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Biomedical subjects

G A Splitter

Publications and source records attributed to G A Splitter.

At least 91 records · Page 5Linked to original sources
PubMed1986

Bovine T lymphocyte response to bovine herpesvirus-1: cell phenotypes, viral recognition and acid-labile interferon production.

The phenotype of bovine cells that proliferate to bovine herpesvirus (BHV-1) were identified by peanut agglutinin (PNA) and monoclonal antibodies with specificity for Pan T cells (B29), a T cell subset (B24), and an MHC class II (Ia-like) antigen (R-1). PNA+ cells but not PNA- cells separated by fluorescent activated cell sorting responded to BHV-1. Virally-activated T cells expressed MHC class II antigens, and possibly antigen specific receptors for virus. Binding of radiolabeled virus increased six-fold beyond the expected value for activated cells suggesting specificity of the lymphocytes for the virus. Finally, cells that responded to BHV-1 produced high levels of acid sensitive interferon. Taken together these results characterize the phenotypes of bovine lymphocytes that interact with BHV-1.

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PubMed1986

Collaboration of bovine T lymphocytes and macrophages in T-lymphocyte response to Brucella abortus.

Brucella abortus-induced bovine macrophage-T-lymphocyte collaboration was studied as a prerequisite for the eventual clearance of this infectious organism. Esterase-positive, peripheral blood monocytes functioned as an adherent antigen-presenting cell population. A dual requirement for expression of bacterial antigens in combination with self major histocompatibility complex class II products was required by adherent cells for the activation of T lymphocytes. Comparison of antigen-presenting cell populations that were either trypsinized or nontrypsinized following B. abortus ingestion substantiated the need for phagocytosis and antigen processing. A monoclonal antibody (H4) directed against major histocompatibility complex class II determinants was able to block or, with complement, to abrogate T-lymphocyte responses. Measurement of both proliferation and interleukin 2 production via [3H]thymidine incorporation confirmed specific activation of an enriched T-lymphocyte population. These results indicate that in vivo-primed T lymphocytes of peripheral blood origin recognize phagocytized bacterial components of the facultative intracellular bacterium B. abortus and may contribute to the removal of the bacteria. Furthermore, bovine peripheral blood-adherent cells function as classic antigen-presenting cells, which suggests that macrophages are capable of processing this bacteria. Therefore, any lymphocyte-mediated dysfunction attributable to B. abortus most likely occurs at some point in the cascade of immune events following initial macrophage-T-lymphocyte collaboration.

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PubMed1986

Localization of antigen-specific lymphocytes following lymph node challenge.

The effect of subcutaneous injections of Brucella abortus strain 19 antigen on the specific localization of autologous lymphocytes in the regional nodes of calves was analysed by fluorescent labelling and flow cytometry. Both in vitro and in vivo FITC labelling of lymphocytes indicated the preferential migration of lymphocytes from a previously challenged lymph node to a recently challenged lymph node. However, lymphocytes from a lymph node challenged with B. abortus failed to localize preferentially in a lymph node challenged with a control antigen, Listeria monocytogenes. Lymph node cells, enriched for T lymphocytes and isolated from primary stimulated or secondary challenged B. abortus lymph nodes, could proliferate when cultured with autologous antigen-pulsed macrophages. The kinetics of [3H]thymidine incorporation in lymphocytes from secondarily challenged lymph nodes occurred earlier and to a greater extent when compared with lymphocytes from primary challenged lymph nodes. Our data show that the accumulation of B. abortus-specific lymphocytes in secondarily challenged lymph nodes is increased by the presence of the specific antigen.

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PubMed1985

Killer cells of feline leukemia virus- and feline sarcoma virus-infected transformed cells: the role of NK, ADCC, and in vitro generated cytotoxic cells.

Feline white blood cells (WBC) manifested a primary in vitro mixed lymphocyte tumor cell culture (MLTC) proliferative response to feline leukemia virus-feline sarcoma virus (FeLV-FeSV)-infected transformed target cells, which reached a peak at Day 15. Furthermore, primary in vitro MLTC cultures generated cytotoxic killer cells capable of killing a variety of targets in non-major histocompatibility gene complex restricted fashion, and effector cells were capable of killing targets introduced repeatedly into cultures over a 49-day period. The presence of feline fibrosarcoma (f-sarc) stimulators was the primary driving force for proliferation and generation of killing because exogenous IL-2 conditioned medium did not appreciably increase the yield of killer cells generated in vitro. WBC cultured without f-sarc stimulators with or without IL-2 supplementation also generated killer (K) cells but at a low level. The killer cell population was composed of approximately 50% lymphocytes and 50% monocytes. Cats had K cells functional in antibody-dependent cell-mediated cytotoxicity against FeLV-coated chicken red blood cells but not against any FeLV-FeSV-infected transformed targets tested. Natural killer (NK) cell activity to any targets tested was not found. Although no evidence was found for K or NK cell activity against FeLV-FeSV-infected transformed cells, feline WBC were readily able to generate killer cells in vitro and it is probable that this cell-mediated immune potential is functionally important in vivo.

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PubMed1985

Lymphokine-induced inhibition of growth of Eimeria bovis and Eimeria papillata (Apicomplexa) in cultured bovine monocytes.

Sporozoites of Eimeria bovis penetrated and developed normally to first-generation meronts in bovine monocytes (BM) and Madin-Darby bovine kidney (MDBK) cells that had been pretreated with culture medium (CM) or supernatant (NS) from nonstimulated bovine T cells. At 240 h after sporozoite inoculation (ASI), the mean percent development (meronts/[sporozoites + meronts]) in CM- and NS-pretreated BM was 52 and 28%, respectively; values for MDBK cells were 36 and 35%, respectively. Pretreatment of BM and MDBK cells with supernatant (ConAS) from concanavalin A-stimulated bovine T cells had no effect on the ability of sporozoites to penetrate cells; however, at 240 h ASI, only 1% of the sporozoites in ConAS-pretreated BM cultures had developed to meronts. In contrast, ConAS had no adverse effect on the ability of E. bovis sporozoites to develop to first-generation meronts in MDBK cells. At 240 h ASI, E. bovis meronts in ConAS-pretreated BM were abnormal in appearance and retarded in development, whereas sporozoites appeared structurally normal by light microscopy. Pretreatment of BM with ConAS had no effect on the ability of sporozoites of Eimeria papillata (Apicomplexa) to penetrate cells. Sporozoites of E. papillata did not develop to meronts in ConAS-pretreated BM and, in contrast to E. bovis, most sporozoites were destroyed intracellularly.

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PubMed1985

Identification of feline monocytes and neutrophils as effector cells in antibody-dependent cellular cytotoxicity: sequential analysis, using light microscopy, histochemistry, and scanning electron microscopy.

Feline monocytes and neutrophils functioned as effector cells in antibody-dependent cellular cytotoxicity (ADCC) against antibody-coated chicken erythrocytes. Using light microscopy, effector cell populations were identified in effector-target cell interactions, with further characterization of these identical individual effector cells by histochemical evaluations and scanning electron microscopy. Monocytes and neutrophils, but not lymphocytes, were observed attacking target cells. Carbonyl iron depletion of monocytes and neutrophils from peripheral blood leukocytes caused a marked reduction from a mean of 62% to 3.6% lysis in ADCC as measured by a 4-hour 51Cr release assay. Effector cells functioning in the ADCC reaction were visualized, using sequential analysis and light microscopy, histochemistry, and scanning electron microscopy.

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PubMed1984

Bovine interleukin 2 (IL-2) production and activity on bovine and murine cell lines.

Long-term growth of T cell cultures requires addition of Interleukin 2 (IL-2). In order to maintain bovine cultures, optimal conditions for bovine IL-2 production were defined using peripheral blood mononuclear cells (PBM). Irradiation and preculture enhanced IL-2 production possibly by reducing suppressor activity. IL-2 activity was also detected in Bovine Herpesvirus Type 1-stimulated cultures. Unlike mitogen-stimulated cultures, a wide variation in IL-2 activity was seen between supernatants produced by virus-stimulated cells from different animals indicating the clonal nature of antigen specific cells from individuals. Bovine IL-2-dependent cells used to quantitate IL-2 activity were characterized as: PNA, esterase negative, H4+ (anti Ia-like), B29+ (anti-pan T cell), and C5- (anti-monocyte). The observations that bovine IL-2 can maintain activated murine cells, CTLL-20 and HT-2, could lead to the replacement of rat IL-2 with bovine IL-2 in long-term murine cultures. Conditions described here result in large volumes of active medium.

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PubMed1983

Transplantation tolerance: evidence for Lyt 1+,2-,Qa 1.2+ Suppressor-inducer T cells in allogeneic thymus-grafted nude mice.

Nude mice, of BALB/c genotype, grafted with thymus stroma become immunocompetent (R. Hong, H. Schultz-Wisserman, E. Jarreth-Toth, S. D. Horowitz, and D.D. Manning, J. Exp. Med. 149, 398, 1979; B. P. Chen and G. A. Splitter, Cell. Immunol. 51, 127, 1980), but are tolerant to the thymus-donor genotype. Using such mice to investigate the mechanism(s) of transplantation tolerance, it was found that maintenance of tolerance required active interactions of three subsets of T cells specific for alloantigens of the thymus-donor genotype: (i) Lyt 1+,2- helper T cells, (ii) Lyt 1-,2+ suppressor T cells, and (iii) Lyt 1+,2-,Qa 1.2+ suppressor-inducer T cells. In mixed-lymphocyte culture, helper T cells could be activated by alloantigens of the thymus-donor genotype, but clonal expansion of these helper T cells was inhibited by suppressor T cells with the same specificity. Furthermore, exogenous interleukin-2 (IL-2) could modulate this suppressor activity, which suggested that one consequence of suppression was to limit IL-2 available to effector T cells. The response of cultures to exogenous IL-2 also indicated that thymus alloantigen-specific helper T cells had functional IL-2 receptors. Last, the presence of Lyt 1+,2-,Qa 1.2+ suppressor-inducer T cells were essential for active suppression, as suppressor T cells could not prevent helper T cells from proliferating to thymus-donor alloantigens when Lyt 1+,2-,Qa 1.2+ cells were removed. Altogether, the data presented in this study indicate a feedback-suppression pathway that led to clonal silencing of effector cells in transplantation tolerance.

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PubMed1983

Transplantation tolerance: Lyt-1+2- helper T cells require a second proliferation signal to overcome Lyt- 1-2+ suppressor T cell activity.

This study investigated why nude mouse T cell precursors that differentiated in an allogeneic thymus graft were rendered tolerant to alloantigens of the thymus genotype. Our results showed that Lyt- 1-,2+ suppressor T cells inhibited clonal expansion of helper T cells reactive to thymus-donor alloantigens as the mechanism of tolerance. This suppressor T cell activity could be modulated in vitro with either exogenous interleukin 2 (IL 2) or anti-Lyt-1 monoclonal antibody as an IL 2 inducer. Monoclonal antibody stimulation of IL 2 production by primed helper T cells was effective even in the presence of suppressor T cells. These results indicate: i) that alloantigen-specific helper T cells were not defective in their ability to recognize alloantigens of the thymus-donor genotype and to produce IL 2; ii) that Lyt-1-,2+ suppressor T cells prevented Lyt-1+,2- helper T cells specific for the thymus-donor alloantigens from producing IL 2; and iii) that signals delivered through binding of anti-Lyt-1 antibody modulated suppressor activity on alloantigen-primed helper T cells and permitted IL 2 production by these helper T cells. Accordingly, a delicate balance of helper and suppressor T cell activity was required for the maintenance of transplantation tolerance. Furthermore, these results argue against clonal deletion of Lyt-1+,2- helper T cells reactive to alloantigens of the thymic genotype as a mechanism of such transplantation tolerance.

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PubMed1981

The bovine major histocompatibility complex (BoLa): close linkage of the genes controlling serologically defined antigens and mixed lymphocyte reactivity.

Detection of linkage between genetic loci in cattle has been hampered by the lack of large full -sib families. A unique source of full-sib families is now available from embryo transplantation. Lymphocytes from six full-sib families, ranging in size from three to seven siblings, were tested for serologically defined BoLA antigens (BoLA-A). In addition, mixed lymphocyte reactivity (MLR) was tested between all paired combinations of cells within each family to distinguish BoLA-D specificities. Serologically identical siblings within each family were reciprocally nonreactive in MLR, and vice versa; thus, no recombinants were detected between the BoLA-A and the BoLA-D loci. Classical genetic linkage analysis revealed that these loci are significantly closer than 11.9 centimorgans.

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PubMed1981

Two molecularly independent surface receptors identify bovine T lymphocytes.

E rosette formation is a commonly used technique to identify T lymphocytes in many species; however, treatment of bovine E rosettes with rhodamine-conjugated F(ab')2 anti-immunoglobulin resulted in 10% (range 3-18%) of these cells exhibiting fluorescence. The failure of E rosettes to identify only non-immunoglobulin bearing cells suggested that an alternative method for T lymphocyte identification was essential. Using a labeled heterologous T cell antiserum and peanut agglutinin (PNA), identical populations of bovine T lymphocytes were identified. Unique and molecularly independent cell surface receptors were detected by dual fluorescent staining experiments differential inhibition of cellular binding of PNA by its carbohydrate ligand and capping experiments. Moreover, both reagents bound to approximately 62% of peripheral blood lymphocytes (PBLs) and virtually all thymocytes. Use of either T cell reagent in combination with rhodamine-conjugated F(ab')2 anti-bovine immunoglobulin provided a rapid, simple and reliable method for simultaneous enumeration of bovine T and B cells and permitted the detection of rare cells (less than 1%) expressing both T and B cell markers. Approximately 90% of all PBLs were identified as either T or B cells.

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PubMed1981

Bovine mixed leukocyte response: effect of selected parameters on cell responses.

The bovine mixed leukocyte culture (MLC) system offers potential benefit for the study of genetic and immunologic mechanisms in this species. Selected parameters of the bovine MLC have been investigated to assess their influence. The findings indicate that maximal MLC response was present at days 5 and 6 with 2 x 10(5) responder to 2 x 10(5) stimulator cells per well or greater. Serum concentrations over a wide range effectively supported cell growth. More importantly, the source and treatment of serum influenced the level of MLC response. Serum autologous to the responder which was heat treated appeared to provide maximal MLC values compared to other sources examined. Antisera to serologically-defined lymphocyte antigens of the stimulating cell population did not affect the response. One-way MLC determinations were consistent when the stimulating cell population was irradiated at 1,000 Rads or greater. Both freshly obtained lymphocytes and lymphocytes held at room temperature for 18 hr provided good sources of MLC responder and stimulator cells; however, lymphocytes held for 18 hr consistently gave higher counts and stimulation indices than freshly obtained lymphocytes. The effect of contaminating RBCs was found to enhance MLC reactivity at lower concentrations and inhibit MLC reactivity when in excess of 7 X 10(9) RBC per well. Stimulator cells were present in both macrophage enriched and depleted populations suggesting several cell populations possess antigens which lymphocytes recognize. Consideration of these parameters were essential in providing optimal, reproducible results.

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PubMed1981

Bovine con A-induced suppressor cells: generation, macrophage requirements and possible mechanisms of regulatory action.

Bovine Concanavalin A-induced suppressor cells were generated from lymphocytes which were non-adherent to anti-immunoglobulin coated dishes and cells possessing receptors for peanut agglutinin. Bovine lymphocytes, preincubated with 25 microgram/ml of Con A for 40-45 hr, could suppress the responses of autologous cells to the mitogens Con A, PHA and PWM as much as 90% when they were cultured together at a ratio of 1:1 (suppressor cell to responder cell) or higher. Suppressor cells were not necessary at the initiation of the mitogenic assay as they could regulate responding cells if added at 48 hr in a 72 hr assay. Allogeneic responder cells could be suppressed at the same level as autologous cells indicating a lack of genetic restriction. Macrophages were not required for suppressor cell generation because peripheral blood lymphocytes (PBL) depleted of macrophages by Sepharose G-10 columns, and subsequently incubated with Con A, could suppress autologous cells to a similar degree as unseparated PBl's. Responder cells depleted of macrophages had normal mitogen responsiveness and were suppressed indicating macrophages were not required in transmission of suppressor signals. Cell to cell contact was not required for suppression connoting a soluble factor(s) as the modulator of suppression.

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PubMed1980

A simple method for specific depletion of B lymphocytes from bovine peripheral blood mononuclear cells.

The treatment of bovine peripheral blood mononuclear cells with rabbit anti-bovine immunoglobulin, goat anti-rabbit immunoglobulin (GAR) and complement resulted in the specific lysis of all surface immunoglobulin (SIg) bearing B lymphocytes. No SIg+ lymphocytes were detected after 12 hours of culture following lytic treatment, whereas cells treated with antibody without complement readily stained for SIg. The percentage of cells forming E-rosettes or binding peanut agglutinin (PNA) increased following lysis. The percentage of latex-ingesting monocytes also increased after lysis even though many of these cells had cytophilic Ig. B lymphocyte-depleted (T cell-enriched) populations cultured with phytohemagglutinin (PHA) and concanavalin A (Con A) were never less reactive than unseparated cells. No differences in background mitosis was observed for these 2 cells preparations. These results suggest that bovine SIg- cells do not require B lymphocytes to respond to PHA and Con A and that lysis of B lymphocytes does not alter the responsiveness of the recovered SIg- cells.

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