Biomedical subjects
J G Sharp
Publications and source records attributed to J G Sharp.
Segregation and characterization of lymphohematopoietic stromal elements.
In the mouse, long-term maintenance of multipotent hematopoietic stem cells in vitro currently requires the establishment of an appropriate adherent layer. When established employing culture medium supplemented with 20% horse serum and 10(-6) M hydrocortisone, the initial adherent layer contains primitive stem cells that are the major contributor of multipotent stem cells (CFUs) assayed subsequently in the supernatant regardless of the addition of fresh bone marrow cells. In contrast, when the adherent layer is established employing 25% fetal calf serum without hydrocortisone, few if any stem cells survive in the adherent layer. Such cultures are dependent upon a recharge with fresh bone marrow as a source of CFUs. A comparison of the latter stem cell-depleted system with the former intact system permits an evaluation of the relative contributions of the adherent layer and stem cells to the long-term maintenance of hematopoiesis in vitro. Studies of the effects of irradiation of the donor animal for the adherent layer and using the intact system demonstrated a reduction in the supernatant CFUs production that was dose- and time-related and evident at doses of 100 and 500 rads. If the adherent layer itself was irradiated immediately before refeeding, a reduction in supernatant CFUs was evident at a dose of 5 rads. These effects, both in vivo and in vitro, cannot be explained solely on the basis of cell killing. Rather, we propose that such doses inactivate, render impotent, or reduce the self-renewal capacity of stem cells that occupy a limited number of "niches" in the adherent layer. Although they are not killed, these impotent stem cells occupy stem cell niches but do not provide an effective contribution of CFUs to the supernatant cells. The adherent layer consists primarily of fibroblasts with significant numbers of macrophages and endothelial cells. The cellular composition of the adherent layer differs between the intact and stem cell-depleted systems in that the latter has a relatively larger proportion of endothelial cells. The composition of the adherent layer influences the type of differentiated cells in the supernatant. The stem cell-depleted adherent layer cultures had a greater proportion of granulocytes among the supernatant cells than macrophages that eventually predominate in the intact system. We have cloned stromal cells from the intact cultures and obtained several stromal cell lines by spontaneous transformation. One of these stromal cell lines (MC1) transfers at least a partial hematopoietic "microenvironment" (granulocytes, macrophages, erythroid cells) to ectopic sites on transplantation to syngeneic recipients.(ABSTRACT TRUNCATED AT 400 WORDS)
The relationship between the compensatory response in the stomach and colon and the extent of small bowel resection in the rat.
The proximal and distal extent of the compensatory response in the gastrointestinal tract of the young adult rat to resection of 10-80 percent of the combined ileum and jejunum was investigated. Thirty days after intestinal resection and subsequent end-to-end anastomosis, rats were injected with tritiated thymidine and sacrificed one hour later. The incorporation of label into colonic crypts or stomach glands was used as an index of the compensatory proliferative response. Although no compensatory response was observed in stomach glands, a significant compensatory proliferative response was found in descending colon after resections involving removal of greater than 60% of the combined ileum and jejunum. These observations imply that the compensatory proliferative response to resection is caused, at least in part, by a humoral mechanism. However, this does not exclude a role for luminal factors, intestinal blood flow, and paracrine effector substances as secondary modifiers of the compensation. The observations that resection of the small bowel can have significant effects distally on cell proliferation in the colon may be important to the planning of combination therapies which involve bowel resection in both animal models and in man.
Morphological assessment of immunologically relevant cells in the thymus.
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Scintigraphy and distribution of labeled antibodies in rats with tumors.
Radioiodinated antitumor (Ab-gamma globulins), non-tumor-specific Ab, and R131ISA were used for imaging radiation-induced intestinal tumors in rats. Each agent detected tumors larger than 2 g, but labeled Ab were most efficient in detecting small tumors. Tissue distribution studies showed that while 'purified' Ab localized specifically in tumors, 'unpurified' Ab concentrated in the tumor by a mechanism not considered immunological. Localization was variable and the concentration of antitumor Ab reached useful levels only in a small number of cases. The use of high specific activity purified Ab unexpectedly decreased the concentrations of label observed in the tumors when compared with the use of the same activity of low specific activity purified Ab. These results indicated the presence of circulating tumor antigens which were capable of binding the injected Ab. Subsequently, these findings have been substantiated. Thus the animal-to-animal variability could be explained on the basis of differing degrees of interaction of injected Ab with circulating tumor antigens. The usefulness of labeled purified or monospecific antitumor antibodies for tumor imaging and therapy would thus be influenced by the extent of such interactions.
Effects of reduced food intake on morphometry and cell production in the small intestine of the rat.
The present study examined the effects of a 60% reduction in food intake on kinetic and morphometric parameters in the small intestine of adult male Lewis rats. We observed that, after 20 days, the wet weight of jejunum and ileum, thickness of muscularis externa of duodenum, crypt depth throughout the intestine, and DPM/mg and DPM/crypt in ileum were decreased in animals on reduced food intake when compared to their paired, normally fed controls. These results demonstrated that reduced food intake caused distinctive effects focused primarily in the ileum, thus opening to question the use of the technique of pair feeding as a control for studies of intestinal cell proliferation in which manipulations of the animals result in altered food intake or body weight.
Evaluation of methods of assessing the intestinal epithelial proliferative response in adaptive situations.
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In vitro evaluation of hematopoiesis in mice treated with busulphan or nitrogen mustard.
Long term bone marrow cultures were established using combinations of untreated marrow and marrow treated with nitrogen mustard or busulphan in vivo. This study showed that 1) adherent cell layers, which morphologically resemble those derived from normal marrow, can be established from mice treated with busulphan or nitrogen mustard, 2) total cellularity in cultures containing a treated marrow is drastically reduced, 3) CFU-S content was slightly below control values in cultures of treated non-adherent cells on normal monolayers, and 4) CFU-S content was severely reduced when the monolayer was derived from drug-treated marrow. The usefulness of this long term culture system to study drug-induced modulation of hematopoiesis has been substantiated by this study.
Modulation of one of three murine bone marrow stromal cell lines to adipose cells by serum and insulin.
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Evidence against a systemic humoral factor controlling the intestinal compensatory response following X-irradiation.
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Initiation, motivation, and cessation of phencyclidine use.
An interview study was conducted on 200 phencyclidine (PCP) users in Northern California. The study population, generated through snowball sampling techniques, was constituted primarily of persons not in treatment programs and included three types of users: heavy chronic, light chronic, and recreational. Differences were found between the motivations and patterns of drug use of the three different user types. Contrary to prior studies, it was found that cessation of PCP use was difficult. Most subjects wanted to stop or cut back their use of PCP but were unable to do so. Many heavy chronic users stated they had a craving for PCP.
Acute effects of phencyclidine (PCP) on chronic and recreational users.
Snowball sampling techniques were used to generate a sample of 200 phencyclidine users from an area with a 10-year history of extensive PCP use. Three types of users were studied: heavy chronic, light chronic, and recreational users. The extent of PCP use varied from less than twice a month for a period of 6 months to daily use for several consecutive years. Each subject participated in a structured interview which lasted an average of 11/2 h. Subjects were asked about the acute effects of PCP, and about their moods before, during, and after using PCP. Scales based on previous research were used to measure the acute effects and moods. Results showed that heavy chronic users were more likely than recreational users to feel energized by PCP, and to experience negative ideations (thoughts about suicide and death). When age was controlled for, heavy chronic users were also more likely to experience violent effects. Analysis of moods over time showed some similar patterns between heavy chronic and recreational users, as well as some striking differences. Overall, heavy chronic users reported greater mood elevations while high on PCP, and a more dramatic drop in mood after the high wore off, than recreational users. Analysis of the results by user types clarified some of the confusion about contradictory descriptions of the effects of PCP, and point to the need to continue distinguishing between user types.
Pharmacokinetics of amantadine hydrochloride in subjects with normal and impaired renal function.
Amantadine is useful for the prevention and treatment of influenza A and for the treatment of Parkinson's disease and drug-induced extrapyramidal disorders. We have compared the pharmacokinetics of amantadine in patients with impaired or negligible renal function to that in normal subjects. The half-life of elimination in subjects with normal renal function was 11.8 +/- 2.1 hours (range, 9.7 to 14.5 h). Eight patients with various degrees of renal insufficiency (creatinine clearance from 43.1 to 5.9 mL/min . 1.73 m2) had half-lives of elimination from 18.5 h to 33.8 days. We also studied 10 patients on thrice-weekly hemodialysis. Assuming complete bioavailability of the drug, less than 5% of the dose was removed by each 4-hour hemodialysis. The mean half-life of elimination during chronic hemodialysis was 8.3 days (range, 7.0 to 10.3). We present guidelines for use of amantadine in patients with impaired renal function, including those on maintenance hemodialysis.
Evaluation of the morphological and functional characteristics of murine thymic non-lymphoid cells grown in vitro.
During the past few years, several investigators have attempted to grow the epithelial component of the murine thymus as a monolayer in vitro in order to study its proposed physiological role in T-lymphocyte differentiation. However, using for the most part identical culture techniques, we have provided morphological, histochemical and functional evidence that the majority of murine thymic nonlymphoid cells grown in culture are epithelioid macrophages and that these macrophages and/or their soluble factors may be responsible for many functions previously attributed to thymic epithelium or "purified thymic hormones". This study emphasizes the absolute necessity and importance of extensive morphological and histochemical evaluation of cultured cells prior to their use in physiological studies. Furthermore, any conclusion regarding the function of thymic non-lymphoid cells in vitro must first take into account numerous species differences in the morphological and histochemical characteristics of these cells as noted both in our laboratory as well as by other investigators.
Evaluation of monolayer, liquid, gelfoam sponge and artificial capillary culture systems for the study of hematopoiesis.
This study evaluated the usefulness of a matrix culture system, the artificial capillary culture system, for the growth of hematopoietic cells, the maintenance of CFUS and the collection of GM-CSF. The system was compared to monolayer, gelfoam sponge and Dexter liquid cultures. Monolayer cultures of adherent lymphohematopoietic stromal cells were prepared from bone fragments, whole bone marrow (Dexter system), spleen, fetal liver and thymus preparations. Morphologically, all the adherent cell populations were similar and tended to accumulate macrophages. Despite these morphological similarities, the ability of bone marrow adherent cell monolayers to maintain CFUS early in the culture period was significantly greater than adherent cells from other sources. Low but significant levels of GM-CSF were detected by bioassay in the supernatants of all but bone marrow adherent cell cultures. However, adherent cells selected from whole marrow cultures by differential trypsinization contained bioassayable levels of CSF suggesting that Dexter cultures contain a supernatant inhibitor/inactivator of GM-CSF. The greatest capability of the artificial capillary culture system was to increase about 500-fold over monolayers the GM-CSF collected from bone fragment derived stromal cells. Unfortunately, CFUS maintenance was poor, the system was more expensive than monolayer cultures and in our hands, suffered many mechanical failures usually resulting in loss of sterility. Histological evaluation suggested there was inadequate matrix for optimal attachment and growth of lymphohematopoietic stromal cells. Even so this system has a much greater potential for development than gelfoam sponges. Overall, Dexter cultures appear to be the most useful system currently available for the study of hematopoiesis and hematopoietic regulatory interactions.
Effects of thymectomy on bone growth in the rat.
The effects of thymectomy (TMX) at 1 or 5 days of age on the subsequent growth of bone have been determined in male and female Holtzman rats. The rats were maintained under aseptic conditions to minimize any effects of infection or wasting disease on bone growth. Femur length and distal epiphyseal cross sectional area were significantly reduced in TMX female rats at 3 months of age. At 1 month of age, the width of the undifferentiated zone was increased and at all subsequent times the columnar zone and growth plate were narrower in TMX rats. The incorporation of 35S into the femur, particularly the proximal and distal growth plates, was uniformly reduced in TMX rats as determined by scintillation counting and autoradiography. Additionally, there was an obvious overall reduction in alcian blue stationing intensity in the growth plates of TMX rats when compared to sham-operated controls. These results suggest a significant reduction in detectable glycosaminoglycan (GAG) content and in bone growth in TMX rats. Although there were minor variations, there were no major differences attributable to sex or timing of thymectomy. At present the causes of this moderate reduction in bone growth in TMX rats are unknown. One explanation might be that the thymus supplies or is in some other way involved in the provision of a hormone and/or cells that are required for normal bone growth. Another possibility is that the thymus is normally a source of immune suppressor cells which are needed to limit auto-immune reactivity against GAG or other antigenic determinants associated with bone. Thymectomy would remove these suppressor cells, thus permitting an auto-immune response which interferes with bone growth. Finally, perhaps the most plausible explanation is that thymectomy reduces cell production in the bone marrow which, in particular, reduces the turnover of monocytes. This reduced output of monocytes in TMX rats might ultimately lead to a reduction in osteoclast activity necessary for normal remodelling and growth of bone. We currently favour this last explanation.
Alterations in bone-marrow cellularity following thymectomy in rats.
The number of nucleated marrow cells was decreased following neonatal thymectomy in rats, and was corrected by administration of syngeneic lymphoid cells, or by implantation of a syngeneic testis. These results suggest that, in the rat, as has been shown previously in the mouse, lymphoid cells exert parital control over bone marrow cellularity and this effect may be further modulated by sex steroids.
Thymectomy modifies androgenizing effects of a testis transplant during critical period for neuroprogramming.
Thymectomy simultaneous with transplantation of a syngeneic testis from a littermate to Fischer 344 rats ameliorated the androgenizing effects of the testis transplant on ovarian morphology at 90 days of age.