Large-scale isolation of CD133+ progenitor cells from G-CSF mobilized peripheral blood stem cells.

We have evaluated the feasibility of large-scale isolation of CD133+ progenitors from healthy mobilized adult donors for potential clinical use in autologous and allogeneic transplantation. A total of 11 healthy volunteer adult donors were mobilized with G-CSF. CD133+ stem cells were isolated from a single leukapheresis using the Clinimacs method. The median percentage of CD133 before positive selection was 0.75% (range 0.39-2.03%). After selection, the median purity and recovery was 94% (range 85.2-98.0%) and 69% (range 44-100%), respectively. The median log10 T-cell depletion obtained by CD133+ positive selection was 4.2 (range 3.8-4.7). The CD133+ progenitors were highly enriched in colony-forming units (CFU) and transplantation into NOD/SCID mice resulted in a high engraftment rate. Transplantation of sorted CD133+/CD34+ cells into NOD/SCID mice showed a higher engraftment compared to CD133-/CD34+ cells. Mobilized peripheral CD133+ stem cells can be purified in large scale for potential clinical use. The biological function of the cells is not impaired. The majority of the NOD/SCID repopulating cells are within the CD133+/CD34+ subpopulation. Therefore, clinical studies using purified CD133+ stem cells can be envisoned to further clarify the role of CD133+ stem cells in hematopoietic reconstitution after transplantation.

A large-scale method for T cell depletion: towards graft engineering of mobilized peripheral blood stem cells.

We have investigated the feasibility and efficacy of large-scale T cell depletion from granulocyte colony-stimulating factor (G-CSF) mobilized peripheral blood stem cells (PBSC). The method is based on the use of a CD3 antibody conjugated to magnetic microbeads and magnetic activated cell sorting (Clinimacs). A total of eight large-scale experiments were performed. In four experiments, CD3(+) T cells were depleted from PBSC obtained from volunteers mobilized with G-CSF whereas, in four experiments, T cells were depleted from PBSC from stem cell donors, in which the CD34(+) stem cells had been removed for allogeneic transplantation by positive selection prior to T cell depletion. The mean number of processed mononuclear cells (MNCs) was 3.3 x 10(10) (range 1.5 x 10(10)-5.1 x 10(10)) with a mean T cell proportion of 35.8% (range 16.7-64.0%). After T cell depletion, the percentage of contaminating T cells was 0.15% (range 0.01-1.01%) with a mean log(10) depletion of 3.4 (range 2.8-4.1). The mean recovery of CD3-negative MNCs after depletion was 76% (range 52-100%). The mean recovery of CD34(+) stem cells in the four evaluable experiments was 82% (range 75-92%). In vitro colony assays and in vivo NOD/SCID repopulation assays showed that this large-scale T cell depletion method has no negative impact on the function of the hematopoietic precursor cells. Therefore, we conclude that this T cell depletion method is a valuable tool for further graft engineering strategies involving mobilized PBSCs.

Effect of an integrated nutrition curriculum on medical education, student clinical performance, and student perception of medical-nutrition training.

BACKGROUND: Ninety-eight percent of medical schools report nutrition as a component of medical education. However, most schools do not have an identifiable nutrition curriculum. Medical schools that do include nutrition have not evaluated its effect on clinical skills. OBJECTIVE: The objective was to determine the efficacy of an integrated undergraduate medical curriculum to increase the quantity of nutrition instruction and to advance nutrition clinical skills demonstrated by medical students. DESIGN: A quasiexperimental design was constructed to determine whether an integrated nutrition curriculum increased the performance on nutrition-oriented clinical examinations of medical school classes that received 1, 2, or 3 y of the curriculum. The evaluation of the curriculum focused on 3 areas: 1) hours of nutrition instruction, 2) the application of nutrition within a clinical setting, and 3) perceptions about the nutrition curriculum. The Objective Structured Clinical Examination (OSCE) nutrition score was compared between graduating classes by use of analysis of variance. Data from the American Association of Medical Colleges were analyzed to determine the change in the proportion of students who reported that the amount of time devoted to nutrition was adequate. RESULTS: The implementation of the integrated nutrition curriculum resulted in a doubling of the total hours of required instruction in the medical curriculum (35 compared with 75 h). The mean (+/-1 SEM) OSCE nutrition score significantly improved after the implementation of the curriculum (41.7 +/- 0.9% compared with 50.6 +/- 1.1%) and the percentage of students who reported that the amount of nutrition taught during medical school was inadequate decreased (68.4% compared with 11.5%). CONCLUSION: Medical students improved their clinical nutrition practice skills through participation in an integrated nutrition curriculum.

Using problem-based learning to promote critical thinking in an orientation program for novice nurses.

To assist novice nurses in making the transition into the acute care setting, a student-centered, small-group, problem-based program was developed and combined with a preceptorship program. Challenged to work through the problems, novice nurses met the program objectives while enjoying the program. Supervisors and clinical specialists working with the graduates noted increased critical thinking, increased interest and ability in seeking information, and increased problem-solving ability.

Atypical infective endocarditis.

BACKGROUND: Although infective endocarditis has changed in the recent past as a result of microbiologic and risk factors, it continues to be clinically challenging. The disease is characterized by the formation of septic masses of platelets on the surfaces of heart valves. Several mechanisms can cause or contribute to the development of endocarditis. Although risk factors for infective endocarditis are well known, patients with atypical signs and symptoms continue to challenge us. METHODS: We describe a case report of a patient admitted to our inpatient service with back pain and presumed pyelonephritis. A MEDLINE literature search was conducted, using the key words "endocarditis," "back pain," and "bacterial," for the years 1986 to the present. RESULTS AND CONCLUSIONS: A 42-year-old woman with a history of intravenous drug abuse was admitted to the family practice service with back pain and pyelonephritis. She developed hypoxia and a new heart murmur and had continued fevers. Blood cultures drawn in the emergency department grew methicillin-resistant Staphylococcus aureus. A bone scan and magnetic resonance imaging led to the diagnosis of epidural abscess. What appeared to be a simple case of pyelonephritis with back pain became a case of infective endocarditis complicated by an epidural abscess.

Teams in a community setting: the AUHS experience.

The Pennsylvania Local Interdisciplinary Team was created to develop and implement an innovative model for the education of students from multiple disciplines and institutions in the theory and practice of quality improvement. The lead poisoning prevention project is presented as an example of the work of an interdisciplinary student team in a community setting in Philadelphia.

A multisite collaborative for the development of interdisciplinary education in continuous improvement for health professions students.

In 1994, The Institute for Healthcare Improvement, in Boston, Massachusetts, formed the Interdisciplinary Professional Education Collaborative (the Collaborative). The mission of the Collaborative is to create an interdisciplinary teaching and learning environment in which future health professionals learn to work together to improve health care delivery. Apart from emphasizing interdisciplinary collaboration, the Collaborative focuses on teaching the methods of continuous improvement (CI), a system of management first developed for manufacturing industries that is increasingly being used in the management of health care delivery. The Collaborative consists of four local interdisciplinary teams (LITs): the Cleveland LIT, the "George" LIT (a collaboration between George Washington University in Washington, D.C., and George Mason University in Fairfax, Virginia), the South Carolina LIT, and the Pennsylvania LIT; and a coordinating committee. This paper describes each LIT's approach to achieving the Collaborative's commitment to give health professions students the opportunity to work in interdisciplinary teams to learn about and practice CI methods, training the Collaborative believes will enable them to be effective providers in a variety of health care systems. The paper describes the overall goals of the Collaborative, presents reports from the four LITs, and discusses common lessons learned.

Mammography and Pap smear screening of Yaqui Indian women.

The Pascua-Yaqui Tribe of Arizona receives its health care services at a local neighborhood health center in Tucson and a satellite clinic located on the reservation. Using a computerized data base from the health center, the authors determined the use rates by Pascua-Yaqui women ages 35-65 of the Papanicolaou smear and mammography screening. Among active users of the health center, 31-36 percent had received a Papanicolaou smear, according to the yearly data bases examined from 1986 to 1990, while 65 percent of the women had received at least one smear test over the entire 5-year period. Regarding mammography screening, 41-43 percent of the women ages 50-65 had received a mammogram in the years studied, and 51-58 percent of the women ages 40-49 had been screened. In all, 67 percent had received at least one mammogram during the 1988-90 period when the center offered mammography. This population of 35-65-year-old American Indian women, for whom financial access is not a barrier, were receiving Papanicolaou smears and mammograms at rates comparable with other segments of the U.S. population but at lower rates than those recommended by the American Cancer Society and National Cancer Institute. The challenge for the health center is to reach those women who are eligible for services but do not use them and to address the nonfinancial barriers to care such as language, transportation, and gender-specific issues.

The service-education linkage: implications for family practice residency programs and community and migrant health centers.

INTRODUCTION: Access to quality primary health care for our country's underserved populations is a challenge for both the government and physicians. The Division of Medicine, through funding priorities and other initiatives, is encouraging family practice educators to train residents and students for work in community and migrant health centers (C/MHCs) in underserved areas. The objective of this research was to study linkages between family practice residency programs and C/MHCs and determine the reasons for affiliation, disadvantages and advantages, predictors of successful linkages, and common errors in the linkage agreement. METHODS: We conducted in-depth telephone interviews with the directors of 13 of the 19 family practice residency programs identified as having linkages with C/MHCs. RESULTS: All interviewees at residency programs indicated that their programs had a mission to serve underserved patients. The most commonly cited constraining factor cited by both residency programs and C/MHCs was financial support for residents, on-site faculty, and support staff. Many programs reported that residents training at the C/MHC were able to gain a community health perspective and practice community-oriented primary care. Finally, financing the relationship involved many different approaches, ranging from the residency paying all of the salaries, to a sharing of salaries by the residency, state, and/or hospital, to C/MHC paying the salaries either through its own funds or through grant support. DISCUSSION: These data provide an assessment of the current issues that family practice residencies must address to implement service-education linkages. They provide an empirical basis to outline the steps involved in forming a linkage between a residency and a C/MHC.

Cytoskeletal events underlying dendrite formation by cultured pigment cells.

In contrast to neurite outgrowth, pigment cell dendrite formation is relatively unstudied. Keratinocyte-conditioned medium (KCM) induces a striking dendricity in human melanocytes and B16 melanoma cells that is detectable within 30 min, maximal in 24-48 hr, and quantifiable by computerized image analysis. Cytochalasin B (CB), known to disrupt actin microfilaments, completely blocks dendrite formation if added to cultures before or with KCM. This effect is rapidly reversible, and dendrites appear within 1 hr after refeeding with KCM alone. In contrast, CB treatment fails to disrupt existing dendrites previously induced by KCM. Agents known to cause microtubule disassembly (colchicine, nocodazole, or vinblastine) do not inhibit dendrite formation if added before or with KCM. In contrast, these agents disrupt established dendrites. Inhibition of protein synthesis with cycloheximide or actinomycin D completely blocks dendrite formation, but if cultures are provided fresh KCM lacking protein synthesis inhibitors, dendrites reappear within 24 hr. Actin microfilaments visualized with a monoclonal antibody or rhodamine-phalloidin are poorly organized in untreated cells, but form numerous fibers localized along dendrites in KCM-treated cells. Microtubules visualized with a monoclonal anti-tubulin antibody are localized in the center of dendrites. These cytoskeletal changes occur without altering beta actin or beta tubulin mRNA levels. Taken together, these data implicate actin microfilaments in dendrite outgrowth, but not in maintenance, and conversely microtubules in dendrite maintenance but not in formation. These keratinocyte-induced changes involving beta actin and beta tubulin polymerization appear to require both new protein synthesis and post-translational regulation. The observed similarities between melanocytes and other neural crest-derived cells suggest that cutaneous pigment cells might serve as an alternative model for studies of neurite outgrowth.

Family physicians' colposcopy practices.

BACKGROUND: The objectives of this study were to determine (1) the extent to which family physicians are performing colposcopy, (2) which colposcopic procedures are performed by these family physicians, (3) demographic characteristics of physicians who perform colposcopy, and (4) whether physicians who do not perform colposcopy plan to do so in the future. METHODS: A questionnaire was mailed to all 757 self-identified family practice physicians in Arizona. RESULTS: The return rate was 72 percent, and the response rate was 55.5 percent. Results indicated that 19.3 percent of respondents were trained to perform colposcopy, and 9.5 percent actually have performed it. For those performing colposcopy, the mean number of procedures performed during the previous 6 months was 25 (range 2-100). CONCLUSIONS: Certain barriers to performing colposcopy were identified: (1) lack of available training, (2) interspecialty "turf battles," (3) quality assurance, and (4) the cost of malpractice liability insurance. Nevertheless, there were no insurmountable reasons why family physicians could not perform colposcopy.

Pigment content of cultured human melanocytes does not correlate with tyrosinase message level.

Tyrosinase is considered to be the rate-limiting enzyme for the biosynthesis of melanin in epidermal melanocytes, and thus tyrosinase activity is thought to be a major regulatory step in melanogenesis. To determine whether the rate of pigment production was controlled at the level of tyrosinase gene expression, we developed a culture system capable of generating large populations of pure human melanocytes and then measured both melanin content as determined spectrophotometrically by absorption at 475 nm and mRNA levels as detected by hybridization with cloned cDNA Pmel 34, encoding human tyrosinase. We examined the relationship between pigment content and tyrosinase mRNA levels among human melanoma and melanocyte lines with very different levels of basal pigmentation; between two clones of a single human melanoma line, one pigmented and one amelanotic; and sequentially in melanocytes before and after simulation with isobutylmethylxanthine to increase melanin content per cell. Using Northern blot analysis and in-situ hybridization we found no correlation between tyrosinase message levels and melanin content, suggesting that posttranscriptional regulation of tyrosinase and/or other events determine the rate of pigment synthesis in human melanocytes.

Cancer risk reduction counseling: a computer-assisted curriculum.

Significant progress has been made in the identification of factors associated with an increased risk of developing cancer. Cancer is increasingly viewed as a preventable disease. Its prevention involves risk reduction counseling. This counseling is an important skill for the family physician but can be difficult to learn and to teach. We used a prototype, computer-assisted cancer risk reduction counseling curriculum with first-year medical students. We found a statistically significant change in both knowledge-based and attitudinal questions and answers after the use of this curriculum.

A microcomputer-based device to simulate biomechanical environments for cultured cells.

We describe software and hardware for a microcomputer-based cyclic strain device which applies programmed cycles of elongation and relaxation to cultured cells. This system has the potential to simulate many of the complex mechanically active environments found in living systems. As a sample application, we use it to simulate the cyclic stresses to which vascular smooth muscle cells in the arterial system are exposed.

Phenolic and tyrosyl ring iodothyronine deiodination by the Caco-2 human colon carcinoma cell line.

Thyroid hormone metabolism was studied in the human Caco-2 colon carcinoma cell line, which at confluence exhibits several functions of differentiated enterocytes. Cells were harvested two to 17 days after reaching confluence. Intact cells and homogenates were tested for deiodination of [125I]-labeled substrates. Small amounts of thyroxine (T4) were converted by homogenates to 3,3',5'-triiodothyronine (rT3), 3,3'-diiodothyronine (3,3'-T2), and 1-, with no detectable production of 3,5,3'-triiodothyronine (T3) by homogenates or cells. rT3 was converted to 3,3'-T2 and 1- with an apparent Michaelis constant (Km) for rT3 of 24 nmol/L; 6-n-propyl-2-thiouracil (PTU) had a 50% inhibitory concentration of 30 nmol/L and abolished rT3 5'-deiodination at 1 mmol/L in the presence of 20 mmol/L dithiothreitol (DTT). T3 was deiodinated to 3,3'-T2 and 3'-monoiodothyronine (3'-T1) with an apparent Michaelis constant (Km) for T3 of 5.7 nmol/L; this reaction was not inhibited by 1 mmol/L PTU. Phenolic and tyrosyl ring deiodinating activities were maximal four and six days, respectively, after the cells reached confluence. Homogenates of cells grown in standard medium containing fetal calf serum had fivefold higher rT3 5'-deiodinating activity than cells grown in a serum-free defined culture medium, reflecting a fivefold difference in the apparent Vmax with no difference in the apparent Km for rT3. There was no difference in T3 5-deiodination rates in homogenates of Caco-2 cells grown in the two media until 12 days postconfluence, when cells grown in standard medium had higher activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Human melanogenesis is stimulated by diacylglycerol.

The intracellular signal pathways that mediate pigmentation in human skin are unknown. We now report that a diacylglycerol (DAG) analogue 1-oleoyl-2-acetyl-glycerol (OAG) 25-100 microns strikingly increased the melanin content of cultured human melanocytes in a dose dependent manner without altering growth rate. The pigment increase occurred within 24 h, was accompanied by increased incorporation of the melanin precursor L-3,4-dihydroxyphenyl alanine (DOPA), required new protein synthesis, and was completely blocked by the protein kinase C (PKC) inhibitors H-7 and sphingosine. A PKC-inactive DAG isomer had no effect on melanin per cell. These results implicate protein kinase C and its effector DAG in melanogenesis.