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C L Clark

Publications and source records attributed to C L Clark.

At least 73 records · Page 4Linked to original sources
PubMed1991

Graft versus host disease in small bowel transplantation.

Quantities of organized lymphoid tissue in small bowel allografts may cause graft versus host disease (GVHD) following transplantation. This study examines the effect of graft mesenteric lymphadenectomy on development of GVHD following small bowel transplantation in rats. GVH reactivity was assessed by measuring the degree of graft cell emigration to the host. In the PVG to DA strain combination, graft mesenteric lymphadenectomy led to a significant reduction in graft cell colonization of host lymphoid tissues from 40-50 per cent to 25-35 per cent. Transplantation from PVG to (PVG x DA)F1 hybrids caused fatal GVHD within 21 days whereas when DA donors were used survival was over 30 days. When mesenteric lymphadenectomy was performed on PVG donors, host survival increased by only 3-4 days. Mesenteric lymphadenectomy in DA donors led to long-term recipient survival with no GVHD. Intensity of GVHD following rat small bowel transplantation is a strain-dependent phenomenon and graft mesenteric lymphadenectomy does not always prevent GVHD. The mucosa may have an important immunological role.

Animals↗
PubMed1991

A comparative study between Michel and Proximate clips for the closure of neck incisions.

A prospective study comparing two types of surgical clip, the Proximate II (Ethicon) staple and the Michel clip, was performed on 30 thyroidectomy (horizontal) and 50 carotid endarterectomy (vertical) incisions with reference to their ease of handling, patient comfort, complication rate, cosmetic result and cost. In each patient half of the wound was approximated with one type of clip and half with the other. The Proximate II staple was more comfortable for the patient in the vertical wounds (P less than 0.02), easier to remove as judged by the nurse in both horizontal (P less than 0.001) and vertical (P less than 0.02) wounds, and by the patient in the horizontal wounds (P less than 0.02) only. The incidence of inflammation was significantly higher with the Michel clip (P less than 0.001). In both the horizontal (P less than 0.05) and the vertical (P less than 0.001) wounds, when the scar was assessed at the time of discharge from hospital, the Proximate II staple achieved a significantly better result. However, assessment of the wounds by patient and researcher 6 weeks postoperatively, revealed no significant difference between the wounds with regard to cosmetic outcome. The Proximate II design, however, costs between seven and ten times more than the Michel clip depending on the size of the dispenser used, and since the late cosmetic result offers no advantage over the latter design, we feel these financial variables deserve consideration.

Adult↗
PubMed1990

Effect of antimicrotubule agents on secretion of relaxin by large luteal cells derived from pregnant swine.

The role of microtubules in the modulation of secretion of the ovarian protein hormone, relaxin, 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 ovine erythrocytes. In the presence of porcine relaxin antiserum and complement, a zone of hemolysis, a plaque, developed around relaxin-releasing LLCs. The rate of development of plaques in time-course experiments (1-12 h) and the detection of discontinuities in the pattern of plaque formation were used in this study as an index of the rate of relaxin release and a method to detect differential responsiveness of individual LLCs, respectively. Monolayers were bathed in medium containing three different antimicrotubule agents that bind to tubulin and induce microtubule loss through depolymerization. Exposure of luteal cell-containing monolayers to colchicine (100 microM), vinblastine (1 microM), and nocadazole (30 microM) resulted in a reduction in the rate of relaxin-induced plaque formation by LLCs, unequivocal evidence of an inhibitory effect of all three of these antimicrotubule agents of the rate of basal relaxin release. However, this suppressive effect on relaxin release was strictly time related. None of the antimicrotubule agents significantly affected plaque formation until 3 h of incubation. Thereafter, a plateau of plaque formation was observed in antimicrotubule-treated monolayers over approximately 3-8 h of incubation, indicating substantial slowing or cessation of relaxin release by LLCs during this period. During this middle phase of the incubation, plaque formation in antimicrotubule-treated monolayers was significantly lower (P less than 0.05) than that in controls. By 8-12 h of incubation plaque formation was still significantly suppressed in vinblastine-treated monolayers, but not, however, in colchicine- or nocadozole-treated monolayers. Treatment with beta-lumicolchicine (100 microM; a form of colchicine that fails to bind to tubulin) resulted in no significant change in the rate of plaque formation compared with that in controls. We conclude that microtubule-assisted transport forms an important intracellular mechanism that assists basal release of an ovarian peptide hormone, relaxin. Moreover, the discontinuity of plaque formation in the middle phase of the incubation is consistent with the view that a subpopulation of LLCs responds preferentially to antimicrotubule agents. However, the exact nature and physiological role(s) of microtubules in the control of relaxin secretion and the interrelationships of microtubules with other intracellular regulatory events remain to be clearly defined.

Animals↗
PubMed1990

High-performance liquid chromatography determination of erythrocyte membrane phospholipid composition in several animal species.

High-performance liquid chromatography (HPLC) was used to determine the phospholipid (PL) composition of ovine, equine, bovine, porcine, and canine RBC membranes. Procedural modifications of established techniques provided for separation of 7 PL within a 15- to 20-minute sample run. Significant (P less than 0.05) differences were detected in RBC membrane PL composition among the various species. The concern for physiologic properties associated with hemolysis and/or sedimentation rate must include evaluation of differences in the PL bilayer structure.

Animals↗
PubMed1989

Analysis of relaxin release by cultured porcine luteal cells using a reverse hemolytic plaque assay: effects of arachidonic acid, cyclo- and lipooxygenase blockers, phospholipase A2, and melittin.

The effect of the obligatory precursor of prostaglandin biosynthesis, arachidonic acid, on the release of relaxin by porcine luteal cells was examined by use of a reverse hemolytic plaque assay. In this assay, luteal cells were cocultured in monolayers with protein-A-coupled ovine erythrocytes. In the presence of porcine relaxin antiserum and complement, a zone of hemolysis, a plaque, developed around relaxin-releasing luteal cells, identified as large luteal cells (LLCs). The rate of development of plaques in time-course studies and the area of plaques were then used as an index of the rate of relaxin release and cumulative amount of hormone released, respectively. Incubation of collagenase-dispersed luteal cells derived from early pregnant pigs with 0.1-100 microM arachidonic acid (AA) resulted in dose-dependent increases in the rate of plaque formation. Despite AA stimulation, however, only 55-65% of all LLCs formed plaques during the experimental incubation period (up to 12 h). Minimally and maximally effective doses were about 1 and 10 microM, respectively. In the presence of 10 microM AA, maximal plaque formation occurred significantly faster (1-2 h) than in controls (4-8 h; P less than 0.05). The percentage of plaque-forming cells (plaque-forming LLCs) was, likewise, significantly greater in 10 microM AA-treated monolayers than in controls during the first 3-4 h of incubation. Similarly, agents that liberate endogenous AA (phospholipase A2 and melittin) also stimulated relaxin release. The stimulatory effect of AA (10 microM) on relaxin release was almost wholly blocked by a cyclooxygenase inhibitor (ibuprofen; 20 microM); but not by a lipooxygenase inhibitor (nordihydroguaretic acid; 20 microM). However, the same dose of ibuprofen (20 microM) failed to modulate the stimulatory effect of prostaglandin E2 (1 microM) or phorbol diester (4 beta-phorbol 12 beta-myristate 13 alpha-acetate; 50 nM) on the rate of relaxin release. These results indicate that a product(s) of the cyclooxygenase pathway of AA metabolism participates in the control of relaxin release, but that this metabolite(s) is not essential to the biological action of at least one stimulatory secretagogue. Moreover, this metabolite failed to influence a subpopulation of nonresponsive LLCs. These data taken in association with our previous demonstration that the pathways of both calcium mobilization and protein kinase-C activation are implicated in the regulation of relaxin release, are consistent with the view that AA liberation may amplify the actions of other signalling mechanisms.

Animals↗
PubMed1988

Inhibitory effect of analogues of cyclic nucleotides and cholera toxin on relaxin release from cultured porcine luteal cells.

The role of cyclic nucleotides (cyclic 3',5'-adenosine monophosphate [cAMP] and cyclic 3',5'-guanosine monophosphate [cGMP]) in the regulation of relaxin release from large porcine luteal cells was examined by use of a reverse hemolytic plaque assay. In this assay, luteal cells are cocultured in monolayers with protein-A-coupled ovine erythrocytes. In the presence of porcine relaxin antiserum and complement, a zone of hemolysis--a plaque--develops around relaxin-releasing luteal cells. The rate of development of plaques in time-course studies has been used as an index of the rate of relaxin release, and the size of plaques formed has been employed as a record of the cumulative amount of relaxin released by each cell. Treatment of monolayers with dibutyryl cAMP (dbcAMP, 60 mM) and dibutyryl GMP (dbcGMP, 15 mM resulted in a prompt inhibition in the rate of plaque formation. In addition, dbcAMP treatment reduced the average size of plaques formed. The stimulatory effect of prostaglandin F2 alpha (PGF2 alpha 10(-6) M) on relaxin release was significantly attenuated by combined treatment with dbcAMP (60 mM). Cholera toxin treatment (500 ng/ml) effectively reduced the average size of plaques formed, but neither this agent nor the beta-adrenergic agonist, isoproterenol (up to 5 X 10(-3) M), influenced the rate of plaque formation. These results--which provide evidence to show that both basal and stimulated relaxin release by large porcine luteal cells can be inhibited by the cyclic nucleotide analogues, dbcAMP and dbcGMP--are consistent with the view that these compounds have the potential to act as a negative regulatory mechanism for relaxin release.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Stimulatory effect of phorbol diester on relaxin release by porcine luteal cells in culture.

The role of protein kinase C (PKC) activation in the modulation of secretion of the peptide hormone, relaxin, by porcine luteal cells was examined by use of a reverse-hemolytic plaque assay. In this assay, luteal cells were cocultured in monolayers with protein-A-coupled ovine erythrocytes. In the presence of porcine relaxin anti-serum and complement, a zone of hemolysis--a plaque--developed around relaxin-releasing luteal cells. The rate of development of plaques in timecourse studies was then used as an index of the rate of relaxin release. The tumor-promoting agent, phorbol 12-myristate 13-acetate (PMA) activates a phospholipid- and calcium-dependent kinase, PKC. This enzyme is present in high concentrations in porcine luteal tissue, although its physiological role(s) is unknown. We report here that PMA exerted a time- and dose-dependent stimulatory effect on relaxin release by enzyme-dispersed porcine luteal cells in culture. Maximum stimulation was achieved by 50nM PMA. In contrast, the non-PKC-activating phorbol ester, 4 alpha-phorbol-12,13-didecanoate, exerted no significant effect on the rate of relaxin in doses up to 1 microM. We further observed that a synthetic 1,2-diacyl-glycerol (1-oleoyl-2-acetyl-rac-glycerol; 125 microM) mimicked the action of PMA in stimulating relaxin secretion. These results are consistent with the view that activation of PKC provides at least one intracellular mechanism that regulates relaxin secretion by porcine luteal cells.

Animals↗
PubMed1988

Regulation of relaxin release from monodispersed porcine luteal cells: effect of calcium ionophore A23187 and calcium channel blockers.

The role of calcium ion mobilization in modulation of secretion of the ovarian protein hormone relaxin by porcine luteal cells was examined by use of a reverse hemolytic plaque assay. In this assay, luteal cells were cocultured in monolayers with protein-A-coupled ovine erythrocytes. In the presence of porcine relaxin antiserum and complement, a zone of hemolysis, a plaque, developed around relaxin-releasing luteal cells. The rate of development of plaques in time-course experiments was used in this study as an index of the rate of relaxin release. Exposure of luteal cell-containing monolayers to the calcium-mobilizing agent A23187 (40 nM to 5 microM) resulted in a dose-related increase in the rate of relaxin-plaque formation. This effect [and the influence of a stimulatory secretagogue, prostaglandin E2 (PGE2; 1 microM)] was suppressed by coculture with Co2+ (5 mM), a calcium channel blocker. These results are consistent with the view that calcium ion redistribution within porcine luteal cells forms a pathway that subserves, at least in part, the rates of basal and stimulated relaxin release in vitro. However, A23187 was equally effective in enhancing the rate of plaque formation when the monolayers were bathed in a low calcium medium (mean +/- SEM, 6.61 +/- 0.92 microM Ca2+), rather than a calcium-replete medium (1.56 +/- 0.09 mM Ca2+). Likewise, neither basal nor PGE2-stimulated (1 microM) relaxin secretion was abrogated by culture of monolayers in low calcium medium. These data suggest that the stimulatory effect of A23187 (and perhaps PGE2) arose predominantly through redistribution of calcium stored within intracellular sites in luteal cells, rather than entrance of calcium into the cell from the extracellular medium. Yet, incompatible with this interpretation, we observed that TMB-8 [8-(N,N-diethylamino)octyl 3,4,5-trimethoxybenzoate hydrochloride; a putative inhibitor of intracellular calcium redistribution] enhanced rather than blocked A23187- or PGE2-stimulated relaxin release. This result is consistent with the possibility that TMB-8 mobilized (rather than blocked) calcium in this cell system or that it acted via calcium-independent mechanisms. We conclude that calcium mobilization has the potential to act as a molecular pathway that transduces secretion of an ovarian peptide hormone, relaxin. However, the exact nature and physiological role(s) of the Ca2+ pathway in the control of relaxin secretion and the interrelationships of this mechanism with other intracellular messengers that may also modulate ovarian peptide secretion remain to be more clearly defined.

Animals↗
PubMed1988

Analysis of release of porcine relaxin by reverse haemolytic plaque assay: evidence for autoregulation.

Relaxin release from monodispersed luteal cells derived from pregnant pigs (days 100-110 of gestation; n = 3) was detected by a reverse haemolytic plaque assay. In this technique, luteal cells were co-cultured in monolayers with ovine erythrocytes coupled to protein-A. In the presence of porcine relaxin antiserum and complement, a zone of haemolysis--a plaque--developed around relaxin-releasing luteal cells. Luteal cells were preincubated with porcine relaxin (up to 1000 ng/ml) or progesterone (up to 0.5 micrograms/ml) for 18 h before the plaque assay. Relaxin (but not progesterone) pretreatment inhibited subsequent relaxin release. The inhibitory effect of relaxin on its own release (autoregulation) has the potential to be a significant part of the local mechanisms which contribute to the overall control of ovarian secretion of protein hormones.

Animals↗
PubMed1987

Detection of relaxin release by porcine luteal cells using a reverse hemolytic plaque assay: effect of prostaglandins E2 and F2 alpha, human chorionic gonadotropin, and oxytocin.

The release of relaxin from cultured porcine luteal cells derived from pregnant sows was detected by a reverse hemolytic plaque assay. In this assay, luteal cells are cocultured in monolayers with protein-A-coupled ovine erythrocytes. In the presence of porcine relaxin antiserum and complement, a zone of hemolysis--a plaque--develops around relaxin-releasing luteal cells. Treatment with prostaglandin E2 (10(-8) and 10(-6) M) significantly accelerated the rate of plaque formation; in contrast, human chorionic gonadotropin (10-1,000 IU/ml) inhibited the rate of plaque formation. Oxytocin (10(-8) to 10(-4) M) had no detectable effect on relaxin release. However, none of these treatments or long-term preexposure to prostaglandin F2 alpha increased the total proportion of large luteal cells that released relaxin, which remained at about 50%. These results are consistent with the idea that prostaglandins of uterine and/or luteal origin and pituitary luteinizing hormone may contribute, alone or perhaps in combination, to the overall regulation of ovarian relaxin release during pregnancy in the sow. In addition, the results indicate that the effects of prostaglandins are restricted to a subpopulation of large luteal cells that release detectable amounts of relaxin in culture.

Animals↗
PubMed1987

Prostacyclin stimulates relaxin release from cultured porcine luteal cells.

The release of relaxin from cultured porcine luteal cells (derived from pregnant sows) was detected by a reverse hemolytic plaque assay. In this assay, luteal cells are cocultured in monolayers with protein-A-coupled ovine erythrocytes. In the presence of porcine relaxin antiserum and complement, a zone of hemolysis--a plaque--develops around relaxin-releasing luteal cells. Treatment with a prostacyclin analogue (carba-prostacyclin; 10 and 100 ng/ml) significantly accelerated the rate of plaque formation in a dose-dependent manner. In contrast, the culture of luteal cells in the presence of cyclooxygenase inhibitors (indomethacin, ibuprofen, and diclofenac sodium) resulted in no change in the rate of basal relaxin release. These results add weight to the view that prostacyclin may contribute alone, or perhaps in combination with other prostanoids, to the overall regulation of ovarian endocrine function and relaxin release during pregnancy in the pig. However, the data are inconsistent with the possibility that prostacyclin arises from relaxin-releasing luteal cells themselves.

Animals↗
PubMed1987

Analysis of relaxin release from cultured porcine luteal cells by reverse hemolytic plaque assay: influence of gestational age and prostaglandin F2 alpha.

Relaxin release from dispersed luteal cells was detected by a reverse hemolytic plaque assay to determine the influence of gestational age on basal relaxin secretion. Monodispersed luteal cells were derived by collagenase treatment of corpora lutea obtained from pigs in early (days 19-26), mid-(days 47-62), and late (days 80-99) gestation. The rate of plaque development under nonstimulated conditions progressively accelerated as gestation advanced, as did the rate of increase in plaque size. These results unequivocally demonstrate that basal relaxin release increases with advancing gestation. However, only about 50% of large luteal cells released relaxin at all stages of pregnancy examined up to day 100. These data indicated not only that basal relaxin release increases during pregnancy, but also that considerable heterogeneity exists with respect to relaxin output by individual cells. In contrast, both the basal rate of relaxin release and the percentage of cells committed to relaxin release declined significantly when luteal cells derived from preparturient sows (days 107-112 of gestation) were examined. Exposure of cultured luteal cells to prostaglandin F2 alpha (10(-8) and 10(-6) M) resulted in a rapid stimulation of relaxin secretion, but this agent did not recruit additional cells into the secretory pool. These data are consistent with the idea that autonomous changes and the action of secretagogues may combine at different times to achieve overall regulation of relaxin release by the corpus luteum. The significance of nonrelaxin-releasing luteal cells remains to be determined.

Animals↗
PubMed1987

Evidence for the existence of a luteal cell type that is steroidogenic and releases relaxin.

Secretion of relaxin from cultured luteal cells derived from pregnant sows was detected by a reverse hemolytic plaque assay. In this method, luteal cells are cultured in monolayers together with protein-A-conjugated ovine red blood cells. In the presence of porcine relaxin anti-serum and complement, relaxin-releasing cells become surrounded by an area of hemolysis--a plaque--which can be microscopically visualized. After fixation, these same luteal cells in monolayers were stained for the presence of 3 beta-hydroxysteroid dehydrogenase, an enzyme marker for steroidogenic cells. Cells could then be classified by their ability to form plaques (relaxin-releasing cells) and/or steroidogenic capability (positive staining). Dual-secretors (large luteal cells that were steroidogenic and released relaxin) could be identified in dispersed luteal cells derived from pigs at all stages of pregnancy examined (Day 22-112 of gestation, n = 9; term is Day 114 +/- 2 days). In addition, luteal cells were detected that were either steroidogenic only or released relaxin, and finally, cells that appeared to possess neither endocrine capability. Frequency analysis of functional subtypes indicated approximately equal representation of each in the first half of pregnancy, but an apparent fall in relaxin-releasing cells in the preparturient period. It is suggested that dual-secretors may represent one mechanism that allows the corpus luteum to express multiple endocrine function during pregnancy without the requirement for increased cell numbers.

3-Hydroxysteroid Dehydrogenases↗
PubMed1986

Ovine prolactin (PRL) and dopamine preferentially inhibit PRL release from the same subpopulation of rat mammotropes.

Autoregulation of PRL release was studied at the single cell level by the use of a reverse hemolytic plaque assay. Monodispersed pituitary cells from adult male rats were first preincubated with test substances and then coincubated with antirat PRL antiserum before development of plaques with complement. At the conclusion of the assay, the percentage of all pituitary cells in culture that formed plaques was evaluated microscopically, and the rate of plaque development was used as an index for the rate of hormone release. In controls, the maximal percentage of pituitary cells formed PRL plaques within a 1.5-h antibody incubation period, and addition of TRH (1 X 10(-7) M) did not increase this proportion. Treatment with ovine PRL (oPRL, 100 ng/ml) or dopamine (1 X 10(-7) M), either alone or in combination, caused a comparable suppression of the rate of PRL plaque development, which was reversed by the presence of TRH. Pretreatment of cells with the lysosomotropic agent chloroquine (1 X 10(-5) M) overrode dopamine inhibition of PRL plaque development, but did not influence oPRL inhibition. Taken together, these results demonstrate that oPRL inhibits basal, but not TRH-induced, PRL release from rat pituitary cells and support the view that PRL can act at the pituitary level to inhibit its own secretion. Moreover, the equipotency and lack of additivity exhibited by oPRL and dopamine coupled with the differential effects of chloroquine suggest that these factors both act upon the same subpopulation of mammotropes to inhibit PRL release, but by separate intracellular mechanisms.

Animals↗
PubMed1986

A novel bioassay for lactogenic activity: demonstration that prolactin cells differ from one another in bio- and immuno-potencies of secreted hormone.

A reverse hemolytic plaque assay for detecting casein release from individual mammary cells in culture was developed as a bioassay for PRL. Treatment with rat PRL caused dose-related increases in the percentage of mammary cells that released casein and the average size of casein plaques that formed. The assay exhibited exquisite sensitivity (156 fg rat PRL per assay slide) and could be used to evaluate the biopotency of PRL released from individual cells. By combining this "plaque bioassay" with a standard plaque assay for measuring the secretion of immunoreactive PRL, it was possible to compare the bio- and immuno-potencies of hormone released from the same pituitary cells. The results of three separate studies revealed major differences among PRL secretors in these potency estimates. Given the existence of PRL variants with different biological and immunological efficacies, these findings suggest that PRL cells differ from one another in the molecular form(s) of hormone released.

Animals↗