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.

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.

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.

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)

Regulation of human melanocyte growth, dendricity, and melanization by keratinocyte derived factors.

Epidermal pigmentation involves the synthesis of melanin in melanocytes and its transfer to surrounding keratinocytes, where it functions in photoprotection. To investigate the possible role of the keratinocyte in regulating pigmentation, human keratinocytes were incubated for 24 h in a defined culture medium, which was then transferred to pure human melanocyte cultures. After 1 week, the conditioned medium produced a fourfold increase in melanocyte yield and a seven-fold increase in total melanin. Increased melanocyte dendricity was clearly visible within 24 h as well. Ultrafiltration of the keratinocyte-conditioned medium suggested approximately one-half of the growth promoting activity as well as most of the dendricity and melanization stimulating activities were of low molecular weight (less than 500 Da). High molecular weight fractions stimulated only melanocyte growth. Of the several known keratinocyte-derived factors tested, none could be implicated as a mediator of the observed effects. Basic fibroblast growth factor, known to stimulate melanocyte growth in some culture systems, failed to stimulate growth, dendricity, or melanin content when added to the complete non-conditioned medium. Interleukin-1 alpha, interleukin-1 beta, 12-hydroxyeicosatetraenoic acid, prostaglandin E2, leukotriene B4, and adenosine 3',5'-cyclic monophosphate analogues also had no effect. These studies demonstrate that keratinocytes in vitro release factors that modulate melanocyte behavior and expand our understanding of controls for human epidermal pigmentation.

Vitamin D, its precursors, and metabolites do not affect melanization of cultured human melanocytes.

Exposure of the skin to sunlight results in both tanning and vitamin D3 production. It has therefore been suggested that vitamin D3 or its active metabolite 1,25-dihydroxyvitamin D3 may be the mediator of UV-induced melanogenesis. To test this hypothesis, newborn foreskin-derived melanocytes were cultured in paired dishes in hormone-supplemented medium with 2% serum containing no detectable vitamin D3 or in the same medium containing 10(-8) or 10(-10) M of either provitamin D3, lumisterol, previtamin D3, vitamin D3, 25-hydroxyvitamin D3, or 1,25-dihydroxyvitamin D3. After 10 days, cell number in cultures containing vitamin D compounds was 93%-140% of unsupplemented controls and melanin content was 60%-120% of control, with no significant difference in either parameter for any compound tested. In separate experiments, human melanocytes and Cloudman S91 melanoma cells were repeatedly irradiated with physiologic doses of simulated sunlight and incubated between irradiations with provitamin D3, previtamin D3, vitamin D3, or 1,25-dihydroxyvitamin D3. Irradiated cultures had a 90%-95% inhibition of cell growth associated with a 200%-800% increase in melanin content per cell relative to controls, but there was no effect of any vitamin D compound on either cell type. Neither cultured human melanocytes nor S91 cells showed evidence of the cytosolic 1,25-dihydroxy-vitamin D3 receptor binding by sucrose density gradient analysis using radiolabeled 1,25-dihydroxyvitamin D3. The combined data strongly suggest that neither vitamin D3 nor its precursors or metabolites directly mediate melanogenesis in these cells.

Inositol is a required nutrient for keratinocyte growth.

Bovine hypothalamus is known to contain a growth-promoting activity for human epidermal keratinocytes. By sequential purification, the substance was isolated and found to be myo-inositol. The identity of the substance as myo-inositol was confirmed by ion modified partition, gas liquid, thin layer chromatography, by mass spectrometry, and quantitative bioassay. The inositol content of the crude hypothalamic extract and of an active acetone precipitate (the first step in the purification) was determined to be sufficient to account for their observed bioactivity. At an optimal concentration of 55 microM (10 micrograms/ml), myo-inositol approximately tripled keratinocyte yield compared to paired cultures in basal medium containing 0.3 microM, although this yield was only half that produced by a crude saline extract of hypothalamus, suggesting that there are additional growth-promoting activities in the tissue extract. No other skin-derived cell type tested was stimulated by supplemental inositol. These results establish that the inositol requirement for cultured human keratinocytes is markedly higher than for any other normal or malignant cell type investigated to date, and expand the list of brain-derived phospholipid precursors known to stimulate epithelial proliferation in vitro. These data suggest that inositol may subserve quantitatively or qualitatively different functions in the keratinocyte than in other cell types.

Effects of iron-, manganese-, or magnesium-deficiency on the growth and morphology of Euglena gracilis.

Iron-, manganese-, or magnesium-deficiency has been induced in Euglena gracilis. Each arrests cell proliferation, decreases the intracellular content of the deficient metal, and increases that of several other metals. Light and electron microscopy of stationary phase cells reveal that Fe-deficient (-Fe) cells are similar in size and shape to control organisms. Magnesium-deficient (-Mg) cells, however, are larger, and approximately 14% are multilobed, containing 2 to 12 lobes of equal size emanating from a central region. Individual (-Mg) cells and each lobe of multilobed cells contain a single nucleus. Manganese-deficient (-Mn) organisms are morphologically more heterogeneous than (-Fe) or (-Mg) cells. Most are spherical and larger than controls. Approximately 15% are multilobed but, unlike (-Mg) cells, contain lobes of unequal size with either zero, one, or several nuclei present in each. Nuclei of (-Mn) cells differ in size and shape from those of control, (-Fe), or (-Mg) cells. All three deficient cell types accumulate large quantities of paramylon. Other cytoplasmic structures, however, appear normal. Addition of Fe, Mn, or Mg to the respective deficient stationary phase cultures reverses growth arrest and restores normal morphology. The results suggest that Fe-, Mn-, and Mg-deficiencies affect different stages of the E. gracilis cell cycle.

Relative responsiveness of cultured human epidermal melanocytes and melanoma cells to selected mitogens.

Early cellular events in the malignant transformation of melanocytes to melanoma are virtually unknown. In vitro investigation of this phenomenon has been hampered by the fastidious nature of the human epidermal melanocyte, which has proven difficult to cultivate. The present study compares responsiveness of cultured human epidermal melanocytes and established melanoma cell lines to serum, cholera toxin, and melanocyte growth factor (MGF), three established melanocyte mitogens. Four of four established human melanoma lines were substantially stimulated by fetal bovine serum, as were newborn foreskin-derived epidermal melanocytes. In contrast, none of the four melanoma lines responded to hypothalamic preparations containing MGF that consistently produced an approximately 30-fold increase in newborn melanocyte cell yield over a 2-week period. Cholera toxin, required for successful establishment of primary melanocyte cultures, had small and variable effects on the melanoma lines, with slight stimulation in one case, moderate inhibition in another, and essentially no effect in two others. These data suggest that transformation of epidermal melanocytes to melanoma often involves at least one phenotypic change resulting in escape from MGF regulation and another associated with insensitivity to cyclic AMP modulation; while at least some of the pathways conferring serum dependence are unaltered. Improved culture systems for the human epidermal melanocyte should facilitate further studies into the mechanism of its malignant conversion and may provide useful insights for the prevention and treatment of human melanoma.

Euglena gracilis chromatin: comparison of effects of zinc, iron, magnesium, or manganese deficiency and cold shock.

The effects induced by Fe, Mn, or Mg deficiency or cold shock on the DNA content and histones of Euglena gracilis have been examined and compared to those produced by Zn deficiency. The DNA content of the stationary-phase organisms used as controls is 2.1 micrograms/10(6) cells. The DNA of stationary-phase iron-deficient (-Fe), magnesium-deficient (-Mg), manganese-deficient (-Mn), zinc-deficient (-Zn), and cold-shocked (CS) cells is increased to 3.0, 4.6, 6.2, 3.8, and 3.8 micrograms/10(6) cells, respectively. The electrophoretic mobilities of proteins solubilized with 0.4 N H2SO4 from CS, -Fe, -Mg, and -Mn cells are nearly identical and are characteristic of the five histone classes, H1, H2A, H2B, H3, and H4. In contrast, no histones are found in the equivalent acid extract from -Zn cells. The effect of micrococcal nuclease on chromatin from control, CS, and -Zn cells was examined. The chromatin of CS cells is 1.2-fold while that from -Zn cells is 10-30-fold more resistant to micrococcal nuclease digestion than is the chromatin of control cells. Thus, the chromatin of cells grown in Zn-deficient conditions differs markedly from that of organisms cultured in media deficient in Fe, Mn, or Mg or exposed to cold shock.

Platelet arachidonate metabolism and platelet function in zinc-deficient rats.

To determine the effect of zinc status on arachidonate metabolism and the initiation of aggregation by a prostaglandin endoperoxide analog (U-44069), immature rats were fed for 5 days a low zinc (less than 1 ppm) or control (100 ppm Zn) diet based on soybean protein. In vitro production of thromboxane B2 (TXB2) by platelet-rich plasma after stimulation by either arachidonate (0.2 mM) or ADP (0.2 microM) was not changed by dietary zinc deprivation, even though bleeding time was prolonged. Oxygenation of exogenous arachidonate, catalyzed by either the cyclooxygenase or lipoxygenase pathway, was not affected by zinc deficiency. Platelet aggregation in response to the prostaglandin endoperoxide analog at both 8 and 16 microM was impaired by dietary zinc deprivation. While short-term zinc deprivation decreased platelet response to minimal levels of aggregating agents, production of arachidonate metabolites was unimpaired. It is postulated that platelet response to the products of arachidonate metabolism is reduced.

Zinc deficiency and impaired platelet aggregation in guinea pigs.

Previous studies have shown that acute zinc deficiency results in impaired platelet aggregation in humans and rats as well as decreased sensitivity to such aggregating agents as ADP, arachidonate and collagen. This study was designed to evaluate the effect of zinc deficiency on platelet function and other pathology in the guinea pig. Guinea pigs of mixed sex were fed a purified diet based on soybean protein (1 ppm Zn) or a similar control diet (100 ppm Zn). In one trial weanling guinea pigs, weighing about 150 g, were fed the diets for 22 days. Those fed the basal diet failed to grow after 2 weeks, and food consumption decreased at this time although it did not become cyclic. They developed skin lesions; zinc concentrations were decreased in plasma, red cells and liver. There was no effect on the packed cell volume. Guinea pigs weighing 350 g and fed the basal diet for 18 days showed little or no effect on growth rate and food intake, but tissue zinc levels were decreased. Plasma zinc dropped significantly within 24 hours. Platelet aggregation in response to minimal levels of ADP and a prostaglandin endoperoxide analog (U-44069) was severely impaired. Aggregation in response to bovine thrombin (1 unit/ml) was significantly delayed, but the partial response in the presence of indomethacin was not affected by zinc deficiency. The results suggest that impaired platelet aggregation is a general sign of zinc deficiency in mammals and that the function of the physiological eicosanoids is impaired.

Effect of acute zinc deprivation on plasma zinc and platelet aggregation in adult males.

In view of earlier results obtained with rodents, the present study was designed to investigate the effect of acute zinc deprivation in man on plasma zinc concentration and the response of platelets to aggregating agents. Three adult men consumed a formula diet based largely on soybean protein for 12 to 14 days. During the control period the diet was supplemented with 12 mg zinc per day. Without supplementation the diet supplied approximately 0.5 mg zinc per 3.0 Mcal; it contained 0.7 ppm zinc, and 0.2% phytate. After removal of the zinc supplement plasma zinc dropped rapidly and reached a minimum by the 5th day. There was a wide diurnal variation in plasma zinc concentration in one subject with the overnight fasting value being the highest and decreasing soon after the morning meal. Platelet aggregation in response to ADP and arachidonate was impaired when plasma zinc was 60 micrograms/dl or less and was restored to normal within 19 h of oral zinc supplementation. These results demonstrate that plasma zinc can be rapidly decreased by dietary zinc deprivation and that extracellular zinc plays an important role in platelet aggregation.

Astatine-211--tellurium radiocolloid cures experimental malignant ascites.

An investigation of the efficacy of astatine-211--tellurium colloid for the treatment of experimental malignant ascites in mice reveals that this alpha-emitting radiocolloid can be curative without causing undue toxicity to normal tissue. By comparison, negatron-emitting phosphorus-32 as colloidal chromic phosphate had no antineoplastic activity. The most compelling explanation for this striking difference is the dense ionization and short range of action associated with alpha-emission. These results have important implications for the development and use of alpha-emitters as radiocolloid therapy for the treatment of human tumors.