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Biomedical subjects

W R Clark

Publications and source records attributed to W R Clark.

At least 109 records · Page 6Linked to original sources
PubMed1984

Modulation of cell-mediated cytotoxicity function after alteration of fatty acid composition in vitro.

Fatty acids incorporated into cytotoxic T lymphocytes (CTL) during in vitro stimulation can enhance or inhibit the subsequent expression of cell-mediated cytotoxicity, depending on the class of fatty acid. Unsaturated fatty acids enhance cytolysis, whereas saturated fatty acids inhibit it. The effects of fatty acids on cytolysis can be mediated in the absence of cell division, thus eliminating relative clonal amplification or contraction as a basis for the observed effects. Nevertheless, the net result of fatty acid alteration is an increase (unsaturated fatty acids) or decrease (saturated fatty acids) in the frequency, in the treated immune population, of CTL capable of lysing target cells. These observations are best explained by a model in which fatty acid incorporated into cell membrane phospholipids results in a direct recruitment into or out of the pool of CTL within the immune population capable of lysing target cells under the conditions employed to assay cytotoxicity.

Animals↗
PubMed1984

The role of class I MHC products in polyclonal activation of CTL function.

The generation of CTL function by agents such as lectins, oxidative chemicals, or serum components has been regarded as independent of class I MHC products, because the CTL thus generated are able to lyse virtually any allogeneic target. However, we show here that CTL activation by lectin requires interaction of the pre-CTL with a class I MHC product on the lectin-presenting cell. The lectin-presenting cell can be an irradiated syngeneic or allogeneic spleen cell or, alternatively, lectin can be seen on a neighboring pre-CTL. In either case, there is an absolute requirement for simultaneous (although probably unlinked) perception of a class I MHC product. These results suggest that restricted clonal CTL activation by antigen, and polyclonal activation by so-called "nonspecific" mitogenic agents, may proceed through similar if not identical pathways.

Animals↗
PubMed1983

HLA-DR and HLA-A, B, C typing of human fetal tissue.

In anticipation of clinical trials of fetal pancreas transplantation we have investigated the feasibility of performing HLA-DR and HLA-A, B, C typing on fetal lymphoid cells other than PBL. Using the standard NIH microcytotoxicity test modified for HLA-DR typing it was possible to demonstrate HLA-DR antigens on subpopulations of bone marrow cells and splenocytes but not on thymocytes or hepatocytes. In contrast, HLA-A, B, C antigens could be detected on all four tissues. Excellent HLA-DR typing, confirmed by maternal typing, was obtained for 19 fetuses (14 to 23 weeks old) using bone marrow cells isolated by two-fold purification on discontinuous Percoll buoyant density gradients. Similar purification of splenocytes resulted in weak reactions with anti-DR sera; however, adherent splenocytes recovered from nylon wool columns proved to be primarily DR-bearing and also provided excellent DR typing. As a corollary to these results, non-adhering splenocytes depleted of DR-bearing cells were ideal for HLA-A, B, C typing since spurious reactions due to DR antigens were greatly diminished, whereas strong specific reactions were obtained with anti-HLA-A, B, C sera. Despite weaker reactions with HLA-A, B, C antisera obtained for thymocytes, reliable HLA-A, B, C typing could be obtained when results from thymocytes were evaluated together with typing from bone marrow cells or splenocytes. The possible benefits of fetal HLA typing for fetal pancreas transplantation are discussed.

Bone Marrow↗
PubMed1983

Normalization of six key hepatic enzymes after fetal pancreas transplantation in diabetic rats.

In diabetic rats transplanted with fetal pancreata we measured the activities of six important enzymes to assess the return of liver metabolism to normal. Comparison was made among the responses of transplanted rats with and without renal-portal vein shunts and of those not transplanted and injected with insulin in varying doses. Insulin supply was not limited since three or four fetal pancreata were first grown in normal rats before transfer into the diabetic animals. Transplantation normalized blood and urine glucose and the rate of disappearance of intravenous glucose. Glucokinase and pyruvate kinase activities in liver rose toward normal at 7 days after transplantation and reached normal levels at 30 and 90 days. The response of the other four enzymes, glucose-6-phosphate dehydrogenase, citric lyase, fructose-1,6-bisphosphatase, and glucose-6-phosphatase, was more rapidly restored to normal at 7 days and remained normal at 30 and 90 days. No difference was observed in the enzyme activities of transplanted-shunted rats to nonshunted animals. Glucokinase activity was restored to normal after 1 wk of daily injections of 1 U of PZI; pyruvate kinase restoration required 3 U/day. Glucose-6-phosphate dehydrogenase and citric lyase required 2 U/day to be restored to normal; 3 U daily resulted in temporary supernormal activities. The gluconeogenic enzymes, fructose-1,6-bisphosphatase and glucose-6-phosphatase, were only partially suppressed toward normal by insulin even with 3 U daily for 3 wk. These findings indicate that pancreas transplantation is a more effective regulator of liver metabolism in diabetes than insulin injections.

ATP Citrate (pro-S)-Lyase↗
PubMed1982

Comparison of glucose, fructose, ascorbic acid and glucosephosphate isomerase enzymatic activity in uterine flushings from nonpregnant and pregnant gilts and pony mares.

In Experiment 1, 40 gilts and 30 pony mares were used to characterize changes in glucose, fructose, ascorbic acid and glucosephosphate isomerase (GPI) enzymatic activity in uterine flushings collected either during the estrous cycle or early pregnancy. Total recoverable glucose was greater (P less than 0.01) in uterine flushings from pregnant gilts, but pregnancy status had no effect on total recoverable glucose in pony mare uterine flushings. Fructose was undetectable in uterine flushings from nonpregnant gilts and pony mares and pregnant gilts and pony mares prior to Day 14, but occurred in increasing amounts between Days 14 and 18 or 20 of pregnancy. In Experiment 2, it was demonstrated that the porcine conceptus is the primary source, if not the sole source of fructose. Total recoverable ascorbic acid in uterine flushings was not affected by pregnancy in gilts, but was greater (P less than 0.01) in pregnant versus nonpregnant pony mares. In both species, total recoverable ascorbic acid was affected (P less than 0.01) by day of the estrous cycle and pregnancy. The GPI enzyme allows for the interconversion of glucose-6-PO4 and fructose-6-PO4. GPI total and specific activities were greater (P less than 0.01) for pregnant than nonpregnant gilts and pony mares. The periods of greatest GPI activity were temporally associated with elevated estrogens of either ovarian or blastocyst origin. Results from Experiment 3 indicated a marked increase (P less than 0.01) in GPI activity in uterine flushings from gilts treated with estradiol valerate. Results of this study indicate that glucose (gilt only), fructose, ascorbic acid and GPI activity are increased in uterine flushings of gilts and pony mares during early pregnancy. The increase in these constituents may reflect increased carbohydrate metabolism in ways which are uniquely beneficial to conceptus development in ungulates.

Animals↗
PubMed1981

The relationship between plasma membrane lipid composition and physical-chemical properties. II. Effect of phospholipid fatty acid modulation on plasma membrane physical properties and enzymatic activities.

The fatty acid composition of plasma membrane phospholipids of the murine T lymphocyte tumor EL4 were systematically modified in an attempt to understand the relationship between lipid bilayer composition and plasma membrane physical and biological properties. Two plasma membrane enzyme activities, adenylate cyclase and ouabain-sensitive (Na+ + K+)-ATPase, were measured in normal and fatty acid-substituted EL4 plasma membrane fractions. The fatty acid effect on enzyme activities was similar to previously reported effects of fatty acids on cytotoxic T cell function. The activity of both enzymes was inhibited by saturated fatty acids, while unsaturated fatty acids had a moderate enhancing effect on both enzyme activities. Using two different nitroxide derivatives of stearic acid, the order parameter and approximate rotational correlation times were calculated from ESR spectra of normal and fatty acid-modified plasma membranes. No significant differences was found in either parameter in these membranes. These results, in conjunction with earlier data from our laboratory and others, suggest that caution should be exercised in inferring changes in membrane 'fluidity' based on lipid modulation of biological membranes.

Adenylyl Cyclases↗
PubMed1981

The relationship between plasma membrane lipid composition and physical-chemical properties. I. Fluorescence polarization studies of fatty acid-altered EL4 tumor cell membranes.

EL4 cels were cultured with exogenous fatty acids under conditions that resulted in their incorporation into membranes phospholipids. The behavior of the fluorescent lipid probes diphenylhexatriene and perylene was monitored in intact EL4 cells and in isolated EL4 plasma membranes. In whole cells substituted with unsaturated fatty acids, there was always a marked decrease in the P value of both probes compared to the P value of the probes in unsubstituted cells. In whole cells substituted with saturated fatty acids, on the other hand, P values for both probes were unchanged compared to unsubstituted cells. In plasma membrane isolated from EL4 cells, no difference in P values for either probe was observed among membranes from unsubstituted, saturated fatty acid substituted or unsaturated fatty acid substituted cells, even when the degree of fatty acid substitution was quite substantial. Most of the fluorescent signal for both probes in whole cells appeared to come from cytoplasmic lipid droplets. The value of techniques such as fluorescent polarization from monitoring physical properties of membranes (such as 'fluidity') is discussed.

Animals↗
PubMed1981

Recovery and function of human fetal pancreas frozen to -196 C.

Human fetal pancreases were dissected into 1- to 2- mm3 fragments and frozen to -196 C using a modification of procedures previously used successfully for cryopreservation of the rat fetal pancreas. Freeze-recovered pancreatic pieces were able to incorporate radioactive amino acids into proteins. When placed into organ culture, nonfrozen control and freeze-recovered pancreas tissue secreted radioimmoassayable insulin in response to glucose plus theophylline, but not to glucose alone. Freeze-killed pancreatic pieces were totally inactive functionally. With respect to those parameters tested, freeze-recovered pancreas tissues were equivalent to nonfrozen tissue.

Cell Survival↗
PubMed1981

Alveolar function following surfactant deactivation.

In vivo cinemicroscopic studies of subpleural alveoli were conducted in dogs for 4 h after pulmonary lavage with 5% Tween 20 and after lavage with normal saline. Saline-lavaged alveoli showed little change in alveolar size during tidal ventilation, whereas Tween lavage resulted both in alveolar recruitment and marked variation in alveolar size from end expiration to peak inspiration, with total collapse frequently occurring by end expiration. Following Tween, alveolar stability decreased, but alveolar capillary perfusion increased. Marked recovery of stability by 4 h was noted in most alveoli. Wilhelmy balance studies on lung extracts showed a decrease in surfactant function 30 min after Tween and a partial recovery of surfactant activity after 4 h. This study provides in vivo evidence that normal surfactant function is critical to alveolar stability and that alveolar stability is markedly restored 4 h after acute deactivation or displacement by Tween. Surfactant deactivation and loss of alveolar stability are associated with increased alveolar capillary perfusion creating significant ventilation-perfusion ratio abnormalities.

Animals↗
PubMed1981

T lymphocyte-mediated cytolysis. II. Role of target cell histocompatibility antigens in recognition and lysis.

Although compelling genetic and serologic evidence implicate target cell (TC) MHC antigens in specific cytotoxic T lymphocyte (CTL)-TC interaction leading to lysis, it is not entirely clear whether TC recognition through an MHC determinant(s) is a prerequisite for lysis to occur. In fact the finding that both specific and nonspecific TC are lysed equally well in lectin-dependent cell-mediated cytotoxicity (LDCC) challenges the necessity for TC MHC involvement in the cytolytic process beyond providing the basis for specificity in direct (nonlectin-dependent) CTL-mediated lysis. In the present paper we present evidence suggesting that even in nonspecific LDCC, as well as in nonspecific lymphocyte-mediated lysis of TC oxidized by periodate treatment or by galactose oxidase (ODCC), TC MHC components are required for lytic interactions with cytotoxic effector cells. This conclusion is based on 3 types of experimental evidence: 1) cells displaying reduced amounts of MHC proteins are poor targets in LDCC; 2) removal of H-2 by papain renders murine target cells refractory to lysis in LDCC, even though Con A binding is only slightly reduced; 3) antisera to target cell H-2-coded products block lysis in both LDCC and ODCC, whereas antisera to other cell surface antigens do not. A theory explaining nonspecific effector target interaction leading to lysis based on involvement of CTL receptor(s) and TC MHC components is presented.

Animals↗
PubMed1981

T lymphocyte-mediated cytolysis. I. A common mechanism for target recognition in specific and lectin-dependent cytolysis.

In this and the accompanying paper we examine the nature of the interactions between effector cells and target cells leading to lysis in T cell-mediated cytolysis reactions. In the first paper, we re-examine the role of lectin (Con A) in the process of lectin-dependent cell-mediated cytotoxicity (LDCC). Lectin has generally been thought to act simply as a bridge between the cytotoxic effector cell (EC) and the target cell (TC), thus bypassing the need for receptor-antigen interaction and accounting for the nonspecific character of this lytic reaction. A role for lectin in the activation of cytotoxic function in the EC has also been suggested. In this paper we confirm that LDCC occurs when TC alone are pretreated with lectin, but not when EC alone are pretreated with lectin, unless free lectin is also present in the assay mixture. In addition, we demonstrate that the failure of lectin-pretreated EC to bind to and lyse TC is not caused by agglutination or self-destruction of the EC. Moreover, lectin-pretreated EC are not inherently deficient in the ability to bind or lyse TC, if the latter are pretreated with lectin, or if free lectin is present in the assay. Finally, when EC are used as both effectors and targets in LDCC, lysis is observed only in the direction of the lectin-pretreated partner. From these results we conclude that in LDCC, as in specific CTL cytolysis, CTL-TC recognition proceeds through interaction of an EC surface receptor (distinct from the lectin receptor), and a TC antigen perhaps modified by, but distinct from, the lectin itself. In the accompanying paper we present evidence that this receptor-determinant interaction involves TC MHC antigen.

Animals↗
PubMed1980

The effect of smoke inhalation on pulmonary surfactant.

This paper details efforts to define the primary pathophysiology of acute smoke inhalation without the variables of infection, burns, or fluid resuscitation. A standard dose of smoke (wood and kerosene) was delivered at 37 C to mongrel dogs. The parameters studied included blood gases, carboxyhemoglobin, pulmonary and systemic hemodynamics, respiratory mechanics, surface tension area curves as an indication of surfactant activity, and in vivo photomicroscopy. The FiO2 of the smoke was 17 volumes per cent; the carbon monoxide 17,000 ppm. Immediately following smoke exposure, dense, nonsegmental atelectasis developed. Hemodynamic changes were insignificant, but the PaO2 fell to 49 mmHg; the right to left shunt rose from 5 to 41%. Surfactant reduction was significant: enough to cause an increase in the minimum surface tension from 7 to 22 dynes/cm. This surfactant loss may explain the atelectasis seen and the marked instability of subpleural alveolar walls. The data collected are consistent and support the acute inactivation of surfactant as one of the primary pathophysiologic events in smoke inhalation. The clinical correlation is good; surfactant loss may explain why victims of smoke inhalation are so vulnerable to fluid administration if they have thermal burns as well effectiveness of medical devices.

Animals↗
PubMed1980

Cryopreservation of human fetal pancreas.

To determine the optimal conditions for successful cryopreservation of the human fetal pancreas, techniques developed for the rat organ were modified. The parameters studied were the conditions of exposure to the cryopreservation agent dimethyl sulfoxide (dmso), the rate of cooling and thawing, and the effect of in vitro culture. A total of 33 pancreases, obtained after fetal death due to prostaglandin-induced abortion, was studied. Survival of the pancreas is based on incorporation of 3H-amino acids into protein by pancreas pieces (2 mm3) during a 4-h incubation compared with nonfrozen control pieces from the same pancreas. Toxicity of DMSO at 37 degrees C was found to be severe after a 4-h exposure. Varying the effects of temperature, time of exposure, and concentrations of DMSO on survival after freeze-thaw revealed that 1.5 M DMSO for 1 h at room temperature was optimal. The cooling rate was 0.22 degrees C/min and thawing was at room temperature. Since these conditions resulted in only 50% survival, a period of culture before exposure to DMSO was added. The optimal duration of culture was 12--16 h. Using this method with addition of culture for 24 h after thawing, survival has varied from 70 to 120% of control. If a functional test for growth and insulin production by the frozen-thawed pancreas is positive, permanent shortage of the human fetal pancreas will be possible.

Amino Acids↗
PubMed1979

Anisotropic molecular motion on cell surfaces.

Photobleaching in a two-dimensional periodic pattern has been used to characterize the motion of succinylconcanavalin A receptors on the surfaces of mouse fibroblasts. These receptors were found to exhibit anisotropic two-dimensional diffusion in adherent cells having parallel stress fibers. Diffusion coefficients of the order of 10(-11) cm2/sec and 10(-12) cm2/sec were measured for diffusive motions parallel and perpendicular to these fibers. The determination of the anisotropic motion used a two-dimensional Fourier analysis. A study was made of the effect of changes in cell shape on the photobleach pattern.

Animals↗
PubMed1979

Importance of hepatic portal circulation for insulin action in streptozotocin-diabetic rats transplanted with fetal pancreases.

The importance of the hepatic portal circulation in the response to insulin was assessed in streptozotocin-diabetic rats transplanted with syngeneic fetal pancreases. Partial reversal of diabetes was accomplished by transplantation of two or three fetal pancreases beneath the capsule of the kidney; complete reversal followed shunting of the venous drainage from the transplants to the liver. Plasma glucose after streptozotocin of 509+/-31 mg/dl (mean+/-SEM) fell after transplantation to 395+/-23 and after the shunt to 143+/-5 mg/dl. Urine volume fell from 84+/-4 to 50+/-5 ml/d and then to normal (17+/-1 ml/d) after the shunt. Glucose excretion which was 8.1+/-0.3 g/d after streptozotocin fell after transplantation to 4.8+/-0.3 g/d and after the shunt completely disappeared from the urine. The disappearance rate of glucose injected into the circulation, which was 0.50+/-0.07%/min in untreated diabetes, increased to 1.39+/-0.38%/min after transplantation and to 2.52+/-0.31%/min after the shunt, not different from normal controls (2.79+/-0.25). Plasma immunoreactive insulin (IRI) was below normal (25-35 muU/ml) and unresponsive to glucose in untreated diabetic rats. After transplantation IRI levels ranged from 73-223 muU/ml and there was no rise after glucose injection. After the shunt both the basal IRI (36+/-5 muU/ml) and the peak response to glucose at 10 min (58+/-7 muU/ml) were the same as in normal controls (42+/-4 and 62+/-7 muU/ml, respectively). The fall in IRI after the shunt is explained by increased extraction of insulin passing into the liver and also diminished secretion. After removal of the transplants plasma glucose and urine values returned almost to pretransplant levels. Secretion of insulin by transplanted pancreases into the liver enhances the effectiveness probably by increased extraction and action and reveals the importance of the normal route for insulin delivery.

Animals↗