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G A Splitter

Publications and source records attributed to G A Splitter.

At least 37 records · Page 2Linked to original sources
PubMed1997

Temperature-dependent non-specific adsorption of recombinant bovine leukemia virus receptor BLVRcp1 in immunoassay.

Recombinant bovine leukemia virus receptor, BLVRcp1, possessed the unusual property of binding plastic plates after blocking nonspecific binding sites. Adhesiveness of BLVRcp1 to blocked plates hindered development of an antigen capture and receptor binding assay with this protein. Unexpectedly, non-specific adsorption of BLVRcp1 was dramatically influenced by temperature. Optimizing incubation temperature and antigen capture at 4 degrees C instead of 37 degrees C and the use of milk as blocking solution removed nonspecific binding of BLVRcp1 allowing development of a functional immunoassay. Thus, the temperature used for antigen capture can be a critical factor that influences performance of the immunoassay.

3T3 Cells↗
PubMed1997

Bovine herpesvirus-1 infects activated CD4+ lymphocytes.

Acute virus infections can induce immune deficiencies, as shown by immunosuppression to a variety of antigens and mitogens. Previously we observed that live bovine herpesvirus-1 (BHV-1) induced considerable lymphocyte death in culture, suggesting that the virus infected one or more cell populations. Our goal was to identify the cells infected by BHV-1 and the mechanism resulting in cell death. ConA activated cells were cultured with BHV-1 and stained with monoclonal antibodies specific for virus envelope glycoproteins (gB, gC and gD) and lymphocyte surface proteins (CD2, CD4 and CD8) and a molecule associated with gamma/delta cells. Two-colour immunofluorescence revealed that virus glycoproteins were preferentially expressed on T lymphocytes of the CD4+ phenotype. Live virus was required for virus glycoprotein expression, and by 48 h considerable loss of CD4 expression was observed. To confirm virus replication, RNA was isolated from cells, reverse transcribed and amplified using primers to a 342 bp region of immediate-early and early genes (IER2.9/ER2.6) or a 392 bp region of an early gene (gD). Immediate-early/early gene products were detected in CD4+T lymphocytes but not in infectious virions. Lymphocyte apoptosis was observed by 7 h post-infection with increasing levels of cell death at 24-48 h after infection. These findings suggest that the loss of proliferating CD4+ T cells during infection or vaccination with modified live vaccines provides the opportunity for secondary infections that commonly occur following BHV-1 infection.

Animals↗
PubMed1997

Polyclonal bovine sera but not virus-neutralizing monoclonal antibodies block bovine leukemia virus (BLV) gp51 binding to recombinant BLV receptor BLVRcp1.

Bovine leukemia virus (BLV), a transactivating lymphotropic retrovirus, is the etiologic agent of enzootic lymphosarcoma or leukemia in cattle. Sera from BLV-infected animals possess high BLV-neutralizing antibody titres. The availability of the recombinant BLV receptor candidate, BLVRcp1, allowed us to determine a mechanism of virus neutralization by polyclonal sera and monoclonal antibodies (MAbs). Bovine sera from animals naturally infected with BLV blocked gp51 binding to recombinant BLVRcp1. In contrast, virus-neutralizing MAbs specific for gp51 F, G, and H epitopes did not prevent gp51-receptor attachment. Furthermore, gp51 neutralization epitopes F, G, and H were accessible to antibodies following gp51 attachment to BLVRcp1. This finding implies that virus neutralization by MAbs to defined BLV gp51 epitopes can occur subsequent to virus engagement of the receptor while polyclonal sera can specifically block virus attachment to the receptor. In conclusion, these data suggest that cell infection by BLV is a multistep process requiring receptor binding (inhibited by polyclonal sera) followed by a second, postbinding event(s) at the cell membrane (inhibited by anti-gp51 MAbs).

3T3 Cells↗
PubMed1996

Optimization of lymphocyte proliferation assay for cells with high spontaneous proliferation in vitro: CD4+ T cell proliferation in bovine leukemia virus infected animals with persistent lymphocytosis.

Evaluation of antigen specific lymphocyte proliferation is extremely difficult with cells displaying high spontaneous proliferation in vitro, such as bovine leukemia virus (BLV) and T cell leukemia virus type I (HTLV-I) infected cells. We introduced modifications to a standard proliferation protocol, and for the first time report antigen specific proliferation in BLV infected animals with persistent lymphocytosis (a benign proliferation of B lymphocytes). Serum (autologous, heterologous or fetal) but not medium was a major factor contributing to spontaneous proliferation. Spontaneous proliferation was strongly serum concentration dependent and reducing serum to 0.5-1% resulted in decreased background proliferation and development of a functional assay. Addition of indomethacin (3-6 micrograms/ml) further increased antigen specific responses not only from persistently lymphocytotic but also from lymphosarcoma animals. Thus, this modified proliferation protocol can be used in infections where background proliferation hinders evaluation of antigen-specific CD4+ T cells.

Animals↗
PubMed1996

Recombinant Brucella abortus proteins that induce proliferation and gamma-interferon secretion by CD4+ T cells from Brucella-vaccinated mice and delayed-type hypersensitivity in sensitized guinea pigs.

Optimal protective immunity to Brucella abortus infection is dependent on a coordinate interaction between different T-cell subsets which leads to an antigen-specific T-lymphocyte-mediated activation of macrophages, the main cellular reservoir for the bacterium. As an initial step in the identification of bacterial proteins that mediate cellular immunity, we have subcloned the B. Abortus ssb, uvrA, GroES, and GroEL genes into the prokaryotic expression vector pMAL-c2 using PCR. Escherichia coli DH5 alpha was transformed with the pMAL-ssb, pMAL-uvrA, pMAL-GroES, and pMAL-GroEL constructs separately, and gene expression was induced by isopropyl-beta-D-thiogalactopyranoside. The resulting fusion proteins were purified by affinity chromatography and confirmed by Western blot analysis using an anti-maltose-binding protein antibody. Furthermore, we have examined the pattern of T helper (Th) cell response from vaccinated BALB/c mice after in vitro stimulation with the recombinant (r) fusion proteins. In addition to T-cell proliferative responses, CD4+ T cells were tested for interleukin-2 (IL-2), IL-4, and gamma interferon (IFN-gamma) secretion. Primed CD4+ T cells proliferated to the rUvrA, rGroES, and rGroEL, but not to rSsb. The cytokine profile of the proliferating cells was characteristic of a Th1 type, as we detected IL-2 and IFN-gamma but not IL-4 in the T-cell culture supernatants. The recombinant B. abortus proteins were also screened in vivo to their ability to elicit DTH reaction in Brucella-sensitized guinea pigs. Moreover, the results of this study suggest that B. abortus rUvrA, rGroES, and rGroEL might be important sources of potentially protective molecules.

Animals↗
PubMed1996

Loss of MHC I transcription trans-activator in the bovine B-LCL, BL3.1.

BL3.1, a variant derived from the BLV infected bovine B cell line, BL3, is distinguished by a loss of expression of MHC class I. All surface MHC I products were down-regulated in BL3.1 compared with BL3 correlating with a diminution in MHC I heavy chain transcription. Class I genes, including regulatory elements, showed no aberrations. The variant, BL3.1, did not differ from the parent cell line in expression of Bovine Leukemia Virus (BLV) or oncogene, c-myc. Transient transfection experiments determined the defect was trans rather than cis, and was due to loss of a trans-activator rather than gain of a trans-suppressor as evidenced by transient heterokaryon fusions. Southwestern blot analysis indicated that two DNA binding proteins associated with the MHC class I promoter were missing in BL3.1 cells. The specific response elements for these proteins in BL3 did not appear to be within the enhancerA region, the major enhancer region of the MHC I promoter.

Animals↗
PubMed1996

Immunization of mice with recombinant L7/L12 ribosomal protein confers protection against Brucella abortus infection.

BALB/c mice were immunized with the recombinant Brucella abortus L7/L12 ribosomal protein fused to maltose binding protein (MBP). Vaccinated animals mounted a specific immune response to the recombinant fusion protein as demonstrated by immunoblot analyses. Additionally, B. abortus L7/L12 ribosomal protein conferred a significant degree of protection when compared to mice vaccinated with adjuvant alone, adjuvant plus MBP or B. abortus. These results indicate that a recombinant B. abortus protein, previously identified as T-cell-reactive, engendered protective immunity to mice against brucellosis.

Animals↗
PubMed1996

Progression to persistent lymphocytosis and tumor development in bovine leukemia virus (BLV)-infected cattle correlates with impaired proliferation of CD4+ T cells in response to gag- and env-encoded BLV proteins.

The mechanism of leukemogenesis and persistent lymphocytosis (PL; benign expansion of B lymphocytes) in cattle infected with bovine leukemia virus (BLV; a retrovirus closely related to human T-cell leukemia virus type 1) is unknown; however, the immune system likely plays an important role in controlling the outcome of infection. In this study, we compared T-cell competence in serologically positive alymphocytotic (AL) animals with T-cell functions in animals with progressive stages of infection, PL and tumor bearing (TB). Dramatic differences were observed in lymphocyte proliferation to recombinant proteins encoded by BLV gag (p12, p15, and p24) and env (gp30, and gp51) genes in different disease stages. Lymphocytes from AL cattle recognized an average of three of five recombinant proteins per animal. Expansion of antigen pulsed lymphocytes in interleukin-2 increased protein recognition to almost five per animal. In contrast, lymphocytes from PL and TB animals failed to recognize any BLV recombinant proteins. Short-term T-cell cultures from the PL group expanded in interleukin-2, as well as the PL and TB cells cultured in indomethacin (3 to 6 microg/ml), increased the average of recognized proteins per animal to one. Cells proliferating to BLV antigens were CD4+ T lymphocytes, as shown by cell depletion studies. The positive effect of indomethacin suggests involvement of prostaglandin E2 as a negative regulatory factor in the later stages of disease. Thus, for the first time, advancing stages of BLV infection were correlated with decreased T-cell competence, providing deeper insight into pathogenesis of retroviral infections.

Animals↗
PubMed1996

Increased interleukin-10 mRNA expression in tumor-bearing or persistently lymphocytotic animals infected with bovine leukemia virus.

Interleukin-10 (IL-10), produced by Th2 helper T cells, B cells, and macrophages, can inhibit cytokine production by Th1 cells and enhance B-cell proliferation and differentiation. Here, we show that peripheral blood mononuclear cells (PBMCs) from bovine leukemia virus-infected animals with late-stage disease express considerably more IL-10 mRNA than animals that are not infected or that are in the early stages of disease. In contrast, the quantities of type 1 cytokines, IL-2 and gamma interferon, decrease with disease progression. In addition, we observed that IL-10 is expressed principally by monocytes/macrophages, not B lymphocytes, in persistently lymphocytotic animals. This observation supports a role for monocytes/macrophages in progression of bovine leukemia virus infection and, of importance, indicates that proliferating B cells are not the source of IL-10 expression. These findings suggest that IL-10 produced by monocytes/macrophages may influence the progression of bovine leukosis in animals that develop persistent lymphocytosis of B cells or B-cell lymphosarcoma.

Animals↗
PubMed1995

The cattle major histocompatibility complex (MHC) class-I possesses HLA-like promoters.

To study the genetic regulation of the cattle major histocompatibility complex (MHC) class-I, a cattle MHC class-I promoter DNA fragment was isolated and characterized for the first time. Semi-degenerate PCR was performed on cattle genomic DNA and the resulting product was isolated, subcloned and sequenced. Sequence comparison of the HLA-A, -B and -C promoters to the cloned product, designated BL3-6prmtr, revealed the cattle MHC class-I promoter to have close homology to human MHC class-I promoters. To address the ability of the cattle MHC class-I promoter to initiate transcription, BL3-6prmtr was subcloned into a luciferase reporter vector and transiently transfected into cattle and human B-lymphoblastoid cell lines. A strong transcription initiation ability of BL3-6prmtr was observed, including the ability of the enhancer A and interferon response sequence (IRS) to upregulate transcription initiation.

Animals↗
PubMed1995

CD8+ type 1 CD44hi CD45 RBlo T lymphocytes control intracellular Brucella abortus infection as demonstrated in major histocompatibility complex class I- and class II-deficient mice.

Genetically engineered mice with a targeted disruption in the beta 2-microglobulin (beta 2-m) gene or the H2-I-A beta chain (A beta) which lack functional CD8+ or CD4+ T cells, respectively, were used to assess the role of T cell subsets in Brucella abortus infection. Murine brucellosis was markedly exacerbated in beta 2-m-deficient mice (beta 2-m-/-) compared to A beta mutant (A beta-/-) or C57BL/6 mice, strongly indicating that optimal resistance to B. abortus requires CD8+ T cells. Splenocytes from Brucella-primed beta 2-m-/-, A beta-/- and C57BL/6 mice exhibited a type 1 cytokine profile marked by elevated IFN-gamma mRNA expression and protein production, and basal levels of IL-2 and IL-4 transcripts. B. abortus did not induce secretion of TGF-beta 1, but substantial IL-10 activity was detected in spleen cell supernatants from all mouse strains studied. CD8+ T cells from A beta-/- and C57BL/6 mice displayed a CD44hi CD45RBlo phenotype and a type 1 cytokine transcription profile featuring high levels of IFN-gamma mRNA. Additionally, we have shown the ability of C57BL/6 CD8+ CTL to kill Brucella-infected macrophages. This study illustrates the predominant role of MHC class I-restricted T cells in controlling B. abortus infection.

Animals↗
PubMed1995

CD8+ lymphocytes that kill allogeneic and xenogeneic major histocompatibility complex class I targets.

CD8+ CTLs generated in a two-way MLR should lyse target cells only if these targets share a class I MHC allele with the original stimulators. Using cattle PBMCs in a two-way MLR, we generated CD8+ CTLs that kill allogeneic and xenogeneic cell lines. We have named these cells MLK cells. PBMCs isolated from two unrelated animals were cultured together. After 14 days microfluorimetry analysis was performed on the MLK cells with results showing > 90% CD8+ cells. RFLP analysis revealed these cells to be predominately of one animal. MLK cells were then used as effector cells in cytotoxicity assays with syngeneic, allogeneic, and xenogeneic target cells. MLK cells were able to kill all targets. Incubating MLK cells with mAbs to CD8 markedly reduced killing, suggesting a TCR-mediated cytolytic pathway. Effective cytolysis of these targets by MLK cells was dependent on class I expression. MHC class I expression-impaired mutants of allogeneic and xenogeneic targets were not susceptible to cytolysis. Comparisons to LAK cells revealed similarities in phenotype and function to the NK1.1-, CD8+ subset.

Amino Acid Sequence↗
PubMed1995

BHV-1 glycoprotein 1 and recombinant interleukin 1 beta efficiently elicit mucosal IgA response.

The mucosal immune response to most soluble antigens administered directly to the mucosal system is low and requires a large amount of antigen and frequent vaccinations. In this study we tested whether immunizing cattle at a site which shares lymphatic drainage with the nasal mucosa could prime local mucosal immunity. We further tested whether recombinant bovine IL-1 beta (rBoIL-1 beta) could potentiate the induction of mucosal immunity. Animals were immunized subcutaneously at the base of the ear (s.e.) with recombinant bovine herpesvirus-1 (BHV-1) envelope glycoprotein I (gI) (35 micrograms animal-1) emulsified in incomplete Freund's adjuvant with or without rBoIL-1 beta (500 ng kg-1) followed by a second immunization 42 days later. Animals were challenged with virulent BHV-1 intranasally 42 days after the second immunization. Mucosal IgA from the nares was induced after only one immunization, and enhanced by boosting. rBoIL-1 beta treated animals had higher levels of BHV-1 specific nasal IgA (p < 0.01) and serum neutralizing antibody (p < 0.05). rBoIL-1 beta-treated animals also had increased numbers of surface IgA+ (p < 0.05) and IgG1+ (p < 0.001) B cells after in vitro antigen (gI) stimulation of peripheral blood lymphocytes suggesting that there was a greater expension of IgA+ and IgG1+ B cells in rBoIL-1 beta treated animals. When challenged with BHV-1, 3 of 4 animals in the gI+rBoIL-1 beta group were fully protected from viral replication in the nares, while only 1 of 4 animals receiving gI alone was protected.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Interferon-gamma inhibits transgene expression driven by SV40 or CMV promoters but augments expression driven by the mammalian MHC I promoter.

The use of mammalian gene expression vectors has become increasingly important for transgenics and gene therapy as well as basic research. Essential for the success of these vectors in medical research applications is the proper choice of promoter linked to the gene of interest. Many mammalian expression vectors use promoter elements from pathogenic viruses, including simian virus 40 (SV40) and cytomegalovirus (CMV). Lymphokines produced by the immune response to proteins expressed by these vectors could inhibit further transcription initiation by viral promoters. Our objective was to determine the effect of interferon-gamma (IFN-gamma) on transgene expression driven by a viral SV40 or CMV promoter/enhancer and the mammalian promoter/enhancer for the major histocompatibility complex class I (MHC I) gene. We transfected the luciferase gene driven by these three promoters into 14 cell lines of many tissues and several species. Luciferase assays of transfected cells untreated or treated with IFN-gamma indicated that, although the viral promoters could drive luciferase production in all cell lines tested to greater or lesser levels than the MHC I promoter, treatment with IFN-gamma caused inhibition of transgene expression in most of the cell lines and amplification of the MHC I promoter-driven transgene expression in all cell lines. These data indicate that the SV40 and CMV promoter/enhancers may not be a suitable choice for gene delivery especially for immune response applications or in patients where IFN levels may be elevated. The MHC I promoter/enhancer, on the other hand, may be an ideal transgene promoter for applications involving the immune system.

Animals↗
PubMed1995

Picornavirus-specific CD4+ T lymphocytes possessing cytolytic activity confer protection in the absence of prophylactic antibodies.

Picornaviruses are a family of positive-strand RNA viruses that are responsible for a variety of devastating human and animal diseases. An attenuated strain of mengovirus (vMC24) is serologically indistinguishable from the lethal murine wild-type mengovirus and encephalomyocarditis virus (EMCV). Immunogen-specific stimulation of vMC24-immune splenocytes in vitro demonstrates preferential activation of CD4+ lymphocytes. vMC24-immune splenocytes adoptively transferred to naive recipients conferred protection against lethal EMCV challenge. Immune splenocytes, expanded in vitro, were > 92% CD4+ T lymphocytes. Interestingly, adoptive transfer of these expanded cells engendered protection against lethal challenge. In vivo depletion of CD4+ T lymphocytes prior to lethal challenge abrogated survival of transfer recipients, confirming that CD4+ T lymphocytes were essential for protection. Subsequent rechallenge of vMC24-immune splenocyte recipients with a greater EMCV dose elicited serum neutralizing antibody titers paralleling the high titers observed in vMC24-immunized mice. Unexpectedly, an augmented humoral response was absent in vMC24-specific CD4+ T-cell recipients after the secondary challenge. Moreover, comparably low serum neutralizing antibody titers failed to protect passive transfer recipients when correspondingly challenged. vMC24-immune splenocytes expanded in vitro (> 94% CD4+) lysed vMC24-infected A20.J target cells. The ability to transfer protection with primed CD4+ T cells, in the absence of primed B lymphocytes or immune sera, is novel for picornaviral infections. Consequently, mechanisms such as CD4+ cytolytic T-lymphocyte activity are implicated in mediating protection.

Animals↗
PubMed1994

Induction of MHC-unrestricted cytolytic CD4+ T cells against virally infected target cells by cross-linking CD4 molecules.

Cross-linking CD4 molecules with the use of specific mAbs induced Ag-nonspecific, MHC-unrestricted killing by human or bovine CD4+ T cells and human allospecific T cell clones against virally infected (bovine herpesvirus-1 and herpes simplex virus-1) target cells. Heat-aggregated goat IgG anti-mouse IgG elevated CD4 cross-linking efficiency, resulting in augmented killing activity by effector cells. However, Ab-activated effector cells failed to kill tumor target cell lines, e.g., K562, Daudi (human), and BL3 (bovine). The result supports the hypotheses that the CD4 molecule plays an important role in activating CD4 cytotoxic cells and that viral glycoproteins participate in conjugating target cells with activated effector cells in two species. The herpesviruses eliciting killing represent potential natural infections for each species. The relationship between a cytolytic response and cell proliferation was independent because cross-linking of CD4 molecules induced cytotoxicity but not T cell proliferation, in contrast to cross-linking CD3 molecules. Our findings of non-MHC-restricted cytotoxic lymphocytes induced by cross-linking CD4 molecules identify an activation mechanism of antiviral cytotoxicity.

Animals↗
PubMed1994

Functional and phenotypic characterization of WC1+ gamma/delta T cells isolated from Babesia bovis-stimulated T cell lines.

Functional studies with WC1+ gamma/delta T cell lines were performed to clarify the role of this subpopulation of gamma/delta T cells in the in vitro immune response to Babesia bovis. As CD4+ T cells decreased and gamma/delta T cells increased in B. bovis-stimulated T cell lines, antigen-specific proliferation declined to background levels. One irradiated gamma/delta T cell line inhibited proliferation of autologous Th1 cells, although unirradiated gamma/delta T cells either synergized with or had no effect on Th cell proliferation. gamma/delta T cells were not cytolytic for bovine alpha/beta T cells, but expressed natural killer (NK)-like cytotoxicity when assayed on xenogeneic NK-sensitive target cells. The gamma/delta T cells were IL-2 dependent and expressed IFN-gamma and TNF-alpha, but not TNF-beta, IL-2, or IL-4 mRNA. Together, these results raise the possibility that WC1+ gamma/delta T cells respond in vitro to autoantigens present on CD4+ T cells or to cytokines secreted by activated CD4+ T cells, resulting in modulation of the CD4+ T cell response and outgrowth of the gamma/delta T cells in parasite-stimulated lines.

Animals↗