstrate reverse transport of dopamine across the cell membrane and to suggest a role of this process in the molecular mechanism of amphetamine.
A multianalyte immunoassay concept based on the geometric separation of different analyte-specific antibodies has been demonstrated. The assay and amperometric detection are done in a cell with two working electrodes controlled at the same potential, and the amperometric signal at each electrode is monitored. The distance between any two adjacent electrodes in this prototype is 2.5 mm, and during the course of amperometric measurement, the product formed at one electrode does not reach the other working electrode within 20 min after the addition of enzyme substrate. Thus, the method relies on the spatial resolution between the different antibodies being such that measurements are taken before cross-interference due to diffusion can occur. Identical enzyme labels (alkaline phosphatase, ALP) and substrates (p-aminophenyl phosphate, PAPP) are used for all analytes. Alkaline phosphatase-conjugated rat anti-mouse IgG was immobilized by passive adsorption. Our studies showed that this concept is feasible and can be applied to the simultaneous measurement of multiple analytes.
The growing importance of polymer membrane-based potentiometric polyion sensors in biomedical research and clinical measurements has brought up the question of how accurate and reproducible these sensors are. Indeed, recent research has revealed that these sensors behave quite differently than classical ion-selective electrodes. This paper explores ways to improve measurement reproducibility and long term potential stability by considering the unique pseudo steady-state response mechanism of the polyion sensors developed so far. Heparin may be stripped out of the phase boundary membrane surface with a high sample NaCl concentration and this characteristic is used to modify the calibration procedure in order to avoid memory effects. It is also attempted to reduce long term potential drifts by continuously stripping heparin out of the membrane at the membrane-inner filling solution side.
A functionalized sol-gel thin film modified glassy carbon electrode has been prepared for the determination of mercury. The tetrasulfide in the sol-gel matrix has a high affinity for mercury species. The chemically integrated functional groups in the sol-gel precursor ensure a high chemical and mechanical stability of the modified electrodes, and therefore a stable and reproducible analytical performance. By medium exchange anodic stripping voltammetry, this modified electrode allows reliable, low cost and interference-free determination of mercury.
An electrochemical method for quantifying beta-lactam antibiotics (cephalexin and ampicillin) and their hydrolysis products is described. Cyclic voltammetry at the water/nitrobenzene interface in a four-electrode system was used. The zwitterionic compounds were ionized to the necessary electrochemical form by pH adjustment. The pH change, however, resulted also in hydrolysis of the antibiotics. Hydrolysis products were characterized across UV-vis spectrum. The various hydrolysis products as well as the ionized antibiotics were studied in voltammetric transfer from water to nitrobenzene using the method of the interface between two immiscible electrolyte solutions (ITIES). It was concluded that this electrochemical method is suitable for the quantification of beta-lactam antibiotics and their hydrolysis products.
We have studied the catalytic two-electron reduction of 3-chloro-2,4-pentanedione by cobalt(I) salen electrogenerated at a glassy carbon cathode in acetonitrile containing tetramethylammonium tetrafluoroborate. When cobalt(I) salen is electrogenerated at -0.65 V (a potential that is 30 mV more negative than the peak potential for the reversible one-electron reduction of cobalt(II) salen), the carbon-chlorine bond of 3-chloro-2,4-pentanedione is catalytically cleaved to form 2,4-pentanedion-3-ate; this anion can be protonated either by adventitious water or by a deliberately added proton donor to produce 2,4-pentanedione, or the anion can be trapped with iodoethane to give 3-ethyl-2,4-pentanedione. However, when cobalt(I) salen is electrogenerated at -0.40 V (a potential at which the rate of generation of cobalt(I) salen is relatively small), the 2,4-pentanedion-3-ate, resulting from the catalytic two-electron cleavage described above, can deprotonate unreduced starting material to form 3-chloro-2,4-pentanedion-3-ate and 2,4-pentanedione. In further work, we have found that 2,4-pentanedion-3-ate can be oxidized directly to form the corresponding radical which couples to yield 3,4-diacetyl-2,5-hexanedione. Chemically produced 2,4-pentanedion-3-ate reacts with electrogenerated cobalt(III) salen to give a dionylcobalt(III) salen species which undergoes a one-electron reduction to liberate cobalt(II) salen and the dionate. In addition, cobalt(II) salen reacts with molecular oxygen to give cobalt(III) salen and superoxide, and the latter reduces 3-chloro-2,4-pentanedione to form chloride ion, the 2,4-pentanedion-3-yl radical, and molecular oxygen.
A new method for evaluating chemical selectivity of agonists for the NMDA subtype of glutamate receptor (GluR) channels is described. The method is based on the magnitude of Ca2+ release from GluR-incorporated liposomes, which is measured by a Ca2+ ion-selective electrode with a thin-layer mode. The partially purified GluRs from rat whole brain were reconstituted into Ca2+-loaded liposomes. Small aliquots (each 50 microl) of the proteoliposomes, in the presence of an antagonist DNQX for blocking non-NMDA subtype, were subjected to potentiometric measurements of Ca2+ release under stimulation by three kinds of agonists, i.e. NMDA, L-glutamate and L-CCG-IV. The amount of the Ca2+ ion flux through the GluR channel induced by the agonists was found to increase in the order of NMDA < L-glutamate < L-CCG-IV, which was consistent with that of binding affinity of the agonists toward the NMDA subtype. However, the range of selectivity of the relevant agonists was much smaller compared with results based on binding affinities. The present method provides physiologically more relevant values for the agonist selectivity of GluRs as compared to that of the conventional binding assay in the sense that the selectivity is based on the very magnitude of Ca2+ flux through the NMDA receptor, i.e. the extent of signal transduction by a given agonist. The evaluation of agonist selectivity based on Na+ release was also investigated by using a Na+ ion-selective electrode, but agonist-induced Na+ release was not detected, because of low permeability of Na+ through the NMDA subtype.
The performance of active graphite and carbon fiber surfaces produced by different mechanical/electrochemical methods of surface activation has been investigated in the amperometric determinations of xanthine and hypoxanthine under physiologically relevant conditions. The electrodes showed better limits of detection (LOD) when used with differential techniques with a capability of discriminating the analytical signal from the background. Square wave voltammetry and cyclic voltammetry showed the most sensitive response. Electrochemically activated carbon fiber ultramicroelectrodes showed the highest sensitivity (58 A M(-1) cm(-2)) and the LOD in the 200 nM range was observed at the rough pyrolytic graphite electrodes by square wave voltammetry. The results demonstrate the feasibility of the development of new electroanalytical methods for the determination of oxypurines in biological samples.
Capillary electrophoresis (CE)/electrochemical detection (EC) for the simultaneous detection of hydrazine, methylhydrazine, and isoniazid has been developed with a 4-pyridyl hydroquinone self-assembled microdisk platinum electrode. Such an electrode has very high catalytic ability for hydrazines and they could be detected even at 0.0 V. The responses for hydrazine, methylhydrazine, and isoniazid are linear over 3 orders of detected concentration and of magnitude of 0.2-400 microM, 0.2-400 microM, 0.5 microM-2 mM, with correlation coefficients of 0.9998, 0.9991, and 0.9982, respectively. And they could be detected to levels of 0.1, 0.1 and 0.2 microM, respectively. This modified electrode was found to be very stable and reproducible when continuously used as detector for capillary electrophoresis for period of at least 4 weeks with no apparent loss of response.
Pulsed electrochemical detection (PED) following reversed-phase chromatography has been applied to the direct detection of sulfur-containing antibiotics, specifically, penicillins, cephalosporins, and lincomycin. The compounds are detected sensitively and selectively without the need for derivatization. Integrated pulsed amperometric detection (IPAD) yields limits of detection lower than UV detection for these compounds. Detection limits using an optimized IPAD waveform are typically 10 ppb or less. The high selectivity of PED for thiocompounds reduces sample preparation. This work is applied to the determination of penicillin and related analogues in various pharmaceutical formulations/preparations, including a chicken feed.
Two simple modifications to a commercially available thin layer electrochemical detector cell permitted the attainment of ultra-low detection levels of two neurotransmitter catecholamines. An ESA model 5041 analytical cell was modified with a glassy carbon embedded ceramic composite electrode to allow the use of a thin 12 microm gasket. Also a capillary HPLC column was connected directly to the detector cell using a 30 microm i.d. fused silica capillary. These modifications permitted the extensive redox cycling of the electrochemically reversible catecholamines. The ensuing amplified analytical signal allowed the detector cell to achieve efficiencies of 1300%. This resulted in a mass limit of detection of 4 fg and a concentration limit of detection of 116 pM for dopamine with an S/N of 3.
Neonatal Sprague Dawley rat brain tissue was extracted with methanol, acetonitrile, acetic acid and trifluoroacetic acids (TFA). Among the extractants tested, 0.1 M TFA gave the highest recovery, 73.4 +/- 5.2% (slope of regression of 'added' vs. 'found' and standard error of the slope) of S-sulfocysteine (SSC). The poorest recovery of SSC was found with acetonitrile and 90% methanol extractions (less than 10%). Possible reasons for the low recoveries have been explored. The recovery of SSC from aqueous standards in 0.1 M TFA is 92 +/- 5%. Detection of picomole quantities of SSC has been demonstrated with a combination of the optimized extraction procedures and our previously developed detection system. Supernatant of rat brain homogenate (0.10 M TFA as extractant) was evaporated to dryness in a vacuum centrifuge. Residues were reconstituted with deionized water. Samples were separated on a reversed phase column. The mobile phase was 20 mM aqueous acetate buffer (pH 5.2) containing 0.40 mM cetyl trimethylammonium p-toluene sulfonate and 2 vol.% methanol. Electrochemical detection used dual series gold-mercury amalgam electrodes. For the first time, S-sulfocysteine was detected in normal neonatal rat brain. Its concentration is 0.99 +/- 0.25 pmol/mg brain tissue. The results indicate that TFA, rarely reported an an extractant, efficiently recovers SSC from rat brain tissues.
Three techniques, constant-potential amperometry, high-speed chronoamperometry, and fast-scan cyclic voltammetry, have been used extensively to investigate the rapid events associated with neurotransmission. These techniques vary in sensitivity, chemical resolution and temporal resolution. Amperometry provides the best temporal resolution but little chemical resolution. Fast-scan cyclic voltammetry provides both good temporal and chemical resolution, while high-speed chronoamperometry offers good temporal resolution and moderate chemical resolution. The amount of chemical information which is needed for a neurochemical measurement depends upon the sample. For single cells, secondary methods, such as HPLC and capillary electrophoresis, offer extensive chemical information about the contents of a cell. With this information, chemical information is not needed during the electrochemical measurement. Therefore, amperometry is employed to obtain the greatest temporal resolution. However, when using more complex biological samples, such as brain slices or in vivo implantation, there is a greater demand for chemical resolution provided by the electrochemical measurement. To bolster results, further confirmation is sought from anatomical, physiological and pharmaceutical evidence. Within this review, the three electrochemical techniques are outlined and compared. Examples are then provided of measurements which have been made in the three predominant biological samples which have been studied: single cells, brain slices and intact animals.
The coupling of enzymes and electrode transducers permits the rapid and simple determination of endogeneous compounds and therapeutic drugs in clinical samples. New developments in the operation, miniaturization and microfabrication of electrochemical biosensors offer exciting possibilities for biomedical and pharmaceutical analysis. This review focuses on the current state of amperometric enzyme electrodes for biomedicine, with emphasis on recent advances, challenges and trends.
The advance of materials chemistry has influenced the design of analytical sensors, especially those using spectroscopic or electrochemical methods for generating the signal. New methods of immobilizing enzymes, chromophores, and electron-transfer catalysts have resulted from initiatives in materials science. Systems based on sol-gel chemistry are especially noteworthy in this regard, but other important materials for chemical and biochemical sensors include zeolites, organic polymers, and various conducting composites. Applications cited include determinations of inorganic ions, gases, neurotransmitters, alcohols, carbohydrates, amino acids, proteins, and DNA.
In recent years, electrochemical detection (EC) methods have become increasingly important for the determination of carbohydrate compounds in a variety of biological and pharmaceutical samples. In this work, recent advances in the design and application of EC approaches are reviewed, with the goal of providing the non-electrochemist with a basic understanding of the most important EC approaches to carbohydrate detection and an overview of their current applications. Two specific EC detection strategies are considered in detail: enzyme electrodes and electrodes used for HPLC or capillary electrophoresis detection.
Sub-micron sized domains of a carbon surface are derivatized with antibodies using biotin/avidin technology. These sites are spatially-segregated from, and directly adjacent to, electron transfer sites on the same electrode surface. The distance between these electron transfer sites and enzyme-loaded domains are kept to a minimum (e.g. less than a micron) to maintain the high sensitivity required for the measurement of enzyme-linked cofactors in an enzyme-linked immunoassay (ELISA). This is accomplished through the use of photolithographic attachment of photobiotin using an interference pattern from a UV laser generated at the electrode surface. This allows the construction of microscopic arrays of active ELISA sites on a carbon substrate while leaving other sites underivatized to facilitate electron transfer reactions of redox mediators; thus maximizing sensitivity and detection of the enzyme mediator. The carbon electrode surface is characterized with respect to its chemical structure and electron transfer properties following each step of the antibody immobilization process. The characterization of specific modifications of micron regions of the carbon surface requires analytical methodology that has both high spatial resolution and sensitivity. We have used fluorescence microscopy with a cooled CCD imaging system to visualize the spatial distribution of enzyme immobilization sites (indicated by fluorescence from Texas-Red labeled antibody) across the carbon surface. The viability of the enzyme attached to the surface in this manner was demonstrated by imaging the distribution of an insoluble, fluorescent product.
Amperometric bienzyme electrodes based on coupled L-glutamate oxidase (GlOx) and horseradish peroxidase (HRP) were constructed for the direct monitoring of L-glutamate in a flow injection (FI)-system. The bienzyme electrodes were constructed by coating solid graphite rods with a premixed solution containing GlOx and HRP crosslinked with a redox polymer formed of poly(1-vinylimidazole) complexed with (osmium (4-4'-dimethylbpy)2 Cl)II/III. Poly(ethylene glycol) diglycidyl ether (PEGDGE) was used as the crosslinker and the modified electrodes were inserted as the working electrode in a conventional three electrode flow through amperometric cell operated at -0.05 V versus Ag¿AgCl (0.1 M KCl). The bienzyme electrode was optimized with regard to wire composition, Os-loading of the wires, enzyme ratios, coating procedure, flow rate, effect of poly(ethyleneimine) addition, etc. The optimized electrodes were characterized by a sensitivity of 88.36 +/- 0.14 microA mM(-1) cm(-2), a detection limit of 0.3 microM (calculated as three times the signal-to-noise ratio), a response time of less than 10 s and responded linearly between 0.3 and 250 microM (linear regression coefficient = 0.999) with an operational stability of only 3% sensitivity loss during 8 h of continuous FI operation at a sample throughput of 30 injections h(-1).Department of Chemistry, Addis Ababa University, Ethiopia.
A gradient high-performance liquid chromatographic (HPLC) method was developed for the analysis of Synercid freeze-dried powder in routine quality control, stability and compatibility studies. This method is suited for a simultaneous assay of drug substances and impurities. The method was validated for precision, reproducibility, linearity, accuracy and limits of detection. The robustness study that was performed according to an experimental design is described.
A sensitive and selective HPLC/electrospray ionization tandem mass spectrometric (LC/ESI/MS/MS) method for the quantitative determination of MTIC (5-(3-N-methyltriazen-1-yl)-imidazole-4-carboxamide), a pharmacologically active hydrolysis product of temozolomide, was developed and validated over a linear range from 10 to 400 ng ml(-1) in dog plasma and from 10 to 500 ng ml(-1) in rat plasma. This HPLC method utilized small plasma volumes (70 microl), rapid sample processing, and isocratic elusion conditions to achieve sensitive and selective MS/MS detection. Samples were processed and analyzed one at a time every 4.5 min in order to compensate for the inherent instability of MTIC. Both MTIC and the internal standard DTIC [5-(3,3'-N,N'-dimethyltriazen-1-yl)-imidazole-4-carboxamide] were quantitated in the positive ion, selected reaction monitoring (SRM) mode. The lower limit of quantitation (LLOQ) was 10 ng ml(-1) in the plasma from both species. Inter-assay accuracy and precision of all calibration standards and quality control (QC) samples were within +/- 11 and 12%, respectively, with the exception of the LLOQ in rat plasma (17%). The validated method was used to determine the time dependent plasma concentration of MTIC in rats and dogs following a single oral dose of temozolomide. The standard curve and the quality control data indicate that the method performed acceptably throughout the sample analysis period.
Severe peak asymmetry--fronting--was observed for oxycodone during elution at 30 degrees C from a C18 HPLC column using a mobile phase consisting of 14.9% MeOH, 84.5% 0.05 M KH2PO4 (pH 3.0), 0.5% MTBE, and 0.1% TEA. Investigation using deuterium-labeled oxycodone and analysis by LC/MS showed that gem diol and hemiketal adducts of oxycodone formed as a result of the equilibrium addition of water and methanol to the C-6 ketone on oxycodone. As a result of slow equilibrium kinetics at room temperature in aqueous solution, the gem-diol and methyl hemiketal eluted as an unresolved broad band in front of the oxycodone peak. Decreasing the column temperature to 0 degrees C decreased the rates of interconversion and allowed the resolution and separation of these species from each other and from oxycodone. Increasing the column temperature to 60 degrees C increased the rates of interconversion with the result that the three species eluted as a single, homogenous peak with greatly improved peak symmetry.
A simple, systematic method was developed for rapidly screening potential capillary electrophoresis (CE) separation conditions for small, amine-containing enantiomers. During method development, 39 pairs of enantiomers were investigated and partial or complete separation was achieved in every case. Baseline resolution was achieved by these initial screening conditions in over half of the cases. The screening strategy uses a bare fused silica capillary and a pH 2.5 amine-modified phosphate buffer containing one of the selected cyclodextrins (CD): dimethyl-beta-CD, hydroxypropyl-beta-CD, hydroxypropyl-alpha-CD, hydroxypropyl-gamma-CD and sulfated-beta-CD. An additional set of compounds have been screened by this approach to demonstrate the validity of the method. The paper outlines the experimental work carried out to develop the screen and describes how one might implement it for a new compound.
High affinity, specific murine monoclonal antibodies have been produced for ranitidine using the novel RIMMS (repetitive immunizations, multiple sites) technique. We demonstrate that this technique can be employed to produce high affinity monoclonal antibodies to drug haptens in approximately 1 month; whereas, conventional techniques typically require 3-9 months. Polyclonal antiserum development typically requires at least 6 months. Consequently, RIMMS has a clear impact allowing reagent antibodies to be available earlier in the drug development process. Isotyping studies demonstrated that the developed antibodies are either IgG1 or IgG2b immunoglobulins which confirms that the technique produces class-switched, affinity matured reagent antibodies. The most promising monoclonal antibody for quantitative applications afforded similar sensitivity, by competitive ELISA, to the established sheep polyclonal anti-ranitidine sera. The calibration range, estimated as the limits between the asymptotic regions of calibration graphs, is 0.5-41.2 ng ranitidine per well. Specificity studies indicated that the monoclonal antibody afforded superior selectivity, yielding only 4.1% cross-reactivity with the ranitidine sulphoxide metabolite; the corresponding value for the antiserum was 8.6%. Both reagents had similar cross-reactivities with the N-oxide metabolite.
Solution conformations of the polypeptides beta endorphin (beta-END) and a cysteine peptide (CYSP) were investigated with the use of particle beam LC/FT-IR spectrometry. Gradient elution HPLC with mobile phases that contained acetonitrile with 0.1% TFA (v/v) and 0.1% aqueous TFA (v/v) were used. The conformations of both polypeptides were studied in 0.9% sodium chloride injection USP, 5% dextrose in water injection USP and sterile water for injection USP. Additional conformational studies over a pH range of 2-10, temperatures of 25, 50, 75 and 100 degrees C and after storage for 24 h were investigated. The studies indicated that the two polypeptides did not behave similarly under identical conditions. It was observed that both beta-END and CYSP had slightly different conformations in the various parenteral solutions. It was also shown that the conformation of CYSP changed with both pH and temperature while beta-END was conformationally stable to both temperature and pH. The identity of the peptides and the conformationally sensitive charge-state intensities of the peptides were investigated with electrospray ionization mass spectrometry (ESI/MS). The combination of IR and MS data allowed an estimation of solution effects on the conformations of the model polypeptides.
A gradient, reversed phase, HPLC method was developed for simultaneous analysis of potassium sorbate, methylparaben, propylparaben, and indinavir in aqueous suspensions that contain a proprietary orange flavoring and Magnasweet sweetener enhancer (MacSanrews and Forbes Company, Magnasweet product brochure). The chromatographic separation is performed on an Eclipse XDB-C8 column using a gradient run with an analysis time of 35 min. The mobile phase consists of acetonitrile and acetonitrile:citrate buffer, pH 4.0 (20:80 v/v). The method successfully separates the three preservatives, indinavir (active ingredient), the orange flavoring, the Magnasweet species, and the indinavir lactone degradate. Recovery, linearity, and precision results for the three preservatives and indinavir are described. The method applies to two types of formulations: Xanthan Gum suspension and NanoSystems suspension.
A sensitive and reliable method based on solid-phase extraction and reversed-phase liquid chromatography was developed and validated for the quantitation of Lidocaine (Lid) in dog plasma. Phenacemide was used as an internal standard (IS) in the extraction which employed C18 solid-phase extraction cartridges. The washing and eluting solutions were 2 ml acetonitrile-pH 9.0 phosphate buffer (10:90 v/v) and 0.5 ml acetonitrile-pH 4.0 phosphate buffer (40:60 v/v). respectively. The eluent obtained from the cartridge was directly analyzed on a reversed-phase ODS column with UV detection at 210 nm. A clean chromatogram and high sensitivity were achieved at this wavelength. The mobile phase was acetonitrile and pH 5.9 phosphate buffer (20:80 v/v). The retention times were 6.4 and 7.2 min for Lid and IS, respectively, at a flow rate of 1.0 ml min(-1). The mean absolute recovery was 96.6% (n = 9) with a CV of 3.8% for Lid and 81.7% with CV of 2.5% (n = 3) for IS. The limit of quantitation was 20 ng ml(-1), with the intra- and inter-day precisions (n = 5) of 4.4 and 3.4%, respectively, and the intra- and inter-day accuracies (n = 5) of -4.3 and -5.0%, respectively. For the analyses of Lid in spiked plasma samples at 20, 100 and 200 ng ml(-1), the overall mean intra- and inter-day precisions (n = 15) were 3.9 and 4.9%, respectively, and the overall mean intra- and inter-day accuracies (n = 15) were -3.7 and -4.6%, respectively. The correlation coefficients for calibration plots in the range 20-1000 ng ml(-1) in plasma were typically higher than 0.998. The suitability of the method was demonstrated by the study in a beagle dog receiving a low intravenous dose of Lid.
A method using pharmacologically based and visual limit of detection criteria to determine the acceptable residue level for Meclizine Hydrochloride (MH) on pharmaceutical manufacturing equipment surfaces after cleaning is described. A formula was used in order to determine the pharmacologically safe cleaning level for MH. This level was termed as specific residual cleaning Level (SRCL) and calculated to be 50 microg 100 cm(-2). The visual limit of detection (VLOD) was determined by spiking different levels of MH on stainless steel plates and having the plates examined by a group of observers. The lowest level that could be visually detected by the majority of the observers, 62.5 microg 100 cm(-2), was considered as the VLOD for MH. The lower of the SRCL and VLOD values, i.e. 50 microg 100 cm(-2), was therefore chosen as the cleaning acceptance criterion. A sensitive reversed-phase HPLC method was developed and validated for the assay of MH in swabs used to test equipment surfaces. Using this method, the mean recoveries of MH from spiked swabs and '180-Grit' stainless steel plates were 87.0 and 89.5% with relative standard deviations (RSD) of +/- 3.3 and +/- 2.4%, respectively. The method was successfully applied to the assay of actual swab samples collected from the equipment surfaces. The stability of MH on stainless steel plates, on cleaning swabs and in the extraction solution was investigated.
An HPLC method for quantifying the putative pharmacologically active constituents: thymoquinone (TQ), dithymoquinone (DTQ), thymohydroquinone (THQ), and thymol (THY), in the oil of Nigella sativa seed is described. Extraction of the constituents from the oil was carried out using C18 PrepSep mini columns followed by quantification of the recovered constituents by HPLC on a reversed-phase muBondapak C18 analytical column, using an isocratic mobile phase of water:methanol:2-propanol (50:45:5% v/v) at a flow rate of 2 ml min(-1). UV detection was at 254 nm for TQ, DTQ, and THY, and at 294 nm for THQ. The above four compounds were separated with good resolution, reproducibility, and sensitivity under these conditions. This analytical method was used to quantify the above four constituents in a commercial sample of N. sativa seed oil, and provides a good quality control methodology for the pharmacologically active components in this widely used natural remedy.
There are few methods available for the rapid and precise quantitation of non-covalent aggregation. The very methods used to measure the aggregation can easily disrupt the weak forces holding an aggregate together. This paper describes the novel application of free solution capillary electrophoresis (CE) for the quantitation of a biologically inactive non-covalent aggregate of C8GLIP (Des-amino-histidine-7-arginine-26 N(epsilon)-octanoyl-lysine-34-human glucagon-like insulinotropic peptide), an acylated peptide. The CE results are compared to a more traditional approach using size exclusion chromatography (SEC). Under the conditions explored in this paper, SEC showed a significantly slower apparent rate of aggregation than CE. This is due to the disruption of the aggregate during the SEC process. The cause of the disruption is complex and is potentially related to the separation process itself, on-column dilution effects, and/or interactions of the aggregate with the column packing or SEC components. Analysis times and dilution are greatly reduced by CE, and, because there is no potentially interactive stationary phase and because both the protein and the walls of the capillary are negatively charged, potential disaggregation due to surface interactions is reduced. Thus, CE is shown to be superior to SEC for this peptide in that disruption of the aggregate is minimized.
The determination of the endothelin (ET) antagonist receptor ABT-627 (I) and related substances is performed by HPLC. I is determined in bulk drug substance and drug formulation using isocratic conditions and an Inertsil ODS-2 column. The determination is stability indicating and detector response is linear from 24 to 118 microg ml(-1) (33-164% of assay level). Intermediate precision for the determination ranged from +/- 0.60 to +/- 1.9% RSD. The measurement is accurate, with quantitative recovery of I from the formulation placebo. Related substances in I and formulated I are determined using the same chromatographic conditions, with a gradient elution profile to elute impurities having varying relative polarities. The detector response for related substances determination is linear for I from 0.60 to 17.8 microg ml(-1) (0.05-1.5% of assay level) with the limit of detection and quantitation estimated at 0.01 and 0.05%, respectively. Comparable precision was obtained in drug substance and drug formulation (RSD values +/- 3.7 to +/- 12% and +/- 5.5 to 16.9%, respectively for impurities ranging from 0.05 to 0.30%). The quantitated impurities agreed well for the same lot of I when assayed as a bulk substance and after the formulation into a drug product.
An analytical HPLC method is reported for simultaneous measurement of low (1.0-100 microg ml(-1)) concentrations of dextran-methylprednisolone succinate (DEX-MPS) and its degradation products methylprednisolone hemisuccinate (MPS) and methylprednisolone (MP). The analytes were detected at 250 nm after resolution using a size exclusion column with a mobile phase of KH2PO4 (10 mM): acetonitrile (3:1) and a flow rate of 1 ml min(-1). The resolution of MP and MPS peaks was substantially affected by the pH of the mobile phase; while MP and MPS co-eluted at pH 3.4, they were baseline-resolved at pH > or = 5. Linear relationships (r > or = 0.997) were found between the detector response and the concentrations of the analytes (1.0-100 microg ml(-1) for MP and MPS and 2.5-100 microg ml(-1) for DEX-MPS). Intra- and inter-run error (< 13%) and precision (CV of < or = 6%) data indicated that the assay could accurately and precisely quantitate all three components in the examined concentration range. The application of the assay to determination of degree of substitution, purity, and stability of DEX-MPS was also demonstrated.
A reversed phase high performance liquid chromatographic (HPLC) method was developed and validated for use as a stability indicating assay (potency and related substances) of paroxetine in paroxetine hydrochloride 20 mg tablets. Assay samples were extracted at a paroxetine concentration of 0.4 mg ml(-1) utilizing mobile phase as the extraction solvent. The chromatographic conditions employed a C18 column (Inertsil, 5 microm, 15 cm x 4.6 mm), isocratic elution with 10 mM 1-decane sulfonic acid sodium salt containing 10 mM sodium phosphate monobasic (pH 3.0)-ACN (60:40, v/v) and ultraviolet (UV) detection at 235 nm.
A simple and accurate liquid chromatographic method was developed for estimation of estradiol valerate and medroxyprogesterone acetate in pharmaceuticals. Drugs were chromatographed on a reverse phase C18 column, using a mixture (30:70) of ammonium nitrate buffer and acetonitrile and eluants monitored at a wavelength of 280 nm. Solution concentrations were measured on a weight basis to avoid the use of an internal standard. The method was statistically validated for its linearity, accuracy, precision and selectivity. Due to its simplicity and accuracy, the authors believe that the method may be used for routine quality control analysis. It does not require any specific sample preparation except the use of a column guard before the analytical column and suitable prefilter attached to the syringe prior to injection.
A case of calcium pyrophosphate dihydrate (CPPD) crystal deposition arthropathy of the temporomandibular joint is reported. The patient presented a 10-year history of swelling and pain of the left preauricular region. Magnetic resonance imaging showed a calcified mass filling the joint space and destroying the roof of the joint. Radiographs showed chondrocalcinosis of other joints. The authors discuss the diagnosis of this arthropathy and the reason why the temporomandibular joint is more affected than the other joints in the patient reported.
The aim of this study was to report a series of extracranial cephalic schwannomas. Fifteen patients with extracranial schwannomas treated between 1981 and 1999 are presented, and their clinical course during a median follow-up of 4.1 years is discussed. There is a female predominance. No specific factors have been identified. Their diagnosis is often delayed (median, 2.6 years). There is no predominant side. The orbit represents the most frequent location of schwannomas (26%). The trigeminal nerve is the most often affected (53%). Computed tomography and magnetic resonance imaging contribute to the diagnosis. Macroscopically, the schwannoma is a well-defined tumor of ovoid form and brownish color. It is formed of soft tissues and is fragmented easily. Diagnosis is often evident on microscopic examination. The only treatment is surgery. It consists of enucleation after opening the epineurium using an operating microscope, without interruption of the continuity of the nerve. The authors have observed only two relapses (the first two patients operated without a microscope). Total excision allows recovery. Nerve injuries have variable prognosis. It is necessary in juvenile populations to search for neurofibromatosis. All schwannomas required surgical treatment using an operating microscope to obtain total recovery.
New instrumentation and techniques for skeletal fixation have been developed in response to concerns regarding variability of the skull base and the calvaria. The fear of intracranial penetration has limited the use of internal or external fixation to the skull base and the calvaria. Despite potential clinical usefulness, limited anthropometric data have been reported on the skull base and the calvaria. The purpose of this study was to measure the average thickness of various points of the skull base and the calvaria of Korean adults. Fifty-one Korean adult skull bases and calvaria, which were cut 2 cm above the supraorbital margin anteriorly and 2 cm above the inion posteriorly, were measured in this study. Thickness of 13 clinically important points were measured with a Teclock GM-21 Calipergauge (Teclock Co., Tokyo, Japan). The mean thickness of the two opposite sides were compared using paired t-tests. They did not differ significantly from the others except for three points. Frontal bone A (located 1 cm above the supraorbital margin and 1.5 cm lateral to the midline) was 6.35 +/- 2.88 mm, frontal bone B (located at the midpoint of the frontal midpoint and the frontoparietal suture on the cut edge) was 5.24 +/- 1.50 mm, frontal bone C (located at the midpoint of the vertex and frontal bone B) was 6.63 +/- 1.77 mm, the pterion was 3.19 +/- 0.85 mm, the midpoint of the squamous temporal was 1.96 +/- 0.65 mm, parietal bone A (located 1.5 cm below the middle meningeal groove) was 3.99 +/- 1.11 mm; parietal bones B and C (divided at the distances between the frontoparietal suture and the occipital midpoint with a ratio of 1:3 and 2:3 respectively on the cut edge) were 4.71 +/- 1.06 mm and 5.35 +/- 0.91 mm respectively, occipital bone A (located 1.5 cm posterolateral from the foramen magnum) was 2.31 +/- 1.00 mm, occipital bone B (located 1.5 cm lateral to the midline and 0.5 cm above the inion) was 5.41 +/- 1.50 mm, occipital bone C (located occipital to the midpoint and 2 cm above the inion) was 8.2 +/- 1.67 mm, the orbital roof was 1.37 +/- 1.08 mm, and the orbital lateral wall was 2.25 +/- 1.06 mm. The data give practical information for determining the position and depth for safe and effective internal or external fixation during craniofacial procedures.
An extensive review of biomaterials in the face was conducted in an American Society of Maxillo-facial Surgeons-sponsored biomaterials symposium. The symposium was held in Boston, MA, immediately preceding the 1998 annual meeting of the ASPRS/PSEF. The scope of the symposium extended from current reconstructive techniques for the facial skeleton, including autogenous bone and biomaterials, to potential application of new techniques in molecular biology that may enable the body's own tissues to be engineered to provide bone and cartilage to reconstruct the facial skeleton. The authors review the presentations and relevant literature on biomaterials in the face. The following topics are reviewed: current reconstructive techniques using autogenous bone grafts, methyl methacrylate cranioplasty, demineralized bone, and hydroxyapatite; biomaterials used for rigid fixation, including metallic and bioabsorbable implants; biomaterials used for facial augmentation, including porous polyethylene, hard-tissue replacement, and ceramic biomaterials; biofilm, or a layered polysaccharide matrix secreted by bacteria on the surface of implants; and potential means of inducing bone formation by directing the body's own tissues through cytokine interaction, gene transfer, and tissue engineering.
During embryogenesis the clefting event unleashes a series of processes that act in concert to deform the soft tissue envelope into a "dysfunctional matrix" within which bone and cartilage subsequently form. Much of secondary cleft deformity results from failure to solve this pathologic soft tissue-bone equation at the primary repair. The sliding sulcus procedure uses the subperiosteal plane to create a centralization of the soft tissue matrix, alleviate tension, and redistribute the forces of growth in a more normal pattern. By incising at the level of the alveolus, this approach preserves the blood supply to the osteogenic cells of the buccolabial periosteum. This approach has interesting theoretical implications for the preservation of alveolar growth potential. Arch stabilization and elimination of fistula may be of long-term benefit to the orthodontist.
Sleep apnea syndrome (SAS) is a complex respiratory disorder that is very difficult to diagnose and to treat surgically as well as medically. SAS can affect growing patients as well as adults. SAS shows a central, an obstructive, and a mixed form. Diagnosis is based on clinical examination of the patient and instrumental examinations such as teleradiography, nuclear magnetic resonance imaging (NMR), three-dimensional computed tomography, polysomnography, rhinomanometry, and spirometry. The patient presented has an obstructive form of SAS in addition to Crouzon's disease. He underwent a Le Fort III osteotomy to obtain an advancement of the orbitomaxillary complex, allowing an increase in volume of posterior airway space at the level of the hypopharynx.
A 4-year-old girl underwent craniofacial reconstruction for giant cystlike encephalocele deriving from the temporo-maxillary region and giving impression of the duplicated head. The case of temporal encephalocele in this report is especially unusual in the extent of encephalocele, the degree to which it had expanded the zygomatic arch, mandible, cranial vault, and the radiologically undetectable bony defect. The use of craniofacial principles in the resection and reconstruction of the temporal encephalocecle are described.
The authors describe a simple technique for reducing zygomatic arch fractures with a towel clip. The orbital rim and zygomatic arch are outlined with a marking pen. The exact fracture site is then marked by palpation. The depressed fracture site is held with a towel clip and pulled outward gently. A clicking noise may be heard. The clip is then released. The contour of the zygomatic arch is compared with the other side. Using this technique the authors reduced 11 fractured zygomatic arches (arch only, displaced medially) in 11 patients and the results were satisfactory in all patients. The towel clip method is one procedure to reduce zygomatic arch fracture.
Juvenile ossifying fibroma is an unusual maxillofacial fibro-osseous lesion characterized by cell-rich osteoid strands. A 7-year-old girl presenting with a massive juvenile ossifying fibroma of the maxilla resulting in facial deformity, orbital displacement, and extension into the anterior skull base is discussed. The importance of combining clinical, radiographic, and histopathological findings for the diagnosis and management of the tumor is discussed.
Combining large-segment orthognathic surgery and unitooth or small-segment surgery is an effective approach to deal with a wide range of dentofacial deformities that have minor to severe occlusal problems. The indications for combining single- or double-tooth osteotomies with traditional orthognathic surgery were patients with dentofacial deformities and malocclusion requiring stable correction within a short overall treatment period. From 1991 to 1998, a total of 36 patients underwent combination single- or double-tooth-segment osteotomy with traditional orthognathic procedures performed at Chang Gung Memorial Hospital. The indications for surgery were maxillary protrusion (N = 5), bimaxillary protrusion (N = 19), mandibular prognathism with maxillary protrusion (N = 11), and noncleft maxillary retrusion (N = 1). The types of osteotomies performed were the Le Fort I, the anterior segmental osteotomies of the maxilla or the mandible, the palatal split, and the posterior segment in combination with single-tooth or double-tooth segments. Follow-up ranged from 12 months to 6 years and showed stability in the movements with no complications. There was no loss of any "osteotomized" segment. The average overall treatment time was approximately 18 months--5 months preoperative and 13 months postoperative orthodontic treatment. This was at least 6 months shorter in duration compared with traditional orthognathic surgery. Combining traditional orthognathic surgery with single- or double-tooth segments allows us to treat complex dentofacial deformities in the vertical, transverse, and sagittal dimensions with differential repositioning of all segments, either major or minor, simultaneously. The authors' experience with 36 consecutive patients evidence good results and demonstrate the procedure to be safe with minimal complications.
Over a 10-year period the authors have performed 92 transethmoidal optic nerve decompressions for the treatment of visual loss due to various pathological processes, including 45 cases of trauma, 32 cases of neoplasm, 2 cases of bacterial pansinusitis, 5 cases of sphenoethmoidal mucocele, 4 cases of aspergillosis (all were immunocompetent patients), 2 cases of Wegener's granulomatosis, and 2 cases of sarcoidosis. Forty-eight patients (52%) had preoperative visual acuity of light perception or better, and in 44 patients (48%) the preoperative vision was no light perception. Sixty-five patients (71%) achieved improvement of vision postoperatively. Twenty-four patients (26%) had no change in vision and 3 patients (3%) had deterioration of vision after surgery. The mean percentage of improvement was 40.7% +/- 6.9% in the trauma group, 61.6% +/- 23.2% in the neoplasm group, 66.4% +/- 25.2% in the infectious/mucocele group, and only one patient in the inflammatory group had slight visual improvement from no light perception to counting fingers. Extracranial optic nerve decompression can result in the improvement of visual function in some patients with optic nerve injury from various causes.
A 1-year-old infant with left hemicoronal synostosis was treated by distraction osteogenesis of the craniofacial skeleton using an internal distraction device. Surgery was performed through a coronal incision. The frontal bone and upper half of both orbits were first osteotomized en bloc after minimal epidural dissection of the supraorbital area and no epidural dissection around the coronal osteotomy site. The lateral one fourth of the frontal bone, including the right lateral half of the orbit, was left intact. The internal distraction device was fixed in the left temporal area. A 0.5-mm per day rate of distraction was performed up to an elongation of 17 mm after a 5-day latency period. The distraction device was removed after a consolidation period of 2 months. The results obtained were satisfactory, with symmetry of the forehead, orbit, and nose achieved without complications. The merits of this procedure are no extradural dead space after the operation (which prevents infection), shortened operative time, reduced blood loss, filling in the bone gap created by advancement with new bone, acceptable cosmesis by the parents during distraction, and no fixation device left after the second operation.
A male infant is described in whom coincident pathologies of metopic synostosis and deformational plagiocephaly were observed. The role in causation of localized pressure (in particular, extreme constraint) is addressed.
The sagittal split ramus osteotomy is the most commonly used procedure to reposition the mandible surgically. Because it is more technically difficult and associated with a higher incidence of complications compared with other mandibular osteotomies, thorough knowledge of the anatomy of the mandibular ramus is a prerequisite. Anatomic measurements related to the mandibular foramen were obtained from 57 formalin-preserved non-Asian hemimandibles. As shown in previous reports, great variability was noted in the position of the mandibular foramen. However, these studies utilized Asian mandibles with a clear discrepancy in key anatomic measurements in comparison with the authors' data. This brings into question the validity of these earlier studies when applying their data to non-Asian groups. The "fade-out" point of the internal oblique ridge was found not to be a reliable anatomic reference for placement of the horizontal osteotomy along the medial ramus. Thus, familiarity with the described relationships of the mandibular foramen will assist in performing properly a sagittal split of the ramus and will reduce the chance for an unfavorable split.
This study analyzed in three dimensions the longitudinal growth pattern of young patients with hemifacial microsomia (HFM) before and after mandibular distraction osteogenesis (DO). Six individuals with HFM (five boys and one girl; age at distraction, 12.5 +/- 2.4 years) were treated with similar procedures (surgery, type and direction of distraction, no functional orthodontic treatment before and after DO). Two individuals who did not undergo DO until late in their growth were used to compare growth patterns. In addition, one individual besides the six previously chosen sample in whom no DO was performed was also used to compare longitudinal growth patterns. Lateral and posteroanterior (PA) cephalograms were utilized preoperatively, spanning a period of 9 years. Computerized three-dimensional models were constructed from the lateral and PA cephalograms using a vector intercept algorithm. In the comparison group, for a period of 8 years on the affected side, the ramus height, body length, and total mandibular length increased at an average rate of 1.3, 1.9, and 3.0 mm per year respectively. On the unaffected side, the ramus height increased by 2.1 mm per year, 1.9 mm in the body, and 2.9 mm per year in total mandibular length. On average, the gonial angle on the affected side was increased by 1 deg per year, yet decreased by 1 deg per year on the unaffected side. The proportions between the affected to the unaffected side were maintained. In the six individuals 18 months after DO, it was found that the ramus height was reduced by 1.0 mm, whereas the body was found to resume its growth with a faster rate on the distracted side, maintaining its proportion. Angular changes demonstrated closing of the gonial angle on both the unaffected (0.5 deg) and distracted (3.5 deg) sides. Observed in three dimensions were the following: (1) On average, unoperated patients with isolated HFM tend to maintain their asymmetrical facial proportions and do not worsen substantially with time. (2) Different treatment effects were seen on the ramus, body, and total length of the mandible: changes in body length > ramus height > total length. (3) Eighteen months after DO, the correction was stable but with some degree of settling back from the initial overcorrection (< 5%). (4) Eighteen months after DO the mandibular body was found to have greater growth than the ramus. (5) Evaluating changes in three dimensions provides an improved understanding of the growth pattern and distraction effects on the mandible and its structural components. (6) Additional studies on the effect of mandibular distraction on other conditions involving mandibular deformities are required. In addition, the effect of various distractor devices should also be evaluated. Three-dimensional evaluation is recommended for improved accuracy.
Distortion of the supratarsal sulcus of the upper eyelid after orbital trauma is a well-recognized and troublesome problem. This is particularly true of the anophthalmic orbit. The authors present two patients in whom this deformity has been addressed using a pedicled pericranial flap. They found this technique provides abundant, well-vascularized tissue that is manipulated easily to conform to the demands of the defect. In addition, the vascularity of the tissue provides predictability of the result when compared with other described techniques such as fat and dermis-fat grafts.