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Biomedical subjects

C L Clark

Publications and source records attributed to C L Clark.

At least 55 records · Page 3Linked to original sources
PubMed1994

Lanthanum can be transported by the sodium-calcium exchange pathway and directly triggers catecholamine release from bovine chromaffin cells.

A comparison of the effectiveness of the trivalent cation, lanthanum (La3+) relative to Ca2+ in causing catecholamine release from bovine chromaffin cells has been made, together with a determination of the pathway by which La3+ enters these cells. In chromaffin cells maintained in tissue culture and permeabilised with digitonin, both La3+ and Ca2+ caused 3H release from cells preloaded with [3H]-noradrenaline; La3+ and Ca2+ caused similar maximal release but the EC50 for La3+ was an order of magnitude less than that for Ca2+. At maximal release caused by either La3+ or Ca2+ (approximately 14% of cell 3H content in 15 min), the other cation caused a small, but significant, further release. At submaximal effective concentrations the effects of the two cations were exactly additive. Using 3H release as an indicator of cytosolic La3+, its route of entry into intact chromaffin cells was investigated. With La(3+)-containing medium there was no release evoked by nicotine or by K(+)-depolarisation indicating that La3+ does not enter either via the nicotinic receptor linked ion channel or via voltage-sensitive (Ca2+) channels. However, in sodium-loaded chromaffin cells (ouabain incubation in Ca(2+)-free medium for 15 min) exposure to bathing media containing either Ca2+ or La3+ caused 3H release. La3+ (0.1 mM) caused a release similar in magnitude to that caused by Ca2+ (about 1 mM). La3+ at low concentrations had an additive (0.1 mM La3+) or synergistic (0.25-0.45 mM La3+) action with Ca2+ (< 3.6 mM) on 3H release. At higher concentrations (> 0.9 mM) the effects of La3+ predominated and prevented the expected effects of Ca2+. In other experiments, La3+ (1 mM) blocked export of 45Ca2+ via both Nao-dependent and independent pathways, i.e. sodium-calcium exchange and the calcium pump. The results indicate that La3+ can enter bovine chromaffin cells via the Nai/Cao exchange pathway independently of, or together with, Ca2+ but, that concentrations above 0.9 mM block the influx or efflux of Ca2+. However, Ca2+, even at 3.6 mM, did not block the influx of La3+. The results further indicate that, within chromaffin cells, La3+ is at least as effective as Ca2+ in triggering catecholamine release and maintaining prolonged release. La3+ also appears to act cooperatively with Ca2+ at the release pathway.

Adrenal Medulla↗
PubMed1994

Evidence for a novel source of relaxin: atrial cardiocytes.

Antibody-directed, complement-induced erythrocyte lysis (reverse hemolytic plaque assay) around atrial cardiocytes was used to determine whether this cell type possesses the capacity to secrete the insulin-like hormone relaxin. After 2h of incubation, 33 +/- 4% (n = 3) of cardiocytes derived from the atria of neonatal rats secreted detectable amounts of immunoreactive relaxin (i.e. formed plaques) when cultured in monolayers. Increased culture time of cardiocytes failed to increase the fraction of cardiocytes that secreted relaxin. The cumulative amount of relaxin secreted after 3h of incubation (plaque area) was 31% greater (P < 0.05) than the amount of hormone present after 1h of incubation, evidence of sustained peptide secretion by cultured cardiocytes. These data suggest that the source of the endogenous ligand for the specific and high-affinity relaxin receptors located in rat atria is the atrial cardiocyte itself. Therefore, relaxin may act via autocrine and/or paracrine routes to regulate cardiovascular structure and/or function.

Animals↗
PubMed1994

Giant uterine fibromyoma producing secondary polycythemia.

BACKGROUND: Although the association between large uterine fibromyomas and secondary polycythemia has been described previously, the mechanism has not been elucidated definitively. Investigators have measured erythropoietin levels in fibromyomas to determine whether these tumors are causing the polycythemia by erythropoietin overproduction; however, these studies were performed before the availability of recombinant erythropoietin assays. CASE: A 59-year-old woman presented with a 3-year history of polycythemia. Pelvic examination revealed a large lower abdominal mass. Laboratory evaluation revealed a hemoglobin of 20.8 g/dL, red blood cell mass of 3300 mL, oxygen pressure of 58 mmHg with an oxygen saturation of 89%, and erythropoietin level of 18 mU/mL. Cardiac echocardiogram showed no evidence of shunt. Computed tomography scan of the abdomen showed a large mass arising in the pelvis and compressing both ureters. The patient was treated surgically with a total abdominal hysterectomy. Pathology confirmed a uterine leiomyoma weighing 2320 g. Two months post-surgery, the patient was asymptomatic with a hemoglobin of 13.9 g/dL and erythropoietin level less than 4.0 mU/mL. CONCLUSION: This case provides evidence for three of the postulated mechanisms by which uterine fibromyomas may cause polycythemia. First, the patient was hypoxic, suggesting shunting within the tumor. Second, the leiomyoma was compressing the ureters, so the kidneys may have been inappropriately producing excess erythropoietin. Third, the tumor itself may have been producing the erythropoietin. In any case, the erythropoietin level in this patient was inappropriately high, providing useful evidence that her polycythemia was secondary to her fibromyoma.

Female↗
PubMed1993

Lipopolysaccharide and a phorbol ester stimulate secretion of tumor necrosis factor-alpha from alveolar macrophages through action on overlapping subsets of cells.

A cell-by-cell analysis of the secretory ability of stimulated, individual alveolar macrophages (AMs) was performed through use of a tumor necrosis factor alpha (TNF-alpha) reverse hemolytic plaque assay. Two functional end points were measured: the percentage of AMs that were TNF-alpha secretors and the cumulative amount of TNF-alpha secreted by AMs (average plaque area, microns 2). Lipopolysaccharide (LPS; 100 micrograms/ml) increased cumulative TNF-alpha release at both 7 and 20 h of incubation. On the other hand, a phorbol ester (phorbol myristate acetate, PMA) stimulated TNF-alpha release at 20 h of incubation but not at 7 h. Under nonstimulated culture conditions, 5-10% of all AMs released detectable TNF-alpha PMA (but not LPS) induced a significant increase in the fraction of AMs capable of releasing TNF-alpha (15.1 +/- 1.1% vs. 9.0 +/- 1.6%, PMA vs. control, P < .05). Differences in the time course of secreted TNF-alpha, together with the recruiting effect of PMA, suggest that LPS and PMA target TNF-alpha-secretory subpopulations of AMs that differ in number and secretory characteristics.

Animals↗
PubMed1993

The effect of rejection and graft-versus-host disease on small intestinal microflora and bacterial translocation after rat small bowel transplantation.

Bacterial translocation and the development of sepsis after small bowel transplantation may be promoted by immunological damage to the intestinal mucosa or by quantitative and qualitative changes in intestinal microflora. This study assessed the effects of rejection, graft-versus-host disease (GVHD) and immunosuppression on intestinal microflora and bacterial translocation after heterotopic rat small bowel transplantation. Isografts, allografts with and without CsA immunosuppression, and the semi-allogeneic parent to the F1 hybrid GVHD model were studied. Intestinal microflora in graft and host loops and bacterial translocation to host organs and the graft mesenteric lymph node were determined. Bacterial colonies were counted and individual colonies identified using API 20E nutrient and fermentation indicator techniques. Colony counts in isografts and allografts were significantly higher than in the native intestine, whereas there was a massive overgrowth in the native intestine in the GVHD group. The species profile for the host and graft loops was similar in animals that had received isografts, allografted animals receiving CsA, and animals undergoing GVHD. However, there was a large increase in Staphylococcus epidermidis in animals with rejection. Bacterial translocation was not detected in isografted animals, but was observed in all other animal groups, with S. epidermidis being the most prevalent organism. These findings demonstrate that rejection and GVHD are associated with shifts in intestinal microflora toward potentially pathogenic organisms and that bacterial translocation into recipient tissues poses a major threat for the development of sepsis.

Animals↗
PubMed1993

Relaxin secretion by porcine large luteal cells: effect of protein synthesis inhibitors.

The purpose of the experiments reported herein was to investigate the relative importance of new hormone synthesis to basal and prostaglandin E2-stimulated rates of relaxin release. A relaxin-reverse hemolytic plaque assay was used to monitor relaxin release from individual large luteal cells (LLC) in which new protein synthesis was inhibited by cycloheximide or actinomycin D. These treatments significantly decreased the rate of relaxin release. In addition, cycloheximide reduced the total fraction of LLC possessing the ability to form plaques by about 10%, suggesting complete suppression of relaxin from this subset of cells. Exposure of inhibitor-treated LLC to prostaglandin E2 (a relaxin stimulatory secretagogue) enhanced relaxin release, and restored suppressed LLC back into the secretory population. Taken overall, these results demonstrate that the majority of relaxin-releasing LLC exploit a mixture of newly synthesized and older, stored hormone to achieve basal secretion. A minority of relaxin-releasing LLC, however, appear to depend wholly on newly synthesized hormone for basal secretion. The differential activity (and interaction) of these pathways in individual LLC may provide a potential explanation for the markedly heterogenous manner of hormone release observed in this (and other) cell types, and for the action of relaxin secretagogues.

Animals↗
PubMed1992

Introduction of a partial shift system for house officers in a teaching hospital.

OBJECTIVES: (1) To introduce a partial shift system to reduce the hours of work of preregistration house surgeons to an average of 64 a week to comply with the New Deal for junior doctors; (2) to test linking the partial shift concept to an existing structure of "on call" firms. DESIGN: Formal assessment after three months of a pilot partial shift system for eight house surgeons on three firms instituted on 1 November 1991, followed by questionnaire and interview evaluation at three and six months of a revised system implemented on 1 February 1992. SETTING: Department of general surgery at St Bartholomew's Hospital, London. SUBJECTS: 24 house surgeons attached to three surgical firms. RESULTS: In eight weeks each house surgeon worked one week (five shifts) of night duty, one week of "cover" (afternoon and evening) duty, and six weeks of normal daytime hours. Each weekday a house surgeon from the firm on call worked an extended daytime on call shift until 10 pm. Weekend duties were split between two house surgeons from the firm on call. A computer generated graphical display of the rota was used to facilitate leave planning. Average working hours were reduced to below 64 per week, including prospective cover, without detriment to patient care and educational standards. Within the shift system individual house surgeons could be on call with their own firm by day and at weekends. Opinions were equally divided among junior staff as to their preference for either on call or partial shift systems. CONCLUSIONS: The principles of this partial shift system are generally applicable and the model can readily be adopted by district general hospitals.

Attitude of Health Personnel↗
PubMed1992

Persistence of allogeneic cells in graft and host tissues after small bowel transplantation.

Small bowel transplantation is associated with a significant risk of graft versus host disease owing to the large amount of organized lymphoid tissue within the graft. This study assessed whether graft lymphoid cells could persist in the long term following fully allogeneic small bowel transplantation when graft rejection was prevented by cyclosporin immunosuppression. Transplantation was carried out between PVG and DA strains of rat. Cyclosporin (15 mg/kg) was given daily from transplantation, and groups of animals were studied at 28 and 56 days after grafting. The proportions of donor- and recipient-derived cells in the graft and in the host gut and lymphoid tissues were assessed using immunohistochemical tissue staining and monoclonal antibodies specific for cells expressing class I antigens from the two strains of rat. Results demonstrated a persisting population of graft-derived T cells which were capable of migration to the host. Therefore, there may be a long-term risk of graft versus host disease after small bowel transplantation under cyclosporin immunosuppression.

Animals↗
PubMed1992

Evidence that basal secretion of relaxin by individual cultured large luteal cells is influenced by mobilization of intracellular calcium: analysis by a reverse hemolytic plaque assay.

Ca2+ redistribution from an intracellular site(s) is a key biochemical event associated with relaxin (RLX) secretion by large luteal cells (LLCs) of porcine origin. However, the functional significance of internal stores of Ca2+ to basal rates of RLX secretion is not well understood. In addition, the identity of the intracellular storage site(s) for Ca2+ within LLCs is not known, nor is it clear if all RLX-releasing LLCs are equally dependent on this pool. In the present study, release of RLX from 24 h cultured luteal cells derived from early pregnant swine was monitored by a reverse hemolytic plaque assay (RHPA). Incubation of cultures in the presence of graded concentrations of thapsigargin (1 nM-1 microM), a plant sesquiterpene lactone that inhibits endoplasmic reticulum Ca(2+)-ATPase and thereby increases cytosolic Ca2+ concentrations, resulted in a dose-related increase in basal RLX secretion. The stimulatory effect of thapsigargin on RLX production was not abrogated by culture in Ca(2+)-free medium. Suppression of Ca2+ release from the endoplasmic reticulum of LLCs, achieved by incubating monolayers in medium containing dantrolene (1-100 microM), resulted in dose-related inhibition of basal RLX release. Taken together, these results suggest that the endoplasmic reticulum serves as a major storage site for Ca2+ redistribution within LLCs and, furthermore, that mobilization from this site is functionally coupled to basal secretion of RLX.

Animals↗
PubMed1992

Reduction of graft-versus-host reactivity after small bowel transplantation: ex vivo treatment of intestinal allografts with an anti-T cell immunotoxin.

A specific T lymphocyte immunotoxin was used to pre-treat small bowel grafts in an attempt to prevent graft-versus-host (GVH) reactivity and GVH disease in a rat transplant model. The immunotoxin used was a conjugate of the anti-CD5 MoAb MRC OX-19 with ricin A chain. The grafts were perfused ex vivo with a standard solution of immunotoxin followed by incubation at 4 degrees C for 1 h before transplantation. In a semi-allogeneic strain combination (parent to F1 hybrid offspring) graft treatment with immunotoxin led to a prolongation of recipient survival compared with groups receiving similar transplants without immunotoxin treatment. An additive effect on survival was observed when the host was treated with cyclosporin. The effect of immunotoxin was greater than that of mesenteric lymphadenectomy in increasing host survival. The effect of graft treatment with the immunotoxin on cellular migration from graft to host lymphoid tissues was assessed in fully allogeneic transplantation (PVG to DA). Host lymphoid tissues were subjected to immunohistochemical analysis using a MoAb specific for donor class I MHC antigens. Graft treatment with the immunotoxin led to a significant decrease in the number of graft cells found in host lymphoid tissues 7 days after transplantation. However, this effect was less marked than that achieved by graft mesenteric lymphadenectomy. With our current protocol graft treatment with a specific T cell immunotoxin can significantly reduce but not abolish GVH reactivity in rat small bowel transplantation.

Animals↗
PubMed1992

Basic fibroblast growth factor inhibits basal and stimulated relaxin secretion by cultured porcine luteal cells: analysis by reverse hemolytic plaque assay.

The role of the basic fibroblast growth factor (basic FGF) in the control of secretion of the ovarian protein hormone relaxin (RLX) by porcine large luteal cells (LLCs) was examined by use of a reverse hemolytic plaque assay. In this assay, luteal cells were cocultured in monolayers with protein-A-coupled sheep erythrocytes. In the presence of complement and porcine relaxin antiserum, a zone of hemolysis (a plaque) developed around a RLX-releasing LLC. The rate of plaque development in time-course experiments was used as an index of the rate of RLX secretion. Monolayers were bathed in medium containing graded concentrations of basic FGF in the presence or absence of a stimulatory secretagogue [0.01 microM prostaglandin E2 (PGE2)]. Exposure of luteal cell-containing monolayers to basic FGF resulted in a significant reduction (P less than 0.05) in the rate of RLX-induced plaque formation, evidence of an inhibitory effect of basic FGF on the rate of basal RLX secretion. This suppressive effect was variable in onset (1-3 h of incubation) and dose related. Minimally and maximally effective doses were about 10 and 30 ng/ml basic FGF, respectively. Higher doses of basic FGF (20 and 30 ng/ml) entirely suppressed RLX from a substantial subset of LLCs (10-20% of all LLCs), an indication of a differentially sensitive subpopulation. Addition of basic FGF (30 ng/ml) to monolayes also treated with PGE2 resulted in a significant (P less than 0.05) attenuation of the stimulatory effect of PGE2 on RLX secretion, evidence that these agents functionally interact in the modulation of RLX. We conclude that these results taken in association with the prior demonstration of the presence of basic FGF in luteal tissue suggest that basic FGF serves as a local inhibitory mechanism that regulates RLX secretion. Furthermore, the ability of basic FGF to counteract the effect of PGE2 implies that intraluteal stimulatory/inhibitory agents may act in concert to achieve fine control of RLX secretion. The observation of a preferentially responsive subpopulation is consistent with the possibility that basic FGF is implicated in heterogenous RLX secretion. Nevertheless, the physiological role(s) of basic FGF in the control of RLX secretion and the interrelationships of basic FGF with other local and systemic secretagogues remain to be clearly defined.

Animals↗
PubMed1992

Discordant secretion of relaxin by individual porcine large luteal cells: quantitative analysis by a reverse haemolytic plaque assay.

Individual large luteal cells (LLC) derived from pregnant swine differ conspicuously in their ability to secrete the peptide hormone relaxin under basal and stimulated conditions--the phenomenon of functional heterogeneity. The purpose of this study was to quantitate knowledge of this phenomenon through use of a reverse haemolytic plaque assay, a technique that utilizes antibody-directed, complement-mediated erythrocyte lysis to detect hormone secretion by single LLCs in culture. Measurement of individual plaque areas (an index of the amount of relaxin secreted) demonstrated an approximate 100-fold range in the amount of relaxin secreted by a single cell under basal conditions. This range was doubled by exposure to the phorbol ester, 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA; 50 nmol/l). Under basal conditions, 50 and 80% of the total amount of relaxin was secreted by approximately 10 and 30% of all LLCs respectively. The size of these fractions was not influenced by the time of incubation (1-8 h), or by the presence of either of two non-specific stimulatory relaxin secretagogues, PMA (50 nmol/l) or arachidonic acid (1 mumol/l). The unimodal frequency distribution of plaque areas (under basal or stimulated conditions) suggests that relaxin-secreting LLCs comprise a discrete functional population of secretory cells, at least under these experimental conditions. We conclude that a remarkably small fraction of LLCs secretes the majority of relaxin, and that the size of this fraction was not influenced by time or secretagogues.

Animals↗
PubMed1992

Transforming growth factor-beta is a potent inhibitor of basal and stimulated relaxin release by porcine luteal cells maintained in monolayer culture.

The effect of transforming growth factor-beta (TGF-beta) on relaxin release by porcine large luteal cells (LLC) was examined by use of a reverse haemolytic plaque assay. In this assay, mixed luteal cells were co-cultured in monolayers with protein A-coupled sheep erythrocytes. In the presence of complement and porcine relaxin antiserum, a zone of haemolysis (a plaque) developed around relaxin-releasing LLCs. The rate of plaque development in time-course experiments and the average size of plaque areas were used to monitor the rate of relaxin release and cumulative amounts of hormone respectively. Monolayers were bathed in medium containing TGF-beta alone, or in the co-presence of a stimulatory secretagogue (prostaglandin E2; PGE2). Exposure of luteal cell-containing monolayers to TGF-beta (1 ng/1-100 micrograms/1) elicited a dose-related inhibition in the rate of basal relaxin release. Minimal and maximal concentrations were approximately 10 ng/1 and 10 micrograms/1 respectively. Treatment with 1 microgram TGF-beta/1 reduced the cumulative amount of relaxin released to 63 +/- 6% of control values (mean +/- S.D., P < 0.05, n = 6; averaged over the whole course of the experimental incubation). Exposure of monolayers treated with TGF-beta to the relaxin-stimulatory secretagogue PGE2 (0.1 mumol) resulted in a significant (P < 0.05) increase in the amount of relaxin released by TGF-beta-suppressed LLCs, and restored rates of hormone release to control levels. This is evidence that TGF-beta and PGE2 interact antagonistically in the modulation of relaxin. The effect of TGF-beta was strictly time-dependent.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗