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G A Splitter

Publications and source records attributed to G A Splitter.

At least 55 records · Page 3Linked to original sources
PubMed1994

Bovine NK and LAK susceptibility is independent of class I expression on B lymphoblastoid variants.

Numerous tumors express low or no class I molecules, resulting in their avoidance of recognition and destruction by different effector cells of the immune system. Using a parent and two MHC class I mutant cell lines, we have tested the role of MHC class I molecules in natural killer (NK) cells, lymphokine activated killer (LAK) cells and cytotoxic T lymphocytes (CTLs). Both class I expressing parent cells and class I loss mutants were insensitive to NK cell lysis as assayed, regardless of the amount of class I molecules on the target cell surface. However, LAK cells demonstrated higher cytolysis on these target cells than NK cells, suggesting different mechanisms of target cell recognition or different levels of lytic activity by these two effector cell populations. Up-regulation of class I expression on the target surface by gamma interferon (gamma-IFN) had little influence on NK and LAK susceptibility, indicating there was no correlation between class I expression and bovine NK or LAK cytolysis. However, allogeneic CTLs mediated a lytic pattern distinct from NK and LAK cells, in which target sensitivity to allogeneic CTLs correlated with the amount of class I molecules expressed on the cell surface. Additionally, effector-target cell conjugation studies demonstrated that target class I expression was not involved in NK and LAK cells binding to targets. These results demonstrate that NK and LAK cytolysis of these two class I mutant cell lines is independent of the amount of class I molecules expressed on the target cell surface.

Animals↗
PubMed1994

Reduction of class I expression in a bovine B-lymphoblastoid cell line is locus specific.

A bovine B-lymphoblastoid cell line, BL3.1.2, defective in MHC class I expression was isolated following gamma irradiation and immunoselection of a previously mutated cell, BL3.1. Flow microfluorimetry demonstrated that BL3.1.2 cells expresed reduced amount of class I molecules on the cell surface when compared with the parent cells. 2D gel electrophoresis showed that two less class I gene products were synthesized by BL3.1.2 cells than the parent cells. However, no detectable RFLP difference was revealed between this mutant and the parent cell lines as analyzed by Southern blots using bovine class I cDNA and locus-specific oligonucleotide probes, suggesting no major deletions, insertions, or translocations within class I genes. Northern blot analysis, using class I cDNA probes, revealed a marked reduction of class I mRNA in BL3.1.2 cells. RNA unblots, probed with loci-specific oligonucleotides, and slot blot analysis demonstrated that absence of class I gene transcription was locus specific. This finding indicates there is separate regulation of each class I locus in bovine cells. Furthermore, recombinant IFN-gamma upregulated class I gene expression on the BL3.1.2 cells, as indicated by flow cytometry and slot blot analysis. These findings indicate that the locus-specific MHC class I gene was functionally downregulated in its expression. The silent class I gene could be activated by cytokines known to activate the cis-interferon-responsive element for subsequent transcription and translation of the newly synthesized class I RNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Truncated bovine herpesvirus-1 glycoprotein I (gpI) initiates a protective local immune response in its natural host.

Current modified live and killed BHV-1 vaccines have not reduced the incidence of bovine herpesvirus-1 (BHV-1), the principal viral agent in bovine respiratory disease complex. The requirement for production of viral proteins for immune study has resulted in the establishment of a cell line which constitutively expresses BHV-1 gpI. A truncated BHV-1 envelope gpI protein was secreted into the culture supernatant of D17 cells transfected with the gpI gene lacking the coding sequence for the transmembrane region (TMR). The transmembrane domain is essential for gpI stability in the envelope, virus infectivity and, most probably, natural killer cell recognition; however, we have tested the possibility that this domain is not required for inducing an adaptive, protective immune response. Immunization of calves with this truncated gpI protein induced gpI-specific nasal IgA, IgG1, serum neutralizing antibodies and gpI-specific peripheral lymphocyte proliferation. All immunized calves were protected from clinical disease after BHV-1 challenge. Further, nine of ten immunized calves had no intranasal viral shedding. One animal shed a minimal amount of virus following challenge, but produced no antibodies to other viral proteins as evidenced by immunoprecipitation of 35S-labelled viral proteins by sera from virus-challenged animals. This study represents the first evidence that a recombinant truncated gpI subunit vaccine can confer local mucosal immunity and establish a strong protective barrier against disease caused by BHV-1 in the natural host. Also, these data demonstrate the feasibility of preventing initial viral replication in the host and distinguishing vaccinated from wild-type virus-infected animals.

Animals↗
PubMed1994

Expression of tfx and sensitivity to the rhizobial peptide antibiotic trifolitoxin in a taxonomically distinct group of alpha-proteobacteria including the animal pathogen Brucella abortus.

Three phylogenetically distinct groups within the alpha-proteobacteria which differ in trifolitoxin sensitivity are described. Trifolitoxin sensitivity was found in strains of Agrobacterium, Brucella, Mycoplana, Ochrobactrum, Phyllobacterium, Rhodobacter, Rhodopseudomonas, Rhodospirillum, and Rhizobium. Strains of Agrobacterium, Brucella, Phyllobacterium, Rhizobium, and Rhodospirillum were capable of producing trifolitoxin upon conjugal transfer of tfxABCDEFG.

Anti-Bacterial Agents↗
PubMed1994

Subcloning and expression of the Brucella abortus L7/L12 ribosomal gene and T-lymphocyte recognition of the recombinant protein.

The Brucella abortus L7/L12 ribosomal gene was amplified by PCR and subcloned into the prokaryotic expression vector pMAL-c2. Escherichia coli DH5 alpha was transformed with the pMAL-L7/L12 construct, and gene expression was induced by IPTG (isopropyl-beta-D-thiogalactopyranoside). The resulting fusion protein was purified by affinity chromatography and confirmed by Western blot (immunoblot) analysis using an anti-maltose-binding protein antibody. Additionally, purified recombinant L7/L12 protein induced T-lymphocyte proliferation of B. abortus-primed bovine peripheral blood mononuclear cells. Phenotypic analysis of the proliferating cell population demonstrated an increase in the percentage of CD4+ T lymphocytes when peripheral blood mononuclear cells were cultured with recombinant L7/L12 compared with cells cultured in medium alone. Subcloning and expression of a B. abortus gene encoding a previously demonstrated immunodominant protein for bovine lymphocytes are important steps in selecting Brucella proteins that have potential as a component of a genetically engineered candidate vaccine.

Animals↗
PubMed1994

Recombinant L7/L12 ribosomal protein and gamma-irradiated Brucella abortus induce a T-helper 1 subset response from murine CD4+ T cells.

Immunity to Brucella abortus crucially depends on antigen (Ag)-specific T-cell mediated activation of macrophages, which are the major effectors of cell-mediated killing of this organism. Ribosomal preparations have been used as vaccines against several pathogens, including B. abortus, conferring a high degree of protection. In the present study, we have examined the pattern of T-helper (Th) cell response from infected BALB/c mice after in vitro stimulation with recombinant (r) L7/L12 ribosomal protein or gamma-irradiated B. abortus. In addition to Ag-specific proliferation, CD4+ T cells were tested for interleukin-2 (IL-2), IL-4 and interferon-gamma (IFN-gamma) mRNA expression and secretion. Detection of cytokine transcripts and secreted cytokines was performed using reverse transcriptase (RT)-polymerase chain reaction (PCR) and specific ELISA assays. Primed CD4+ T cells proliferated to the recombinant protein or whole B. abortus. The functional cytokine profile of the proliferating cells was typical of a Th1 cell phenotype, as we detected transcripts for IL-2 and IFN-gamma but not IL-4. Among the cytokines analysed, only IFN-gamma produced in the Th cell culture supernatants was detected by ELISA when bacteria or recombinant protein were used. Thus, rL7/L12 ribosomal protein and gamma-irradiated B. abortus preferentially stimulated IFN-gamma-producing Th1 cells after in vitro stimulation. The results of this study provide for the first time an explanation of why ribosomal vaccines may protect against intracellular infections, and an experimental basis for identifying polypeptides from a pathogen which stimulates the desired cytokine profile and Th cell response crucial for the design of genetically engineered candidate vaccines.

Animals↗
PubMed1993

Isolation of Brucella abortus ssb and uvrA genes from a genomic library by use of lymphocytes as probes.

Brucella abortus proteins from virulent S2308 expressed from a pBluescript II SK- genomic library stimulated peripheral blood mononuclear (PBM) cell proliferation from cattle vaccinated with B. abortus S19. The method described here permits a rapid and directed approach to isolate genes encoding antigens of B. abortus that interact with lymphocytes primed to the living bacterium. The supernatants from the bacterial host JM109 (DE3) were cultured with freshly isolated bovine PBM cells. A total of 300 clones were evaluated. Ten clones were identified that stimulated T-lymphocyte proliferation. Among them, one clone with a 2.5-kb insert stimulated T-lymphocyte proliferation in all three animals, suggesting that the proteins encoded by genes within this fragment may represent immunodominant antigens. DNA sequencing of this clone reveals two large open reading frames (ORFs). ORF II has a high degree of similarity to the Escherichia coli ssb gene, which codes for the single-stranded DNA binding protein. ORF I, in the opposite direction to ORF II, shows similarity to the N terminus of the E. coli uvrA gene, which codes for one of the three subunits of the E. coli ABC excision nuclease. The observation that the PBM cells recognized and proliferated in response to proteins expressed from single clones provides a novel strategy to select bacterial antigens that may prove useful in designing alternative vaccines against brucellosis.

Amino Acid Sequence↗
PubMed1992

Impairment of MHC class I transcription in a mutant bovine B cell line.

To better define the regulation and expression of bovine major histocompatibility complex (MHC) antigens, the bovine B lymphoblastoid cell line, BL3, was exposed to gamma-irradiation and surviving cells were immunoselected for MHC class I antigen loss. The resulting class I expression loss variant, BL3.1, was characterized at both the protein and genetic levels to ascertain the nature of the defect. Microfluorimetry analysis revealed a 3--5-fold surface density reduction of all class I products on BL3.1 cells relative to the parental BL3 cells. This decreased surface expression was specific for MHC class I and not for MHC class II or the non-MHC-linked gene product, immunoglobulin (Ig). Northern and quantitative slot blot analyses demonstrated a corresponding diminution of class I RNA in BL3.1 suggesting a transcriptional level defect. Nuclear run-off and transcription inhibition experiments confirmed no post-transcriptional changes while Southern blot analysis provided no evidence for alterations within or near the class I genes. To help elucidate the mechanism of altered class I expression, the parent, BL3, and variant, BL3.1, were cultured with factors known to enhance MHC class I transcription. Interferon (IFN)-gamma, lipopolysaccharide (LPS), and activated peripheral blood lymphocyte (PBL) supernatant cultured with both cell lines induced MHC class I transcription and surface expression 2--3-fold greater than the untreated controls. It is likely, therefore, that a genetic alteration outside of the class I genes has occurred within BL3.1 impairing expression of MHC class I.

Animals↗
PubMed1992

Bovine major histocompatibility complex class I specific monoclonal antibodies characterized by flow cytometry, one- and two-dimensional electrophoresis, western blot and inhibition of cytotoxic T lymphocyte function.

To identify and characterize the bovine major histocompatibility complex (MHC) class I molecules, a panel of 11 monoclonal antibodies (mAbs) were analyzed. The mAbs reacted with bovine MHC class I antigens, as assessed by flow cytometry and immunoprecipitation followed by one- and two-dimensional gel electrophoresis. Analysis by flow cytometry revealed that class I molecules were expressed less on a class I mutant B-lymphoblastoid cell line than on the parent cell line. The relative molecular weights of the proteins identified by these mAbs were similar to those reported previously for cattle and humans. Nonequilibrium pH gradient two-dimensional gel electrophoresis showed that RH16C recognized four different class I gene products, indicating this mAb reacts with a conserved epitope present on different class I molecules. These mAbs effectively blocked cytotoxic T lymphocyte killing of allogeneic lymphoblasts, suggesting the functional importance of beta-2m in this process. These mAbs should be useful reagents for studying bovine MHC class I molecules.

Animals↗
PubMed1992

Sodium dodecyl sulfate- and salt-extracted antigens from various Brucella species induce proliferation of bovine lymphocytes.

One- and two-dimensional gel electrophoresis and cellular immunoblotting were used to compare the protein profiles and immunogenic capabilities of salt- and sodium dodecyl sulfate-extracted components isolated from different Brucella species. Cellular immunoblotting demonstrated that freshly isolated bovine peripheral blood mononuclear cells proliferated to similar molecular mass components from the various Brucella extractions. One- and two-dimensional gel electrophoresis showed similarities in protein profiles among the different Brucella species that correlated with lymphocyte reactivity. The results presented in this study provide preliminary evidence that common proteins that induce lymphocyte proliferation are present among different Brucella species, suggesting that a genuswide subunit vaccine may be feasible.

Animals↗
PubMed1992

Antigens of Brucella abortus S19 immunodominant for bovine lymphocytes as identified by one- and two-dimensional cellular immunoblotting.

Cellular immune responses are influential for protection against intracellular bacteria such as brucellae. Therefore, identification of Brucella abortus antigens that activate primed bovine lymphocytes is fundamental for discerning the breadth of cellular response in bovine brucellosis. Potentially antigenic components of B. abortus S19 were isolated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by nitrocellulose blotting. Specific one-dimensional blot segments induced proliferation of peripheral blood lymphocytes from all 25 of the vaccinated cattle tested and were defined as immunodominant. Individual proteins that stimulated lymphocyte proliferation were further characterized by two-dimensional cellular immunoblotting by two different approaches. Individual one-dimensional stimulatory blot segments were eluted, concentrated, and then subjected to two-dimensional cellular immunoblotting. Alternatively, entire two-dimensional gels containing all of the B. abortus components were blotted and nitrocellulose sections containing individual proteins were assayed for lymphocyte activation. Thirty-eight Brucella proteins that induced lymphocyte proliferation were resolved by both procedures. Phenotypic analysis of the proliferating cell population demonstrated the presence of CD4+, CD8+, and immunoglobulin M+ lymphocytes. Two immunogenic proteins, 12 and 31 kDa, identified by two-dimensional cellular immunoblotting, were subjected to partial N-terminal amino acid analysis. The 12-kDa protein was within the area of greatest lymphocyte proliferation, while the 31-kDa protein was chosen for comparison with a 31-kDa protein previously reported by others. A search of the National Biomedical Research Foundation protein data bank showed that the sequences were not homologous with other known proteins. Identification of Brucella proteins immunogenic for bovine lymphocytes provides an important step in distinguishing the various proteins involved in pathogenicity and/or disease resistance.

Amino Acid Sequence↗
PubMed1992

Constitutively expressing cell lines that secrete a truncated bovine herpes virus-1 glycoprotein (gpI) stimulate T-lymphocyte responsiveness.

The desire to obtain authentically glycosylated viral protein products in sufficient quantity for immunological study has led to the use of eucaryotic expression vectors for protein production. An additional advantage is that these protein products can be studied individually in the absence of their native viral environment. We have cloned a complementary DNA (cDNA) encoding bovine herpes virus-1 (BHV-1) glycoprotein 1 (gpI) into the eucaryotic expression vector, pZipNeo SVX1. Since this protein is normally embedded within the membrane of BHV-1 infected cells, we removed sequences encoding the transmembrane domain of the native protein. After transfection of the plasmid construct into the canine osteosarcoma cell line, D17, or Madin-Darby bovine kidney (MDBK) cells, a truncated BHV-1 (gpI) was secreted into the culture medium as demonstrated by radioimmunoprecipitation and SDS-PAGE. Both a CD4+ T-lymphocyte line specific for BHV-1 and freshly isolated T lymphocytes could recognize and respond to the secreted recombinant gpI. Further, recombinant gpI could elicit both antibody and cellular responses in cattle when used as an immunogen. Having established constitutively glycoprotein producing cell lines, future studies in vaccine evaluation of gpI will be facilitated.

Animals↗
PubMed1991

The bovine p150/95 molecule (CD11c/CD18) functions in primary cell-cell interaction.

The monoclonal antibody (MAb), C5B6, recognizes the CD11c/CD18 molecule on the surface of bovine peripheral blood monocytes. C5B6 was reactive with 69-83% monocytes, all granulocytes, and less than 5% of lymphocytes from cattle. Of the lymphocyte series, the antibody had specificity for large lymphocytes and two lines expressing T cell markers, but was not reactive with small lymphocytes, thymocytes, a tumor cell line of B-cell lineage, an interleukin 2 (IL2)-dependent T cell line, fibroblasts, or human, sheep, goat or pig peripheral blood mononuclear cells. No dual fluorescence was seen using C5B6 and antibodies to bovine IgM, CD2, CD4 or CD8. Immunoprecipitation of 125I labeled peripheral blood mononuclear cells with C5B6 antibody defined two bands: 150,000 and 95,000 Da. Antibody to the beta chain (CD18) of the leukocyte adhesion receptor family precipitates the 95 kDa beta subunit and the three associated alpha subunits (180, 165 and 150). The bands obtained using MAb C5B6 correlated with the p150/95 bands observed using an antibody that precipitated the alpha and beta chains of the leukocyte adhesion receptor family. Functionally, the primary but not the secondary proliferative response to alloantigens was inhibited by C5B6 MAb. No effect was seen using C5B6 MAb in cytotoxicity assays or in the secondary proliferative response to Brucella abortus or bovine herpes virus type 1.

Animals↗
PubMed1991

Effect of monoclonal antibodies on in vitro function of T-cell subsets.

We have examined the functional heterogeneity of 43 monoclonal antibodies which recognised CD1, CD2, CD4, CD5, CD6 or CD8. Blocking of the proliferative response to alloantigen, virus, and phytoheamagglutinin (PHA) was studied. The majority of anti-CD2 and CD4 antibodies could inhibit an alloantigen response. Some of the anti-CD5 and CD8 antibodies could also block this response. Only a few antibodies to CD2 and CD4 could prevent proliferation of lymphocytes to PHA or virus.

Animals↗
PubMed1991

Biochemical analysis of bovine T cell antigens with workshop monoclonal antibodies.

The relative molecular weight (Mr) of the CD1, CD2, CD4, CD5, CD6, CD8 and additional molecules identified by monoclonal antibodies (mAb) submitted to the workshop was determined using 125I surface-labelled leukocytes. Of 37 mAb precipitating lymphocyte surface molecules, 33 had Mr correlating with their clusters of differentiation. Further, we were able to determine Mr for three "undefined T lymphocyte" antibodies. Of the 20 mAb that did not precipitate a molecule from the surface of leukocytes, one was non-reactive as determined by flow microfluorimetry. The inability to precipitate a molecule from the remaining cells was probably due to lower affinities of those antibodies and conformational changes in the antigen(s) following lysis of the leukocytes.

Animals↗
PubMed1991

Analysis of workshop antibodies to null cells on a non-T/non-B lymphocyte population.

Non-T and non-B lymphocytes (null cells) were obtained by Sephadex G-10 depletion followed by treatment with mAbs to CD2 and MHC class II and complement. The enriched cells were principally CD5dim and contained greatly increased numbers of null lymphocytes. This methodology will be useful for null lymphocyte enrichment in examining cell surface molecules and functional attributes of null lymphocytes.

Animals↗
PubMed1990

CD5dim peripheral blood lymphocytes proliferate to exogenous IL-2 in the absence of antigen and kill in an NK-like manner.

Bovine CD5 lymphocytes can be categorized into "bright" and "dim" populations on the basis of CD5-specific monoclonal antibody (mAb) binding. CD5dim lymphocytes enriched by negative selection using mAbs to CD2 and MHC class II were examined phenotypically and functionally. CD5dim-enriched lymphocytes were 50-74% T19+ and 5-36% CD8+. Fewer than 13% expressed CD4, IgM, or MHC class II on their surface, and 6-20% expressed low levels of CD2. These cells did not proliferate to alloantigen or uv-inactivated BHV-1, but did proliferate to Con A and IL-2 in the absence of antigenic or mitogenic stimulation. Following IL-2 stimulation, the T19+ CD5dim lymphocytes proliferated and CD11c expression was induced. Freshly isolated CD5dim lymphocytes failed to kill in a bovine NK-like assay; however, following culture CD5dim-enriched lymphocytes exhibited non-MHC restricted cytotoxicity. We suggest that the CD5dim-enriched lymphocyte population contains the precursors of bovine LAK killing; however, a more extensive phenotype of these precursors in the bovine is yet to be determined.

Animals↗