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Biomedical subjects

L W Clem

Publications and source records attributed to L W Clem.

At least 73 records · Page 4Linked to original sources
PubMed1988

Temperature-mediated processes in teleost immunity: homeoviscous adaptation by channel catfish peripheral blood cells.

1. Channel catfish peripheral blood erythrocyte, thrombocyte, T cell and B cell membranes were assayed by fluorescence depolarization using the fluorescent probe 1,6-diphenyl-1,3,5 hexatriene (DPH) to determine the effects of in vivo temperature acclimation on membrane viscosity and the kinetics of homeoviscous adaptation. 2. Erythrocyte membranes did not undergo homeoviscous adaptation during the 8 week time period studied and were more rigid compared with those of the other cell types. 3. The kinetics of homeoviscous adaptation exhibited by membranes from T cells, B cells and thrombocytes differed: B cells required 1-3 weeks while T cells and thrombocytes each required 3-5 weeks. Membranes from T cells, B cells and thrombocytes from fish acclimated for relatively short times (less than or equal to 3 weeks) exhibited similar membrane fluidities. 4. T cells from channel catfish appeared not only to be sensitive to temperature but also to a factor(s) independent of temperature but correlated to long term in vivo acclimation, i.e. T cell membranes underwent additional decreases in membrane viscosity between 3 and 5 weeks. 5. In conclusion, it appears that low temperature-mediated immunosuppression of T cell functions in channel catfish is probably not due to an inherent non-adaptability or rigid nature of the T cell membranes.

Adaptation, Physiological↗
PubMed1988

Temperature mediated processes in teleost immunity: differential abilities of channel catfish T and B lymphocytes to cap membrane antigen.

1. The effect of both in vivo acclimation temperature and in vitro assay temperatures on channel catfish T and B lymphocyte membrane antigen (mAg) capping were investigated to determine if capping might be the temperature sensitive step involved in the low temperature immunosuppression of channel catfish T cell responses. 2. Flow cytometry was used to monitor the kinetics of capping induced by a mouse monoclonal antibody (mAb 11G3) specific for a common antigenic determinant present on channel catfish T and B cells. Results indicated that the kinetics of mAg capping were dependent on in vitro assay and in vivo acclimation temperatures and the length of time of in vivo acclimation. 3. T cells from fish appropriately acclimated to 27 degrees C cap mAg more efficiently at low assay temperatures than do B cells. 4. Activation energies were 32 and 47 kcal/mol for B and T cells, respectively, from fish acclimated to 17 degrees C for 3 weeks, but were significantly lower (14 and 22 kcal/mol, respectively) after acclimation for 5 weeks. 5. In summary, it appears that after appropriate in vivo acclimation, channel catfish T cells are better able to cap mAg at low assay temperatures than are B cells. These results suggest that mAg capping is not the low temperature sensitive step involved in T cell immunosuppression in channel catfish.

Acclimatization↗
PubMed1987

Conformational changes in the DNA of hybridoma cells from pristane treated mice.

The effects of pristane on the DNA of hybridoma cells propagated as ascitic tumors in pristane-primed BALB/c mice were determined using flow cytometric analyses. Hybridoma cells maintained in vitro or cell isolates from solid tumors which developed in unprimed mice injected with hybridoma cells exhibited similar propidium iodide (PI) staining characteristics. In contrast, PI stained cells isolated from ascites which developed in pristane-primed mice injected with the hybridoma cells displayed significant decreases in fluorescence intensity. Diphenylamine studies and analyses of pH 10 treated cells indicated that the actual DNA content of the hybridoma cells was not altered by exposure to pristane. Furthermore, the altered staining characteristics of the ascitic tumor cells were reversible in that the fluorescence intensity after serial in vitro passage of the ascites cells was similar to that of the parent cell line which had not been exposed to pristane. In addition, there was a direct correlation between the altered PI staining characteristics and the presence of cell-associated pristane as determined by gas-liquid chromatography analyses of cell extracts. Collectively these results suggest that pristane may have a direct effect on the DNA conformation of hybridoma cells which may in turn enhance their growth as ascitic tumors. The possible role of such an altered DNA conformation in hybridoma cells on the in vivo development of ascites is discussed.

Animals↗
PubMed1987

Phylogeny of lymphocyte heterogeneity: identification and separation of functionally distinct subpopulations of channel catfish lymphocytes with monoclonal antibodies.

The purpose of this study was to identify monoclonal antibodies reactive with channel catfish T cells. Since a variety of commercially available anti-human and mouse T cell reagents failed to react with channel catfish leucocytes, the development of anti-fish T cell monoclonal antibodies was undertaken. One of these reagents, designated mAb 13C10, reacted with channel catfish surface immunoglobulin negative, but not positive, lymphocytes. This reagent, when used in "panning" protocols, was able to isolate those channel catfish lymphocytes which provide helper activity for antibody synthesis to a thymus dependent antigen. In addition, this antibody was observed to react with most thymocytes, neutrophils, and thrombocytes, few hepatocytes, and with some brain cells (possibly neurons). It was concluded that this antibody reacts with relatively high molecular weight antigens on channel catfish T cells and that it may be a pan anti-T cell reagent (possibly akin to Thy-2) for this species.

Animals↗
PubMed1987

Blood serum chemistry measurements of normal and acutely stressed channel catfish.

1. An automated blood serum chemistry analytical system designed for human usage was employed to establish the levels of 26 different components present in sera obtained from various experimental groups of channel catfish. 2. Comparisons of samples from feral and commercial production pond fish during warm months indicated statistically significant differences in the serum levels of sodium, CO2, urea nitrogen, direct bilirubin, cholesterol, creatinine and protein. 3. Laboratory acclimated and production pond fish exhibited differences in serum electrolytes (sodium, potassium, chloride, phosphorus), serum metabolites (urea nitrogen, creatinine, triglycerides), serum enzymes [gamma-glutamyl transferase, glutamate-oxaloacetate transaminase (GOT), lactate dehydrogenase (LDH), alkaline phosphatase, and amylase], and serum iron. 4. Seasonal (temperature?) differences in production pond fish were noted for 12 serum components including potassium, magnesium, CO2, glucose, creatinine, albumin, iron, alkaline phosphatase, and glutamate-pyruvate transaminase (GPT). 5. Comparisons of samples obtained from laboratory-acclimated fish before and 18 hours after acute handling and transport stress revealed significant differences in only three serum parameters: glucose, LDH, and creatine phosphokinase (CPK). 6. These studies suggest that "normal" values established by any method of sera analysis may be different in the same species depending on the diet, season, and presence of environmental stressors.

Acclimatization↗
PubMed1987

Temperature-mediated processes in teleost immunity: the effects of temperature on membrane immunoglobulin capping on channel catfish B lymphocytes.

1. In order to better understand ligand-induced redistribution of membrane receptors and lymphocyte activation in ectothermic vertebrates, flow cytometry was used to monitor the effects of both in vivo acclimation temperature and in vitro assay temperatures on the kinetics of monoclonal antibody-induced membrane immunoglobulin (mIg) capping on channel catfish lymphocytes. 2. It was observed that the kinetics of mIg capping were dependent on in vitro assay temperatures, in vivo acclimation temperatures, and the length of time of in vivo acclimation. In the latter situation in vivo acclimation of fish to 27, 22 and 17 degrees C was considered complete after 3 weeks, while acclimation to 12 degrees C required a minimum of 5 weeks. 3. The energies of activation required for mIg capping ranged from 33 to 24 kcal/mol; lower energies of activation were observed with lower temperature acclimation. 4. It was also noted that the lower energies of activation were associated with concomitant decreases in cellular phospholipid saturated/unsaturated fatty acid ratios. 5. It appears that channel catfish B cell mIg capping, presumably a requisite for immune function, can be significantly affected by environmental temperatures; most likely such effects are attributable to changes in plasma membrane viscosities.

Acclimatization↗
PubMed1987

Cortisol-induced hematologic and immunologic changes in channel catfish (Ictalurus punctatus).

1. Intravenous injections of physiologic doses of cortisol resulted in both hematologic and immunologic changes in channel catfish peripheral blood leucocytes. These changes mimicked those seen when catfish were acutely stressed by handling and transport. 2. Eighteen hours after the administration of cortisol, decreases in the number of circulating lymphocytes and concomitant increases in the number of circulating neutrophils were observed, i.e. to the same levels seen previously in stressed fish. 3. Functional analysis of peripheral blood leucocytes from cortisol-injected fish indicated that the remaining lymphocytes were no longer capable of responding to mitogenic stimuli. 4. This suppression of mitogenic stimuli was not seen when peripheral blood leucocytes were cultured in vitro with physiologic doses of cortisol. 5. This latter observation suggests that the cortisol alone was probably not directly responsible for the loss of responsiveness but possibly acted in vivo as an initiator of other events that eventually resulted in the observed immunosuppression.

Animals↗
PubMed1986

The effects of in vivo acclimation temperature on the fatty acid composition of channel catfish (Ictalurus punctatus) peripheral blood cells.

Channel catfish were acclimated in vivo to 12, 17, 22 or 27 degrees C and their peripheral blood erythrocytes, thrombocytes, T lymphocytes and B lymphocytes assayed for cellular fatty acid composition. Excepting cells from 12 degrees C acclimated fish, all cells responded to acclimation to lower temperatures by exhibiting increased levels of phospholipid unsaturated fatty acids. Although temperature independent differences were observed between erythrocytes, thrombocytes and lymphocytes, no differences between T lymphocytes and B lymphocytes were seen.

Acclimatization↗
PubMed1986

Phylogeny of lymphocyte heterogeneity: the cellular requirements for the mixed leucocyte reaction with channel catfish.

Vigorous mixed leucocyte reactions (MLR) were obtained using channel catfish peripheral blood leucocytes (PBL) when equal numbers of responder and stimulator cells (5 X 10(5) cells each) were cocultured. The use of 2000 rads of X-irradiation was sufficient to block subsequent proliferative responses of the stimulator cells. The cellular requirements for channel catfish MLR responses were assessed by using three functionally distinct leucocyte subpopulations isolated from the PBL. B cells (sIg+ lymphocytes) and T cells (sIg- lymphocytes) were isolated by an indirect panning procedure employing a monoclonal antibody specific for channel catfish Ig. A third population, monocytes, was isolated or depleted by adherence to baby hamster kidney cell microexudate-coated surfaces or adherence to Sephadex G-10, respectively. The results indicated that only the T cells were able to respond in the fish MLR, with monocytes being required as accessory cells. In contrast, all three cell types could function as stimulator cells. In addition, it was observed that low in vitro culture temperatures inhibited the generation of channel catfish MLRs, thereby supporting the contention that low temperature immunosuppression in fish results from a preferential inhibition of the generation of primary T-cell responses.

Animals↗
PubMed1986

Temperature-mediated processes in teleost immunity: binding and mitogenic properties of concanavalin A with channel catfish lymphocytes.

Low (non-permissive) temperatures inhibit the proliferation responses of channel catfish T cells to stimulation with Con A. The study reported here was undertaken to ascertain if failure to bind Con A at non-permissive temperatures could explain the observed suppression at such temperatures. The influence of temperature on the binding of fluorescein-labelled succinyl Con A to channel catfish T and B cells was studied by cytofluorography. The results indicated that Con A bound equally well at both mitogenically permissive (22 degrees, 27 degrees and 32 degrees) and non-permissive (12 degrees and 17 degrees) temperatures. Hence, temperature effects on mitogen-binding to cell surface receptors cannot readily explain the observed suppression of channel catfish T-cell proliferative responses at non-permissive temperatures.

Animals↗
PubMed1985

Phylogeny of lymphocyte heterogeneity: the cellular requirements for in vitro antibody responses of channel catfish leukocytes.

Three functionally distinct leukocyte subpopulations were isolated from the peripheral blood of channel catfish. Surface immunoglobulin-positive (sIg+) and sIg- lymphocytes were isolated by an indirect "planning" procedure employing monoclonal antibodies specific for channel catfish Ig. A third population composed of macrophages was isolated by adherence to baby hamster kidney cell microexudate-coated surfaces. Functional features of these three cell types were established by assessing their role(s) in primary in vitro anti-hapten PFC responses to known murine thymus-dependent (TD) and thymus-independent (TI) antigens. The results indicated that anti-hapten PFC responses to a TI antigen required the presence of sIg+ lymphocytes and macrophages. In contrast, all three cell types were required for responses to TD antigens. Furthermore, the results of studies involving the depletion of antigen-reactive lymphocytes demonstrated that both hapten-specific sIg+ cells and carrier-specific sIg- cells were required for anti-hapten responses to TD antigens. These studies provide direct evidence that catfish have separable B cells (sIg+ lymphocytes), T helper cells (sIg- lymphocytes), and accessory cells (macrophages) quite similar to those seen in higher animals.

Animals↗
PubMed1984

Microsystem for in vitro primary and secondary immunization of channel catfish (Ictalurus punctatus) leukocytes with hapten-carrier conjugates.

Methods are described for the in vitro generation and detection of antibody-secreting cells (PFC) from channel catfish. Hapten-specific PFC can readily be enumerated by an indirect plaque assay employing rabbit antiserum to catfish Ig and guinea pig complement. A modified Mishell-Dutton-type culture system was developed for effectively generating significant in vitro anti-hapten PFC responses with catfish leukocytes at 27 degrees C. The classical hapten-carrier effect and primary responses to both TI and TD antigens were demonstrable with catfish cells. Variables found to be important with catfish cells included the serum supplement, cell densities and, to a lesser extent, antigen form. Optimistically these methods will prove useful in attempts to delineate the functional roles of different lymphocyte subpopulations in fish.

Animals↗
PubMed1984

Recognition of xenogeneic proteins by the blue crab: dissociation of the clearance and degradation reactions and lack of involvement of circulating hemocytes and humoral factors.

Blue crabs clear foreign proteins from the circulation to the gills in a manner that can be explained by postulating the participation of a finite number of naturally occurring quasi-specific receptors. The rate of this clearance reaction is not increased by previous exposure to the foreign protein although recovery to the normal rate of clearance after high level blockade is quite rapid. The cleared foreign proteins are degraded in the gills in a relatively nonspecific fashion suggestive of a clear distinction between the clearance and degradation reactions. The results of perfusion experiments indicate that neither circulating hemocytes nor specific humoral factors are responsible for the foreign protein clearance reaction in blue crabs. Thus it is concluded that fixed cells, most likely in the gills, are responsible for this means of dealing with nonself proteins in blue crabs and possibly other crustaceae .

Animals↗
PubMed1984

Temperature-mediated processes in teleost immunity: differential effects of in vitro and in vivo temperatures on mitogenic responses of channel catfish lymphocytes.

The in vitro mitogenic responses of channel catfish peripheral blood leucocytes to ConA and LPS were differentially affected by both in vitro and in vivo temperatures. The magnitude of the response to LPS was relatively independent of both in vitro culture temperature and in vivo acclimation temperature. The magnitude of the response to ConA was suppressed at lower in vitro temperatures although this suppression could be reduced by lower in vivo acclimation temperatures. In vitro temperature-shift experiments indicated that channel catfish PBL could respond to ConA at a lower in vitro temperature if first stimulated with ConA at a higher in vitro temperature. The converse, however was not true in that channel catfish PBL did not respond at a higher in vitro temperature after an initial stimulation with ConA at a lower in vitro temperature. This latter failure to respond could not be attributed to the induction of a suppressor cell (or factor) by exposure to ConA at a lower temperature. These studies, when coupled with other available data on channel catfish PBL subpopulations, are interpreted as supporting the hypothesis that low temperature immunosuppression in fish may result from preferential inhibitory effects on T cells rather than B cells.

Animals↗
PubMed1984

The molecular mechanism of cryoimmunoglobulin precipitation--II. Thermodynamic basis for self-association as determined by fluorescence polarization.

Protein MAT is a homogeneous human IgM (lambda) cryoprecipitating cold agglutinin wherein the Fabmu and Fcmu5 fragment interaction is facilitated at low temps. Thermal dependence of the association of 2,5 DNS labeled Fab MAT with unlabeled Fcmu5 MAT was examined by fluorescence depolarization. The association data, treated by the method of van't Hoff, showed a nonlinear increase in binding with decreased temp, suggestive of certain dynamic changes in the system. Temperature effects on the rotational diffusion of five different 1,5 DNS labeled Fabmu fragments (including two derived from cryoglobulins) were also examined by fluorescence polarization. The linear nature of the Perrin plots derived from the data failed to reveal temp-induced hydrodynamic changes in any of the Fabmu fragments studied. Involvement of carbohydrates in the low-temp self-association of protein MAT was established by the finding that glycopeptides isolated from another IgM molecule (BAZ) could inhibit (as judged by depolarization of fluorescence) the interaction of 2,5 DNS labeled Fabmu MAT and unlabeled Fcmu5 BAZ fragments. These findings indicate that, although cryoprecipitation of protein MAT seemingly involves an antigen-antibody-like reaction between a site on the Fab region and carbohydrate moieties on the Fcmu5 region, no direct evidence for a low-temp-induced conformational change in the Fab region was obtained.

Agglutinins↗
PubMed1984

A new method for the cytochemical staining of cells immobilized in agarose.

A quick, simple and inexpensive technique utilizing an isotonic matrix of semi-solid agarose for immobilizing channel catfish (Ictalurus punctatus) peripheral blood leucocytes onto microscope slides before cytochemical staining is described. The technique was demonstrated to be superior to the more conventional means of immobilizing cells for procedures requiring either fixed (Sudan Black B and nonspecific esterase stains) or viable (Nitro-Blue Tetrazolium stain) cells. Although the agarose immobilization technique was developed with channel catfish leucocytes, its utility with other fragile cell populations seems likely.

Animals↗
PubMed1984

Temperature-mediated processes in teleost immunity: differential effects of temperature on catfish in vitro antibody responses to thymus-dependent and thymus-independent antigens.

An in vitro culture system was employed to ascertain the effects of different temperatures on the anti-hapten antibody-secreting cell responses of channel catfish leucocytes to murine thymus-dependent (TD) and thymus-independent (TI) antigens. The magnitudes of primary responses to a TI antigen (TNP-LPS) and secondary responses to a TD antigen (DNP-KLH) were relatively independent of in vitro culture temperature. The kinetics of each of these responses as a function of temperature was characterized by a Q10 of 2. In contrast, the magnitudes of primary responses to TD antigens (DNP-KLH and DNP-HoSA) were suppressed at lower in vitro temperatures. Furthermore, it was observed that some of the low temperature suppression of primary responses to TD antigens could be abrogated by appropriate low temperature in vivo acclimation. These findings are interpreted as supporting the hypothesis that low environmental temperatures immunosuppress fish by virtue of differential inhibitory effects on the generation of carrier-specific helper cells.

Animals↗