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G A Splitter

Publications and source records attributed to G A Splitter.

At least 73 records · Page 4Linked to original sources
PubMed1990

Bovine natural killer-like cell responses against cell lines expressing recombinant bovine herpesvirus type 1 glycoproteins.

The murine retrovirus shuttle vector pZipNeo SV(X)1 was used to construct plasmids encoding the three major surface glycoproteins of bovine herpesvirus-1 (BHV-1). Each plasmid was transfected into D17, a canine osteosarcoma cell line sensitive to lysis by bovine NK-like cells when infected with BHV-1. After selection in G418 sulfate, cell lines expressing the recombinant gene products were sorted by flow microfluorimetry, radioimmunoprecipitated, and analyzed by SDS-PAGE for fidelity as compared to the native viral glycoproteins. Two of the three genetically engineered cell lines (gI and gIV) could successfully serve as targets to detect bovine NK-like cytolysis. These findings support and extend a previous study from our laboratory indicating a role for BHV-1 glycoproteins in the cytolytic response by bovine NK-like cells. Additionally, this study demonstrated that individual proteins are recognized by these effector cells, and that recombinant glycoproteins can direct cytolytic activity in the absence of host cell infection-associated proteins. This is the first known report of Ag directed cytotoxicity by bovine null (non-B, non-T) cells.

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PubMed1990

Recombinant herpesviral proteins produced by cell-free translation provide a novel approach for the mapping of T lymphocyte epitopes.

The availability of viral genes in cloned DNA forms, in addition to the parallel development of an efficient cell-free system capable of producing viral proteins in vitro, has provided new opportunities for the identification of important T cell Ag encoded within viral genomes. We have cloned bovine herpesvirus type 1 surface glycoproteins gI, gIII, and gIV into the RNA transcription vector pE5LVPO. After translation of the resulting viral mRNA in rabbit reticulocyte lysates, each cloned viral protein specifically stimulated bovine T lymphocytes previously primed to BHV-1. In addition, a panel of specifically truncated peptides has allowed the identification of at least two T lymphocyte epitopes encoded within gIII of BHV-1. Our results represent a novel approach for viral Ag synthesis and subsequent detection of lymphocyte recognition. These findings will likely have important implications in the mapping of T lymphocyte epitopes.

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PubMed1990

A novel population of cells in the peripheral blood of a cow with a neurofibrosarcoma.

An unusual population of leukocytes was observed in the peripheral blood of a cow with a large tumor burden, using flow microfluorimetry. This new population accounted for 50% of the total cells in the peripheral blood of this animal. These cells expressed the p150,95 molecule (bovine CD11c equivalent), identified by the monoclonal antibody C5B6, a molecule found on myeloid cells and activated lymphocytes. The new population did not express the pan T molecules BoCD2 (the bovine T11 equivalent), BoCD5 (the bovine CD5 equivalent) or surface IgM. Isolated peripheral blood mononuclear cells maintained in bulk culture were able to kill autologous tumor cells and BHV-1 infected A549 in an NK-like assay. In vitro cytotoxicity by cells cultured from the peripheral blood of this animal was augmented 2- to 4-fold by the addition of IL-2.

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PubMed1990

Biochemical and functional characterization of 8-10C5 (gp120,90), a novel molecule on the surface of bovine T leucocytes.

An unclustered molecule, 8-10C5, present on bovine leucocytes, was characterized using a monoclonal antibody (mAb) prepared at the University of Wisconsin-Madison, and the functional and biochemical characteristics of that molecule compared to defined molecules. This molecule was present on a large percentage of peripheral blood cells (84%), including T and B lymphocytes, monocytes and neutrophils, but was absent from immature thymocytes. mAb 8-10C5 blocked proliferation to alloantigen and bovine herpesvirus-1; however, antibody to the 8-10C5 molecule was unable to stimulate proliferation alone or in combination with PMA or a cross-linking agent. These results indicate that this molecule has a role in T-cell activation, but probably not signal transduction. mAb 8-10C5 was unable to block cytotoxic T lymphocyte (CTL) or natural killer (NK)-like lysis. mAb 8-10C5 identifies a non-covalently bound heterodimer present on most peripheral lymphocytes with a molecular weight (MW) of 120,000 for the alpha chain (10% glycosylation) and 90,000 MW for the beta chain (16% glycosylation). The beta chain is an acidic molecule (pI = 4.5-5.0). Biochemical studies included the CD2 and CD5 molecules and further demonstrated their similarity to equivalent molecules in other species, confirming the phylogenetic importance of these molecules in immune responses. This functional and biochemical information will allow further characterization of these molecules at the RNA and DNA levels.

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PubMed1989

Identification of two independent MHC class II antigens in a bovine lymphoblastoid cell line.

Bovine major histocompatibility complex (MHC) class II antigens were investigated using monoclonal antibodies (MoAbs) with known MHC class II specificities in other species. Thirty-four MoAbs were tested for reactivity with bovine peripheral blood mononuclear (PBM) cells and the bovine lymphoblastoid cell line, BL3, by flow cytometry. Twenty-seven of 31 MoAbs tested, reacted with BL3 cells, and 22 of 25 MoAbs tested with PBM cells were reactive. MoAbs that reacted with BL3 cells were used to immunoprecipitate class II molecules from BL3 lysate labeled with [35S]methionine. Using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography, many MoAbs were found to immunoprecipitate a single band of approximately 31,000 relative mass (Mr). MoAbs yielding successful immunoprecipitations and with known antigen specificity in other species were then used in sequential immunoprecipitations and two dimensional (2-D) non-equilibrium pH gradient electrophoresis (NEPHGE). The HLA-DR specific MoAb H4 and the predominantly HLA-DQ specific MoAb CC11.23 were used to identify the presence of two independent antigens in BL3 cell lysate. These class II molecules consist of alpha and beta chains.

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PubMed1989

Characterization of bovine mononuclear cell populations with natural cytolytic activity against bovine herpesvirus 1-infected cells.

Freshly isolated or overnight cultured peripheral blood mononuclear cells from immune or nonimmune animals had natural cytolytic activity against bovine herpesvirus 1 (BHV-1)-infected tumor target cells. No lysis was demonstrated against tumor target cells alone. This natural cytolytic activity was present in mononuclear cells from the spleen, lymph node, and peripheral blood but little or no cytolytic activity was detected in bone marrow or thymus cells. When monoclonal antibodies and complement to deplete bovine mononuclear cell subpopulations from the nonadherent cells were used, results indicated the effector cell was not a T cell, B cell, or activated monocyte. From nonadherent populations separated on density gradients, it was determined that the effector cells were large, low density mononuclear cells. These results indicate the nonadherent effector cells mediating lysis of BHV-1-infected xenogeneic adherent target cells were large null lymphocytes and/or immature monocytes.

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PubMed1989

Brucella abortus regulates bovine macrophage-T-cell interaction by major histocompatibility complex class II and interleukin-1 expression.

T-cell activation is dependent on nominal antigen associated with major histocompatibility complex (MHC) class II molecules and interleukin-1 (IL-1), both provided by antigen-presenting cells. We have studied the effects of Brucella abortus and recombinant bovine gamma interferon (IFN-gamma) on bovine macrophage expression of MHC class II and IL-1 molecules and subsequent T-cell proliferation in response to B. abortus. When peripheral blood mononuclear cells were cocultured with B. abortus and IFN-gamma, increasing amounts of IFN-gamma, from 1 to 100 U/ml, down regulated T-cell proliferation. Expression of MHC class II molecules on macrophages was increased independently by IFN-gamma or B. abortus treatment. Thus, class II molecule expression was not the cause of down regulation of T-cell proliferation as observed in other systems. However, B. abortus-IFN-gamma-treated macrophages obtained from overnight cultures had minimal membrane IL-1 compared with macrophages treated with B. abortus alone. Loss of membrane IL-1 required IFN-gamma and the o-polysaccharide of the lipopolysaccharide. IFN-gamma at 1 U/ml in combination with B. abortus produced a 32% decrease in T-cell response, while IFN-gamma at 100 U/ml added to B. abortus-treated cultures produced an 82% reduction in T-cell response. Membrane IL-1 levels were not altered when recombinant bovine IFN-alpha or the rough strain 45/20 of B. abortus, which lacks the o-polysaccharide, was used. Secreted IL-1 levels were unaffected by IFN-gamma and B. abortus treatment. The addition of recombinant bovine IL-1 beta (0.001 to 0.1 ng/ml) to B. abortus- and IFN-gamma-treated cultures failed to provide a signal necessary for T-cell proliferation. These data suggest that membrane IL-1 has a key role in T-cell activation in response to B. abortus. When the o-polysaccharide of B. abortus lipopolysaccharide is combined with IFN-gamma at an inappropriate time during an immune response, T-cell proliferation is prevented and cannot be restored by the addition of exogenous IL-1.

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PubMed1989

Development of a diagnostic test for Johne's disease using a DNA hybridization probe.

A DNA probe, M13 mpHAW71, that detects Mycobacterium paratuberculosis in the fecal material of infected animals was developed for use in the diagnosis of Johne's disease. The probe detected as few as 10(5) M. paratuberculosis when hybridized under stringent conditions to total genomic DNA purified from bovine fecal material. When the probe was used diagnostically, it did not differentiate members of the Mycobacterium avium-M. intracellulare-M. paratuberculosis complex. Compared with culturing, the DNA probe identified 34.4% more mycobacterium-containing fecal samples, and testing took only 72 h to complete.

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PubMed1989

Bovine naturally cytolytic cell activation against bovine herpes virus type 1-infected cells does not require late viral glycoproteins.

Freshly isolated or overnight cultured bovine peripheral blood mononuclear (PBM) cells lysed bovine herpes virus 1 (BHV-1)-infected allogeneic and xenogeneic target cells but not non-infected target cells. To determine if late viral genes contribute to target cell lysis, phosphonoacetic acid (PAA), an inhibitor of DNA polymerase activity, was used to block DNA replication that is required for expression of late viral proteins. Both adherent and non-adherent (NA) cell populations mediated lysis against PAA-treated BHV-1-infected target cells in both 4- and 20-hr assays, indicating recognition and killing occurred in the absence of expression of late BHV-1 glycoproteins. Thus recognition of BHV-1 by bovine natural cytolytic effector cells does not require recognition of late BHV-1 glycoproteins for killing virally infected target cells.

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PubMed1989

Monoclonal antibodies to ovine SBU-T8 and SBU-T6 bind analogous molecules on bovine lymphocytes.

Monoclonal antibodies (mAb) to ovine T-lymphocyte molecules SBU-T8 (Maddox, Mackay & Brandon, 1985), the cytotoxic T lymphocyte, human CD8 equivalent, and SBU-T6 (Mackay et al., 1985), the immature T lymphocyte human CD1 equivalent, were reported to cross-react with bovine cells. We have confirmed this cross-reactivity and shown that the percentage of T lymphocytes in thymus, lymph node and peripheral blood is equivalent to that reported in sheep. In addition, we have shown that the molecular weight of the molecules identified by these antibodies (T8, 34,000-38,000; T6, 44,000 and 13,000) is similar to that reported in sheep and humans. The monoclonal antibody to SBU-T8 effectively removes bovine cytotoxic cells in the presence of complement and when used alone blocks killing of allogeneic concanavalin A (Con A) lymphoblasts by mixed lymphocyte-generated cytotoxic T lymphocytes. These results confirm that the anti-SBU-T8 antibody 38-65 binds BoCD8 and anti-SBU-T6 antibody 20-27 binds BoCD1 and that these reagents will be useful in the study of the bovine immune system.

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PubMed1988

Lytic function of bovine lymphokine-activated killer cells from a normal and a malignant catarrhal fever virus-infected animal.

Lymphokine-supplemented long-term cultured bovine lymph node lymphocytes were characterized functionally and phenotypically. Lymphocytes from a normal and a malignant catarrhal fever (MCF) virus-infected animal were maintained without the addition of antigen or feeder cells. Lymphocyte cell lines obtained from both animals: (i) killed allogeneic fibroblasts and allogeneic and xenogeneic cultured tumor cell lines as measured in a 4-h 51Cr release assay, (ii) expressed the same T cell subset marker based on flow cytometry using monoclonal antibodies, and (iii) produced a lytic factor upon stimulation. In contrast, only cells from the MCF virus-infected animal could be maintained for more than 5 months supplemented with 2% Con A-generated lymphokine-containing supernatant. These results suggest that herpesvirus infection enhanced the proliferative capabilities of the cultured lymphocytes from the infected animal. Considering the proliferative and cytotoxic activity together with the T cell phenotype, these data indicated that effector cells are lymphokine-activated killer cells.

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PubMed1988

Determination of bovine lymphocyte responses to extracted proteins of Brucella abortus by using protein immunoblotting.

Isolation and identification of Brucella antigenic determinants important to cellular responses have been difficult. In this study, bovine peripheral blood mononuclear (PBM) cells from cattle vaccinated with Brucella abortus 19 proliferated to extracted bacterial proteins blotted onto nitrocellulose. Proteins were extracted from gamma-irradiated B. abortus 19 with a sodium dodecyl sulfate extraction buffer. The extracted proteins were separated electrophoretically by sodium dodecyl sulfate-polyacrylamide gel electrophoresis prior to electroblotting onto nitrocellulose. Nitrocellulose sections corresponding to individual lanes of the gel (containing all separated proteins) were then cultured with the PBM cells. Primary and secondary stimulation responses of the PBM cells with the whole protein blots were similar kinetically to the responses of the PBM cells stimulated with whole irradiated B. abortus 19 or with whole irradiated B. abortus 19 blotted onto nitrocellulose. Although lipopolysaccharide was determined to be associated with the extracted proteins and transferred onto the blots, the lipopolysaccharide did not stimulate cellular proliferation, as indicated by the antigen-specific secondary responses. Stimulating PBM cells with portions of the blot containing high (greater than 45,000)-, medium (25,000 to 45,000)- or low (25,000)-molecular-weight proteins demonstrated that the responding cells were specific only to the proteins of corresponding molecular weights. These results indicate that cellular responses to individual proteins can be studied without cloning the bacterial genes or purifying the individual proteins.

Animals↗
PubMed1988

Cloned bovine cytolytic T cells recognize bovine herpes virus-1 in a genetically restricted, antigen-specific manner.

The inability to demonstrate bovine herpes virus-1 (BHV-1)-specific lymphocyte responses from BHV-1-infected cattle has been a major difficulty in confirming the importance of cellular effector mechanisms during BHV-1 infection. We have examined the capacity of bovine cytolytic T-cell clones to lyse BHV-1-infected, concanavalin A-stimulated blast cells. Cytolysis was as high as 58% at an effector to target (E:T) ratio of 1:1. All cytolytic T-cell clones produced were genetically restricted in killing cells of the autologous genotype. Cytotoxic T-lymphocyte (CTL) clones were specific for BHV-1 but not related herpes viruses, i.e. BHV-2 and pseudorabies virus. These results provide evidence that cytolytic T lymphocytes have an antigen-specific role in the immune response of cattle against BHV-1, and that CTL may serve as effector cells in the identification of glycoproteins useful in recombinant vaccine preparation since only determinants of BHV-1 were recognized.

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PubMed1987

The development and analysis of species specific and cross reactive monoclonal antibodies to leukocyte differentiation antigens and antigens of the major histocompatibility complex for use in the study of the immune system in cattle and other species.

We examined the potential of developing a set of species specific and cross reactive monoclonal antibodies (MoAbs) for use in the study of the phylogenetic and functional relation of class I and class II antigens of the major histocompatibility complex (MHC) and leukocyte differentiation antigens in cattle and other species. Comparing immunization strategies demonstrated the number of hybrids producing cross reactive antibodies can be increased by hyperimmunization of mice with lymphoid cells from multiple species. Comparing various methods of assay (antibody-complement mediated cytotoxicity [CT], enzyme linked immunosorbent assay [ELISA] and flow microfluorimetry [FMF]), revealed FMF is the most useful technique for the primary assay of hybridomas producing MoAbs of potential interest. By using dual parameter and dual fluorescence analysis, we could determine whether a given MoAb reacted with mononuclear cells (lymphocytes and monocytes) and/or granulocytes, and also whether any two MoAbs of different isotype and specificity recognized antigens present on identical or separate populations of leukocytes. Comparing the patterns of MoAb reactivity with leukocytes obtained from cows, goats, sheep, pigs, horses and humans, as well as comparing the patterns of reactivity with a panel of lymphoid cell lines derived from cattle (with enzootic bovine leukemia) and humans (with various forms of leukemia), revealed sets of MoAbs reactive with unique antigenic determinants present on BoLA class I (15 MoAbs) and class II (9 MoAbs) antigens, and also MoAbs reactive with determinants present on leukocyte differentiation antigens (36 MoAbs). Dual fluorescence analysis demonstrated the antigens detected by some MoAbs are predominantly expressed on one lineage of leukocytes while others are expressed on two or more lineages of leukocytes. Dual and single fluorescence analysis also demonstrated the PNA receptor(s) is: expressed on T cells, granulocytes and class II antigen monocytes and absent or expressed in low amount on sIgM+ B cells and a newly defined Non T/Non B population of cells. The strategies described for identifying and analyzing the specificity of MoAbs demonstrate the feasibility of developing a set of cross reactive MoAbs for identifying homologous molecules in multiple species and delineating their functional and phylogenetic relation.

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PubMed1987

Rapid lysis technique for mycobacterial species.

The lysis of mycobacterial cells typically has been difficult and time-consuming. We report a method for the physical rupture of Mycobacterium paratuberculosis and several other members of the family Mycobacteriaceae using a Mini-Beadbeater cell disrupter (Biospec Products; Bartlesville, Okla.) and zirconium beads, a process which yields DNA and RNA of high molecular weight and in greater quantity than that obtained by rupture in a Ribi pressure cell.

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PubMed1986

Differences in cellular immune response to L-glutamic acid60, L-alanine30, L-tyrosine10 (GAT) in full sibling embryo transfer cattle: examination of requirements for cell proliferation.

Pairs of full sibling embryo transfer cattle that expressed identical MHC class I and II products were tested for their in vitro proliferative response to GAT. Peripheral blood mononuclear cells from these cattle were either high or low responders to GAT. Cells from certain pairs of MHC identical siblings gave opposite responses. Low responder animals were further tested to determine if they might respond to GAT with different kinetics, with secondary in vitro restimulation, or with exogenous help provided by interleukin 2. Also, the role of antigen presenting cells and suppressor T cells from low responder animals was investigated. Using appropriate in vitro conditions, cells from all animals tested could respond to GAT. However, MHC identical animals tested under similar conditions exhibited differences in their response to GAT which suggests the proliferative immune response was influenced by factors in addition to MHC coded products.

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