Biomedical subjects

L W Clem

Publications and source records attributed to L W Clem.

At least 55 records · Page 3Linked to original sources

PubMed1992

Chelation affects the conformation, lability and aggregation of channel catfish (Ictalurus punctatus) phosphorylcholine-reactive protein (PRP).

1. Phosphorylcholine-reactive protein (PRP) affinity-purified from channel catfish (Ictalurus punctatus) serum on phosphorylcholine-Sepharose, eluted from Bio-Gel A-5M as a 94.6 +/- 2.4 kDa protein when the gel filtration column buffer (Tris-saline) contained 25mM ethylenediaminetetraacetic acid (EDTA). 2. PRP chelated with EDTA immediately after affinity purification and gel-filtered in Tris-saline-EDTA, eluted as a 75.5 +/- 2.67 kDa protein referred to as fast-PRP (F-PRP). 3. PRP and F-PRP were identical on SDS-PAGE. Both resolved as a broad band of protein (ca 86-100 kDa) on non-reducing gels or as a ca 100 kDa protein after reduction with 2-mercaptoethanol (2-ME). 4. After gel-filtration in Tris-saline-EDTA, nearly complete reduction of 100 kDa PRP was achieved on SDS-PAGE. However, the protein regained its resistance to reduction upon storage at -60 degrees C. 5. SDS-PAGE and native PAGE also revealed that during storage, PRP and F-PRP combined to form 3 different aggregates referred to as aggregated-PRP (aggPRP). These aggregates are readily dissociated in the presence of 2-ME, suggesting a covalent interaction between adjacent pentamers comprising decameric aggPRPs. 6. PRP, F-PRP, and aggPRP have similar amino acid compositions.

Animals↗

PubMed1992

Cellular pathway(s) of antigen processing and presentation in fish APC: endosomal involvement and cell-free antigen presentation.

Studies were conducted to address further the role(s) of antigen processing and presentation in the induction of immune responses in a phylogenetically lower vertebrate, specifically a teleost, the channel catfish. In particular, studies were aimed at determining the subcellular compartments involved in antigen degradation by channel catfish antigen-presenting cells (APC) as well as ascertaining the reexpression of immunogenic peptides on the surfaces of APC. The results showed that exogenous protein antigens were actively endocytosed by APC as detected by flow cytometry. Use of radiolabeled antigen and subcellular fractionation protocols also showed that antigen localized in endosomes/lysosomes. Furthermore, there was an apparent redistribution of antigen between these organelles and the plasma membrane during the course of antigen pulsing. Functional assays for the induction of in vitro antigen-specific proliferation of immune catfish peripheral blood leukocytes (PBL) showed that membrane preparations from antigen-pulsed autologous APC were highly stimulatory. The magnitude of responses elicited with such membrane preparations was very similar to that of PBL cultures stimulated with native antigen-pulsed and fixed intact APC or prefixed intact APC incubated with a peptide fragment of the nominal antigen. Current data further corroborate our previous findings that steps akin to antigen processing and presentation are clearly important in the induction of immune responses in lower vertebrates like fish, in a manner similar to that seen in mammalian systems. Consequently, it would appear that many immune functions among the diverse taxa of vertebrates are remarkably conserved.

Animals↗

PubMed1991

Spontaneous development of functionally active long-term monocytelike cell lines from channel catfish.

During the course of studies involving the in vitro manipulation of channel catfish peripheral blood leukocytes, spontaneous proliferation was observed with unexpectedly high frequency. Propagation of these spontaneously proliferating cells has resulted in the development of long-term (greater than 11 mo.) cell lines which stain positively for nonspecific esterase and peroxidase, are phagocytic for latex beads, and morphologically resemble mammalian monocytes or macrophages. These long-term cell lines also exhibit two important additional functional features. First, induction with lipopolysaccharide results in the secretion of relatively high levels of catfish high and low molecular weight species of interleukin-1 active on channel catfish and mouse T cells, respectively. Second, these cell lines are efficient antigen-presenting cells to autologous peripheral blood leukocytes for antigen specific in vitro proliferative and antibody responses. This antigen-presenting function is blocked by inhibitors known to prevent antigen processing and presentation by mammalian monocytes. Allogeneic mixtures of cell line (used as antigen-presenting cells) and responding peripheral blood leukocytes, however, resulted in strong mixed leukocyte reaction but not in specific antibody responses. The availability of such cell lines should facilitate further studies on accessory cell functions in fish immune responses.

Animals↗

PubMed1991

Proteolytic fragmentation of channel catfish antibodies.

Specifically purified anti-DNP antibodies from channel catfish were digested with pepsin or trypsin under various conditions and the resultant fragments analyzed in terms of their physico- and immunochemical properties. The results indicated that pepsinolysis of channel catfish antibodies to small peptides was exceedingly rapid and failed to yield stable recognizable fragments under any conditions used. However, trypsinolysis was considerably more successful. In particular, tryptic digestion at 37 degrees C gave good yields of ligand binding material that undoubtedly represented Fab fragments. In addition, it was observed that limited trypsinolysis at 4 degrees C yielded somewhat larger Fab-like material, tentatively designated as Fab' fragments. Hence, it would appear that in spite of their previously reported peculiar covalent tetrameric structure, channel catfish antibodies do exhibit an intramolecular architecture or organization similar to that seen in more conventional polymeric antibodies from other species.

Animals↗

PubMed1991

Temperature-mediated processes in teleost immunity: in vitro immunosuppression induced by in vivo low temperature in channel catfish.

In an attempt to understand the interrelationships between environmental temperature and immune competence, channel catfish in the laboratory were subjected to a rapid change in water temperature in order to mimic conditions which might be encountered in commercial ponds during the winter months and subsequently examined for a variety of immune parameters. The results indicated that lowering the water temperature from 23 to 11 degrees C over a 24 h period suppressed both B and T cell functions for 3-5 weeks as assessed by in vitro responses. Furthermore, this form of suppression was not a typical stress-induced response, i.e. blood serum chemistry and lymphocyte and neutrophil compositions did not change in a manner reminiscent of transport-induced stress. Collectively these results indicate that channel catfish are probably immunocompromised during the winter months and consequently it seems plausible that many of the fish losses associated with the syndrome termed "winter kill" may be attributable, at least in part, to a low temperature-induced immuno-deficient state.

Adaptation, Biological↗

PubMed1991

Correlation between low-temperature immunosuppression and the absence of unsaturated fatty acid synthesis in murine T cells.

1. Studies were performed to determine if the inability of murine T cells to provide primary helper function at low temperature (27 degrees C) could be correlated with their inability to synthesize unsaturated fatty acids (UFAs). 2. In the absence of exogenous oleic acid (18:1), splenocytes responded to a T-dependent (TD) Ag (trinitrophenyl-keyhole limpet hemocyanin, TNP-KLH) at 37 degrees C but not at 27 degrees C. The addition of 150 microM 18:1 almost completely restored plaque-forming cell (PFC) responses to TNP-KLH at 27 degrees C but markedly suppressed PFC responses to the TD Ag at 37 degrees C. 3. During incubation at 27 degrees C, B cells converted 3- to 5-fold more stearic acid (18:0) to 18:1 and showed a greater accumulation of monounsaturated phospholipid molecular species than did T cells. 4. Following incubation in the presence of a rescuing dose of 18:1 (150 microM), both B and T cells accumulated large amounts of dioleoyl PC. 5. It is proposed that the absence of 18:1 synthesis in T cells is responsible for the unique low temperature susceptibility of this lymphocyte population.

Animals↗

PubMed1991

Phylogeny of immune recognition: processing and presentation of structurally defined proteins in channel catfish immune responses.

This work was undertaken to investigate whether or not antigen processing and presentation are important in channel catfish in vitro secondary immune responses elicited with structurally defined proteins, namely, pigeon heart cytochrome C (pCytC), hen egg lysozyme, and horse myoglobin. The use of in vitro antigen-pulsed and fixed B cells or monocytes as antigen presenting cells (APC) resulted in autologous peripheral blood leukocytes (PBL) responding with vigorous proliferation and antibody production in vitro. In addition, several long-term catfish monocyte lines have been found to function as efficient APC with autologous but not allogeneic responders. Subsequent separation of the responding PBL into sIg- (T-cell-enriched) and B (sIg+) cells subsets showed that both underwent proliferative responses to antigen-pulsed and fixed APC. Moreover, allogeneic cells used as APC were found to induce only strong mixed leukocyte reactions without specific in vitro antibody production. Initial attempts at identifying the immunogenic region(s) of the protein antigens for catfish indicated there are two such regions for pCytC, namely, peptides 66-80 and 81-104.

Animals↗

PubMed1991

Phylogeny of immune recognition: role of alloantigens in antigen presentation in channel catfish immune responses.

Previous work from this laboratory demonstrated the importance of antigen processing and presentation in the generation of secondary in vitro immune responses of channel catfish to both simple and complex T-dependence antigens. Further, the usage of functionally active long-term monocyte lines as antigen-presenting cells (APCs) revealed 'putative restriction' of immune responses by peripheral blood leucocytes (PBL) as responders. In addition, these long-term cell lines proved to be effective antigens for the production of specific alloantisera. Studies were therefore conducted to determine the importance of these alloantigens in the immunological recognition of foreign T-dependent antigens in the channel catfish. The addition of homologous, but not heterologous, alloantisera to cultures of antigen-pulsed cell line APCs and autologous immune PBL responders resulted in quantitative decreases in the antigen-specific proliferative responses. Taken together, these results provide evidence that alloantigens [presumably major histocompatibility complex (MHC) or MHC-like molecules] govern antigen presentation and putative restriction of teleost immune responses similar to the situation in mammals.

Animals↗

PubMed1990

Phylogeny of immune recognition: antigen processing/presentation in channel catfish immune responses to hemocyanins.

Studies were conducted to address the role(s) of antigen (Ag) processing/presentation in channel catfish immune responses. Vigorous and specific secondary in vitro proliferative and antibody (Ab) responses were obtained to keyhole limpet and Limulus polyphemus hemocyanins with peripheral blood leukocytes (PBL) from catfish previously primed in vivo with Ag. In addition, such antigen-specific in vitro proliferative and Ab responses were efficiently elicited by antigen-pulsed and subsequently paraformaldehyde-fixed autologous PBL used as putative antigen-presenting cells (APC) but not by APC fixed prior to Ag pulsing. Treatment of these putative APC with lysosomotropic agents, protease inhibitors, or the ionophore monensin prior to or during pulsing with Ag significantly inhibited both in vitro responses. Furthermore, the use of radiolabeled protein indicated that both untreated and inhibitor-treated PBL but not erythrocytes take up Ag; however, only untreated PBL were able to degrade Ag. Immune restriction was indicated by the use of allogeneic PBL as APC in that only strong MLRs were generated with no detectable antibodies produced in vitro. Finally, the employment of isolated leukocyte subpopulations demonstrated that both catfish B (sIg+) lymphocytes and monocytes were efficient Ag presentors.

Ammonium Chloride↗

PubMed1990

The immunoglobulin M heavy chain constant region gene of the channel catfish, Ictalurus punctatus: an unusual mRNA splice pattern produces the membrane form of the molecule.

The immunoglobulin (IgM) heavy chain constant region gene of the channel catfish, Ictalurus punctatus, has been cloned and characterized. The gene contains four constant region domain-encoding exons (CH1 to CH4) expressed in the secreted form of the immunoglobulin, and two exons encoding the transmembrane (TM) domain utilized in the lymphocyte membrane receptor form of the immunoglobulin. The sequence of a cDNA clone encoding the 3' region of the message for the membrane receptor form of the mu chain indicates that the TM1 exon is spliced directly to the CH3 exon, and not into a site within the CH4 exon, as occurs in the mammals, a shark and an amphibian. This unusual pattern of splicing, which produces a membrane heavy chain that is characteristically smaller than the secreted heavy chain, may be common to all teleost fish.

Amino Acid Sequence↗

PubMed1990

Temperature-mediated processes in immunity: differential effects of low temperature on mouse T helper cell responses.

A low culture temperature of 27 degrees C inhibited mouse primary in vitro anti-hapten plaque-forming cell responses to a thymus-dependent (TD) antigen (Ag) (trinitrophenyl-keyhole limpet hemocyanin, TNP-KLH). In contrast, the magnitudes of secondary responses to TNP-KLH or primary responses to a thymus-independent (TI) Ag (TNP-lipopolysaccharide (LPS)) were unaffected. The low-temperature-sensitive step in the primary TD response occurred relatively early and preceded interleukin 2 (IL-2) secretion. Furthermore, the low-temperature-induced suppression could be obviated (rescued) by recombinant IL-2 or IL-4, but not by IL-1. Thus, the low temperature appeared to inhibit the function of virgin Th cells by preferentially affecting T cell-derived interleukin synthesis/secretion and not other cellular activities. These results also imply fundamental differences between the activation requirements of memory and virgin Th cells.

Animals↗

PubMed1990

Channel catfish as an unconventional model for immunological studies.

The channel catfish, Ictalurus punctatus, is an economically important species which is readily available, acclimates well to the laboratory setting, and is amenable to considerable experimental manipulation. Although the channel catfish is still a relatively circumscribed species in terms of comprehensive physiologic and/or endocrinologic studies, our current understanding of the basic immunobiology and immunochemistry of the channel catfish is significantly further advanced than for any other teleost species. In this respect the channel catfish is not only proving useful in the general areas of comparative immunology but it is also showing considerable promise as a model system for definitive studies on problems which bridge the fields of immunology and endocrinology, i.e., understanding the effects of environmental temperature and stress on the immune system.

Animals↗

PubMed1990

A monoclonal antibody specific for neutrophils in normal and stressed channel catfish.

A murine monoclonal antibody, designated mAb 13C5, was found to react specifically with channel catfish neutrophils based upon its ability to identify a subpopulation of leukocytes that are phagocytic and stain positive with both Sudan Black B and nitro-blue tetrazolium. Cytofluorographic analysis with mAb 13C5 was used to assess trafficking of neutrophils in various tissues of catfish subjected to transport stress. Since no prestress neutrophil reservoir was apparent, it seems likely that stress-induced neutrophilia in catfish, as in endotherms, may result from demargination of a pool of capillary-bound neutrophils. MAb 13C5 was also used to successfully "pan" for neutrophil-enriched and depleted populations of peripheral blood leukocytes from transport stressed channel catfish. "Panning" indicated that the continued physical presence of elevated numbers of neutrophils is not responsible for the suppression of T and B cell in vitro proliferation responses to mitogens observed in stressed fish.

Animals↗

PubMed1990

Differential effects of temperature and exogenous fatty acids on mitogen-induced proliferation in channel catfish T and B lymphocytes.

1. Exogenously supplied, BSA complexed saturated and unsaturated fatty acids were compared for their effects on mitogen-induced DNA synthesis in channel catfish T and B lymphocytes. 2. At "permissive" in vitro temperatures (27 degrees C), high concentrations (greater than or equal to 240 microM) of all the fatty acids used were inhibitory. However, at lower concentrations (80-160 microM), differences were noted in the ability of some fatty acids to modulate mitogen responses. While palmitic acid (16:0) and linoleic acid (18:2) had little effect on LPS-induced B cell- or Con A-induced T cell proliferation, stearic acid (18:0) suppressed while oleic acid (18:1) enhanced T cell responses only. 3. Adding equimolar amounts of 18:0 and 18:1 obviated the effects of singularly added fatty acids on T cell mitogenesis. 4. 18:1 was used to successfully "rescue" approximately 60% of the Con A-induced T cell proliferation normally inhibited at "nonpermissive" in vitro temperatures (17 degrees C). 5. While B cells readily appear to desaturate 18:0 and synthesize unsaturated fatty acids, T cells accumulate comparatively large amounts of 18:0 in membrane associated phospholipids. 6. It is proposed that 18:1 enhances T cell responses at permissive high temperatures and rescues suppressed T cell responses at nonpermissive low temperatures by increasing membrane fluidity.

Animals↗

PubMed1989

Amonabactin, a novel tryptophan- or phenylalanine-containing phenolate siderophore in Aeromonas hydrophila.

Aeromonas hydrophila 495A2 excreted two forms of amonabactin, a new phenolate siderophore composed of 2,3-dihydroxybenzoic acid, lysine, glycine, and either tryptophan (amonabactin T) or phenylalanine (amonabactin P). Supplementing cultures with L-tryptophan (0.3 mM) caused exclusive synthesis of amonabactin T, whereas supplements of L-phenylalanine (0.3 to 30 mM) gave predominant production of amonabactin P. The two forms of amonabactin were separately purified by a combination of production and polyamide column chromatographic methods. Both forms were biologically active, stimulating growth in iron-deficient medium of an amonabactin-negative mutant. Of 43 additional siderophore-producing isolates of the Aeromonas species that were tested, 76% (19 of 25) of the A. hydrophila isolates were amonabactin positive, whereas only 19% (3 of 16) of the A. sobria isolates and all (3 of 3) of the A. caviae isolates produced amonabactin, suggesting a predominant synthesis of amonabactin in certain Aeromonas species.

Aeromonas↗

PubMed1989

Unsaturated fatty acids inhibit IL-2 production in thymus-dependent antibody responses in vitro.

Albumin-complexed unsaturated fatty acids such as oleic acid (18:1) exerted a dose-dependent inhibitory effect on in vitro primary anti-TNP plaque-forming cell (PFC) responses to trinitrophenyl keyhole limpet haemocyanin (TNP-KLH), but did not affect primary PFC responses to trinitrophenyl lipopolysaccharide (TNP-LPS). The addition of 150 microM 18:1 at the initiation of thymus-dependent (T-D) antibody cultures inhibited the subsequent PFC response by 85%, and removal of the fatty acid after 24 hr did not reverse its inhibitory effect. By contrast, delaying the addition of 18:1 until 3 or 4 days after culture initiation abrogated its inhibitory effects. T-D antibody cultures displayed maximum production of interleukin-2 (IL-2) on the third day after culture initiation and a 24-hr exposure to 18:1 resulted in a dose-dependent inhibition of IL-2 production. Lastly, the addition of exogenous IL-2 reversed the inhibition of PFC responses in cultures transiently exposed to 18:1. These findings suggest that unsaturated fatty acids inhibit in vitro T-D PFC responses by selectively interfering with early stages of the antibody response, particularly those events leading to IL-2 production by T-helper cells.

Animals↗

PubMed1988

Phylogeny of lymphocyte heterogeneity: the thymus of the channel catfish.

The number of thymocytes (approximately 3 x 10(7)) that were recoverable from fingerling channel catfish remained constant from about 3 to 10 months of age, i.e. from September to April following hatching the previous June. Between 11 and 12 months, i.e. May and June, the thymus dramatically increased in size with 3 x 10(9) thymocytes being recoverable from the tissue of individual fish. The thymus remained enlarged for several months (throughout the summer) but at about 15 months (in September) began to involute such that by 17 months (November) no thymus tissue could be seen macroscopically. This natural involution could be accelerated by subjecting the fish to handling and transport stress. Thymocytes of channel catfish aged 4 to 16 months exhibited reactivity with monoclonal antibodies against peripheral T cells but not B cells. Thymocytes responded to the mitogen Concanavalin A only in the presence of added accessory cells (peripheral blood monocytes) or a monocyte-derived supernatant (presumably containing IL-1) at permissive temperatures (27 degrees C). Thymocytes could also be induced to divide at nonpermissive temperatures (17 degrees C) when incubated in the presence of the following combinations of stimulants, a) the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and the calcium ionophore A23187, b) TPA and ConA, or c) A23187 and ConA. In those cases where TPA or A23187 were used, accessory cells or their products were not needed. Collectively, these results support the notion that channel catfish thymocytes functionally mimic those lymphocytes in the peripheral blood previously designated as T cells.

Age Factors↗

PubMed1988

Ligand binding by murine IgM antibodies: intramolecular heterogeneity exists in certain, but not all, cases.

The ligand binding properties of eight hybridoma-derived murine anti-DNP IgM(kappa) antibodies were analysed by equilibrium dialysis. Four of these proteins exhibited the expected valances of approximately 10 and relatively low affinities (less than or equal to 2.2 x 10(5) M-1). The remaining four proteins exhibited valences of considerably less than 10 (less than or equal to 8) and relatively high affinities (greater than 10(6) M-1). When these proteins were subjected to two cycles of lyophilization, those of the former group were observed to still exhibit approximately 10 sites per molecule with homogeneous affinities similar to those of the respective untreated molecules. However, molecules in the latter group (valences of less than or equal to 8) were observed to exhibit only five to six binding sites subsequent to lyophilization with no changes in affinities. When the reductive subunits from each of the IgM(kappa) proteins were subjected to trypsinization, two different patterns were observed in terms of the yields of Fab mu fragments. Each of the proteins originally exhibiting approximately 10 binding sites yielded greater than 90% of the expected Fab mu fragments. In contrast each of the proteins exhibiting less than or equal to 8 binding sites yielded only approximately 50% of the expected Fab mu fragments. Collectively these results indicate the existence of at least two different forms of murine IgM molecules, those with approximately 10 homogeneous, relatively stable sites and those with only approx. five stable sites. It is suggested that these intramolecular functional differences may be attributable to intramolecular conformational differences.

Animals↗