Biomedical subjects

W C Brown

Publications and source records attributed to W C Brown.

At least 55 records · Page 3Linked to original sources

PubMed1999

Bovine CD4(+) T-lymphocyte clones specific for rhoptry-associated protein 1 of Babesia bigemina stimulate enhanced immunoglobulin G1 (IgG1) and IgG2 synthesis.

Optimal protective immunity against babesial infection is postulated to require both complement-fixing and opsonizing antibodies in addition to gamma interferon (IFN-gamma)-mediated macrophage activation. The rhoptry-associated protein 1 (RAP-1) of Babesia bigemina induces partial protective immunity and is a candidate vaccine antigen. Previous studies demonstrated that cattle immunized with native protein that were subsequently protected against challenge had a strong IFN-gamma and weaker interleukin-4 (IL-4) response in immune lymph node lymphocytes that reflected the cytokine profile of the majority of CD4(+) T-cell clones obtained from peripheral blood. RAP-1-specific T helper (Th) cell clones that coexpress IFN-gamma and IL-4 are typical of numerous parasite-specific clones examined. However, the function of such cells as helper cells to enhance immunoglobulin secretion by bovine B cells has not been reported. In cattle, both immunoglobulin G1 (IgG1) and IgG2 can fix complement, but IgG2 is the superior opsonizing subclass. Therefore, studies were undertaken to ascertain the functional relevance of RAP-1-specific, CD4(+) Th0 cells as helper cells to enhance IgG1 and/or IgG2 production by autologous B lymphocytes. For comparison, Th0 clones specific for the metazoan parasite Fasciola hepatica that expressed relatively more IL-4 than the B. bigemina-specific Th cells were similarly assayed. B. bigemina RAP-1-specific clones could enhance production of both IgG1 and IgG2 by autologous B cells, whereas Th cell clones specific for F. hepatica enhanced predominantly IgG1 production. The capacity to enhance IgG2 production was associated with production of IFN-gamma by Th cells cocultured with B cells, antigen, and IL-2. The in vitro helper T-cell activity of these T-cell clones was representative of the in vivo serologic responses, which were composed of a mixed IgG1-IgG2 response in B. bigemina RAP-1 immune cattle and a biased IgG1 response in F. hepatica-immune cattle.

Animals↗

PubMed1999

Emergence of Anaplasma marginale antigenic variants during persistent rickettsemia.

Anaplasma marginale is an ehrlichial pathogen of cattle, in the order Rickettsiales, that establishes persistent cyclic rickettsemia in the infected host. Within each rickettsemic cycle, A. marginale expressing antigenically variant major surface protein 2 (MSP2) emerge. By cloning 17 full-length msp2 transcripts expressed during cyclic rickettsemia, we determined that emergent variants have a single, central hypervariable region encoding variant B-cell epitopes. The N- and C-terminal regions are highly conserved among the expressed A. marginale variants, and similar sequences define the MSP2 homologues in the agent of human granulocytic ehrlichiosis (HGE). This is in contrast to the MSP2 homologues in ehrlichial genogroup I pathogens, Ehrlichia chaffeensis, Ehrlichia canis, and Cowdria ruminantium, that have multiple hypervariable regions. By defining the variable and conserved regions, we were able to show that the single hypervariable region of A. marginale MSP2 encodes epitopes that are immunogenic and induce variant-specific antibody responses during persistent infection. These findings demonstrate that the MSP2 structural variants that emerge during each cycle of persistent rickettsemia are true antigenic variants, consistent with MSP2 antigenic variation as a mechanism of A. marginale persistence.

Amino Acid Sequence↗

PubMed1999

Biased immunoglobulin G1 isotype responses induced in cattle with DNA expressing msp1a of Anaplasma marginale.

Immunization with the native major surface protein 1 (MSP1) (a heterodimer containing disulfide and noncovalently bonded polypeptides designated MSP1a and MSP1b) of the erythrocytic stage of Anaplasma marginale conferred protection against homologous challenge (G. H. Palmer, A. F. Barbet, W. C. Davis, and T. C. McGuire, Science 231:1299-1302, 1986). The MSP1a polypeptide possesses a conserved neutralization-sensitive epitope. In the present study, the immune response to DNA-mediated immunization using msp1a was studied. The plasmid pVCL/MSP1a, which encodes the complete msp1a gene of A. marginale under the control of human cytomegalovirus immediate-early enhancer/promoter and intron A, was constructed. The immune responses elicited by immunization with pVCL/MSP1a into cardiotoxin-induced regenerating muscle were evaluated in mice and cattle. Antibody reactive with native MSP1a was detected in pooled sera of immunized BALB/c mice 3 weeks following primary immunization. Two calves seronegative for A. marginale were immunized four times, at weeks 0, 3, 7, and 13, with pVCL/MSP1a. By 8 weeks, both calves responded to MSP1a with an antibody titer of 1:100, which peaked at 1:1,600 and 1:800 by 16 weeks after the initial immunization. Interestingly, immunoblotting with anti-immunoglobulin G1 (anti-IgG1) and anti-IgG2 specific monoclonal antibodies revealed a restricted IgG1 anti-MSP1a response in both animals. T-lymphocyte lines, established after the fourth immunization, proliferated specifically against A. marginale homogenate and purified MSP1 in a dose-dependent manner. These data provide a basis for an immunization strategy to direct bovine immune responses by using DNA vaccine vectors containing single or multiple genes encoding major surface proteins of A. marginale.

Anaplasma↗

PubMed1998

CD40 ligand-dependent signaling of bovine B lymphocyte development and differentiation.

The role of IFN-gamma in B cell differentiation in cattle has not been completely elucidated. We have previously investigated the role of IFN- in the control of antibody production by bovine B cells using anti-bovine IgM antibody in the solid-phase as a source of costimulation. Using this mimic of a T1-2 antigen, we demonstrated that IFN-gamma can enhance the production of IgG2 but not IgG1 from sIgM+ cells. The positive effects of IFN-gamma were enhanced by co-addition to cultures of rboIL-2. Under these activation conditions, the frequency of cells expressing mRNA for the IgG2 heavy chain also increased at least two-fold. In these studies, we investigated the role of IFN-gamma in antibody expression under T-dependent (TD) activation conditions using mouse fibroblasts transfected with boCD40L as a surrogate T cell. Under TD conditions, IFN-gamma had less dramatic effects on the production of IgG2 with IgM predominating in the cultures. Interestingly, the production of IgA was modestly enhanced with little effect on the production of IgG1 above baseline levels obtained with medium alone. In comparison to results with T1-2 conditions of activation, IL-2 did not increase total amounts of antibody above two-fold. Our results suggest that TH1 cells in cattle may be limited in their ability to provide B cell help to levels obtainable in a TH2 cytokine microenvironment due to the effects of IFN-gamma on bovine B cells co-activated via CD40.

Animals↗

PubMed1998

Bovine type 1 and type 2 responses.

The Th1/Th2 paradigm has provided a useful framework for understanding the observed bias in immune responses that are often dominated by either cell-mediated or humoral responses, and for devising therapeutic strategies to stimulate T cell- or antibody-mediated immunity. However this paradigm is an oversimplification of a much more complex immunoregulatory network. Studies with bovine Th cell clones and immunoregulatory cytokines support this viewpoint. This paper highlights the progress that has been made in defining type 1 and type 2 responses in cattle. Evidence is presented for the presence of different subtypes of antigen-specific Th cell clones of cattle which constitute a spectrum of cell phenotypes, and for cytokine-mediated regulation of Th cell responses that differs from that observed in mice. The majority of over 60 parasite antigen-specific Th cell clones coexpress IL-4 and IFN-gamma, and polarized cytokine profiles were rarely observed. Furthermore, IL-2 and IL-10 expression was not restricted to IFN-gamma or IL-4-producing cells, respectively. This lack of coordinate regulation of "Th1" and "Th2" cytokines strengthens the emerging viewpoint that Th1 and Th2 responses, per se, do not typify the immune response to most pathogens. In addition, we provide evidence that major regulatory cytokines, IL-4, IL-10, and IL-12, do not selectively exert their negative (IL-4 and IL-10) or positive (IL-12) effects on Th1-like cells.

Animals↗

PubMed1998

Cloning and expression of caprine interferon-gamma.

Caprine interferon-gamma (IFN-gamma) cDNA was cloned from mitogen stimulated peripheral blood mononuclear cell (PBMC) RNA utilizing the reverse transcription-polymerase chain reaction (RT-PCR). The cDNA open reading frame (ORF) is 498bp, encoding a putative 166 amino acid (aa) protein (19327Da). The predicted aa sequence homology of caprine IFN-gamma and the corresponding ovine, bovine and cervine cytokine is 98.8%, 95.2% and 92.8%, respectively. IFN-gamma cDNA was subcloned and expressed in two different plasmids under the control of either the human cytomegalovirus (CMV) immediate early promoter or the caprine arthritis-encephalitis virus long terminal repeat (CAEV LTR). Recombinant caprine IFN-gamma (rCaIFN-gamma) secreted by transfected COS-7 cells shared at least two antigenic epitopes with recombinant bovine IFN-gamma (rBoIFN-gamma) and exhibited biological activity in the vesicular stomatitis virus (VSV) cytopathic effect reduction assay. In-vivo expression of IFN-gamma cDNA promoted by the CAEV LTR was confirmed by the intramuscular (IM) injection of Balb/C mice with plasmid followed by Western blot analysis of mouse serum against purified rCaIFN-gamma produced in E. coli.

Amino Acid Sequence↗

PubMed1998

Kinetics of expression and subset distribution of the TNF superfamily members CD40 ligand and Fas ligand on T lymphocytes in cattle.

CD40 and Fas are members of the tumor necrosis factor receptor (TNFR) superfamily. CD40 and Fas play key roles in T cell-B cell interactions. Cross linkage of these molecules induces cell activation and cell death, respectively. The interaction of CD40 with its ligand (CD40L), which is expressed on activated T cells, plays a pivotal role in the generation of the T-dependent (TD) immune response, and FasL-bearing T cells, which have been shown to be predominantly of either the TH0 or TH1 type, have the potential to induce the apoptotic death of Fas expressing B cells. We investigated bovine CD40L mRNA expression in established T cell clones by RT-PCR and Southern blotting. T cells analyzed included CD4+ TH0 and TH1 cell subpopulations, CD8+, and gamma/delta T cells stimulated with either specific antigen or Con A. All CD4+ clones but not all CD8+ or gamma/delta T cell receptor (TCR)-bearing clones expressed mRNA for CD40L. To determine the activation requirements for CD40L expression in cattle, we examined the kinetics and induction requirements for CD40L transcription in peripheral blood T cells using a phorbol ester and/or ionomycin, immobilized mouse anti-bovine CD3, or Con A. Our results demonstrate that CD40L mRNA appears relatively early after activation (1 h) and peaks at 2-4 h poststimulation. A rise in intracellular calcium concentration mediated by ionomycin treatment alone was sufficient to induce CD40L mRNA expression at relatively high levels. Ionomycin treatment in combination with other agonists (anti-CD3, PMA) did not enhance CD40L mRNA expression above levels obtained with ionomycin alone. The bovine Fas ligand gene was partially cloned and mRNA expression determined by RT-PCR in a panel of T cell clones. Our results demonstrate that TH0 and TH1 bovine T cell clones expressed Fas ligand transcripts although only one gamma/delta T cell clone did. This expression was upregulated within 3 h after mitogen stimulation and reduced by 24 h.

Animals↗

PubMed1998

Phylogeny of benign Theileria species from cattle in Thailand, China and the U.S.A. based on the major piroplasm surface protein and small subunit ribosomal RNA genes.

The major piroplasm surface protein and small subunit ribosomal RNA genes of benign Theileria species isolated from cattle in China, Thailand and the U.S.A. were amplified by polymerase chain reaction, cloned and sequenced. The major piroplasm surface protein genes of these three isolates were more than 89% identical at amino-acid level. Several deletions in the gene from the Thai isolate led to considerable structural change through frame shifts of the major piroplasm surface protein. Phylogenetic analyses based on both of the major piroplasm surface protein and small subunit ribosomal RNA genes suggest that there may be a second cosmopolitan benign Theileria species infecting cattle in addition to the Theileria sergenti/buffeli/orientalis complex.

Amino Acid Sequence↗

PubMed1998

Effects of type I/type II interferons and transforming growth factor-beta on B-cell differentiation and proliferation. Definition of costimulation and cytokine requirements for immunoglobulin synthesis and expression.

In this report, we sought to determine the role of selected type I interferons [interferon-alpha (IFN-alpha) and interferon-tau (IFN-tau)], IFN-gamma and transforming growth factor-beta (TGF-beta) in the regulation of bovine antibody responses. B cells were stimulated via CD40 in the presence or absence of B-cell receptor (BCR) cross-linking. IFN-alpha enhanced IgM, IgG2 and IgA responses but did not enhance IgG1 responses. BCR signalling alone was more effective at inducing IgG2 responses with IFN-alpha than dual cross-linking with CD40. Recombinant ovine IFN-tau was less effective at inducing IgG2 responses when compared with IFN-alpha, though IgA responses were similar in magnitude following BCR cross-linking. At higher concentrations, IFN-tau enhanced IgA responses greater than twofold over the levels observed with IFN-alpha. Previous studies have shown that addition of IFN-gamma to BCR or pokeweed mitogen-activated bovine B cells stimulates IgG2 production. However, following CD40 stimulation alone, IFN-gamma was relatively ineffective at stimulating high-rate synthesis of any non-IgM isotype. Dual cross-linking via CD40 and the BCR resulted in decreased synthesis of IgM with a concomitant increase in IgA and similar levels of IgG2 production to those obtained via the BCR alone. We also assessed the effects of endogenous and exogenous TGF-beta on immunoglobulin synthesis by bovine B cells. Exogenous TGF-beta stimulates both IgG2 and IgA production following CD40 and BCR cross-linking in the presence of IL-2. Blocking endogenous TGF-beta did not inhibit the up-regulation of IgG2 or IgA by interferons.

Animals↗

PubMed1998

Trophoblast IFN-tau differentially induces lymphopenia and neutropenia in lambs.

Type I interferons (IFN), including IFN-alpha and IFN-beta, cause severe lymphopenia, resulting from altered lymphocyte recirculation and redistribution. IFN-tau, a product of trophectoderm of ruminant conceptuses and new member of the type I IFN family has not been examined for its effect on leukocyte recirculation. Additionally, differential effects of type I IFNs on the redistribution and recirculation of subsets of T cells have not been reported. The present study determined the effects of IFN-tau on the redistribution and recirculation of ovine leukocytes and T cell subsets. Total peripheral blood leukocytes, lymphocytes, and segmented neutrophils were reduced (p < 0.05) following treatment of lambs with IFN-tau. Furthermore, administration of IFN-tau caused an acute, differential reduction in peripheral blood CD4+ T cells (p < 0.05), CD5+ cells (p < 0.05), and gammadelta TCR+ (p < 0.01) T cells but had no effect on CD8+ T cells (p > 0.05). IFN-tau reduced the percentage of gammadelta T cells by 8-fold and that of CD4+ T cells and CD5+ cells by <2-fold in peripheral blood when compared with control lambs. The reduction in leukocytes, lymphocytes, and neutrophils was observed as early as 6-12 h after administration of IFN-tau, but levels returned to control values within 48 h. These results indicate that IFN-tau, like other members of the type I IFN family, can have immediate effects on leukocyte recirculation and redistribution. The present study is the first to demonstrate that IFN-tau differentially regulates T cell recirculation with the greatest effect on gammadelta TcR+ T cells.

Analysis of Variance↗

PubMed1998

Amphetamines in hair by enzyme-linked immunosorbent assay.

Human hair was collected from the occipital crown region of the head from several subjects; these hair samples were presumptively positive for amphetamines by a previously evaluated immunoassay. Hair was washed briefly with methanol to remove external contamination, then extracted with hot methanol for 2 h to recover the drugs. The extracts were evaporated to dryness, reconstituted in buffer, and analyzed using a new enzyme-linked immunosorbent assay (ELISA) technique adapted for the detection of amphetamines in hair. Gas chromatography-mass spectrometry was used as the reference technique. Cross-reactivity of several related compounds was evaluated by equating the inverse of the ligand concentration at 50% antibody binding to the affinity constant for each compound. The ratio of a compound's affinity constant to that for d-methamphetamine was used to derive percent crossreactivity. These experiments yielded values of 30.8% for d-amphetamine, 7.4% for I-methamphetamine, 4.3% for phentermine, 2.9% for I-amphetamine, and <1% for ephedrine, methylenedioxyamphetamine, and methylenedioxymethamphetamine. Cross-reactivity of unrelated compounds was found to be non-existent. The optimum cutoff concentration was determined by receiver operating characteristic curve analysis to be 300 pg/mg and the observed limit of detection was 60 pg/mg. Intra-assay precision at 300 pg/mg was 3.3% (coefficient of variation, CV), and the interassay CV was 10.5%. The sensitivity and specificity of the method were 83% and 92%, respectively.

Amphetamine-Related Disorders↗

PubMed1998

CD4(+) T-lymphocyte and immunoglobulin G2 responses in calves immunized with Anaplasma marginale outer membranes and protected against homologous challenge.

Protective immunity against the ehrlichial pathogen Anaplasma marginale has been hypothesized to require induction of immunoglobulin G2 (IgG2) antibody against outer membrane protein epitopes and coordinated activation of macrophages for phagocytosis and killing. In the present study, cell-mediated immune responses, including induction of IgG isotype switching, were characterized in calves immunized with purified outer membranes of the Florida strain of A. marginale. Importantly, these calves were subsequently shown to be protected upon experimental challenge with the Florida strain, and calves which developed the highest IgG2 titers were completely protected against infection. Peripheral blood mononuclear cells (PBMC) obtained after immunization proliferated strongly in response to both whole A. marginale homogenates and purified outer membranes, and this responsiveness persisted until the time of challenge. Responding cells were shown to be CD4(+) T cells, and CD4(+) T-cell lines cultured for 2 to 4 weeks also proliferated specifically in response to A. marginale and produced high titers of gamma interferon. The helper T-cell response included recognition of conserved epitopes, as PBMC proliferation was stimulated by the homologous Florida strain, four genetically distinct A. marginale strains, and Anaplasma ovis. The outer membrane proteins stimulating the PBMC responses in protected calves included major surface proteins (MSPs) MSP-1, MSP-2, and MSP-3, which were previously shown to induce partial protection against infection. These studies demonstrate, for the first time, potent helper T-cell responses in cattle protectively immunized with outer membranes against A. marginale challenge and identify three MSPs that are recognized by immune T cells. These experiments provide the basis for subsequent identification of the helper T-cell epitopes on MSP-1, MSP-2, and MSP-3 that are needed to evoke anamnestic antibody and effector T-cell responses elicited by protein or nucleic acid immunization.

Anaplasma↗

PubMed1998

The repertoire of Anaplasma marginale antigens recognized by CD4(+) T-lymphocyte clones from protectively immunized cattle is diverse and includes major surface protein 2 (MSP-2) and MSP-3.

Major surface proteins of Anaplasma marginale are vaccine candidates. We recently demonstrated that immunization of calves with outer membranes of the Florida strain of A. marginale resulted in protective immunity that correlated with a memory CD4(+) T-lymphocyte response specific for major surface protein 1 (MSP-1), MSP-2, and MSP-3 (W. C. Brown, V. Shkap, D. Zhu, T. C. McGuire, W. Tuo, T. F. McElwain, and G. H. Palmer, Infect. Immun. 66:5406-5413, 1998). As immunogens, these proteins have been shown to induce complete or partial protection against homologous challenge. To further define the T helper (Th) cell response to these and other A. marginale antigens and to determine conservation of Th cell epitopes among genetically distinct A. marginale strains, Th cell clones obtained prior to challenge from three immunized calves were characterized for antigen-specific responses. Nine distinct antigenic profiles were defined by 11 Th cell clones derived by stimulation with the Florida strain. Several clones responded to MSP-2, MSP-3, or both. All of these MSP-2- or MSP-3-specific clones and the majority of other clones that did not respond to MSPs recognized all bovine blood-passaged strains of A. marginale. These results demonstrate conservation of certain Th cell epitopes between MSP-2 and MSP-3 and show that Th cell epitopes in MSP-2, MSP-3, and undefined antigens are conserved among strains of A. marginale. Of seven clones that responded to the blood-passaged Virginia strain, two did not recognize antigen prepared from this strain cultured in tick cells, suggesting differences in the antigenic composition between these stages. Analysis of the cytokines expressed by the Th cells revealed that all clones expressed gamma interferon and tumor necrosis factor alpha, and most coexpressed interleukin-4. Our results provide a rationale for identifying Th cell epitopes conserved among different strains of A. marginale for inclusion in a nucleic acid or recombinant protein vaccine.

Anaplasma↗

PubMed1998

DNA and a CpG oligonucleotide derived from Babesia bovis are mitogenic for bovine B cells.

DNAs from bacteria and variety of nonvertebrate organisms, including nematodes, mollusks, yeasts, and insects, cause polyclonal activation of murine B lymphocytes. Similar studies have not been reported for bovine B cells, and to date no studies have reported mitogenic properties of protozoal DNA for any species. However, we and others have observed that protozoal parasite antigens can induce the proliferation of lymphocytes from nonexposed donors. Extending these studies, we now show that the mitogenic property of protozoal antigen preparations is in part attributable to parasite DNA and that Babesia bovis DNA is directly mitogenic for bovine B cells. DNase treatment of B. bovis extracts abrogated B. bovis-induced proliferation of peripheral blood mononuclear cells from nonexposed cattle. Like DNAs from other organisms that were mitogenic for murine B cells, B. bovis DNA is largely nonmethylated and induced a dose-dependent proliferation of bovine B cells, which was reduced upon methylation. Furthermore, B. bovis and E. coli DNAs enhanced immunoglobulin secretion by cultured B cells, inducing moderate increases in immunoglobulin G1 and stronger increases in immunoglobulin G2. Because certain nonmethylated CpG motifs present in bacterial DNA are known to stimulate proliferation of murine and human B cells, an 11-kb fragment of B. bovis DNA was analyzed for CG dinucleotide content and for the presence of known immunostimulatory sequences (ISS) centered on a CG motif. The frequency of CG dinucleotides was approximately one-half of the expected frequency, and several CpG hexameric sequences with known activity for murine B cells were identified. An oligodeoxynucleotide containing one of these ISS (AACGTT), which is present within the rhoptry-associated protein-1 (rap-1) open reading frame, was shown to stimulate B-cell proliferation. These ISS may be involved in host immune modulation during protozoal infection and may be useful as vaccine adjuvants.

Animals↗

PubMed1998

Helper T-cell epitopes encoded by the Babesia bigemina rap-1 gene family in the constant and variant domains are conserved among parasite strains.

Among important candidates for babesial vaccines are apical complex proteins, including rhoptry-associated protein 1 (RAP-1) from Babesia bovis and B. bigemina, which have been shown to induce partial immunity. Four variant B. bigemina rap-1 transcripts identified in a clone of the Mexico strain have highly conserved sequence in the central region but vary in sequence at the amino and carboxy termini (NT and CT) of the predicted proteins, resulting in different combinations of NT and CT domains in the individual gene products. Cattle were immunized with native protein consisting of the RAP-alpha1 variant, which contains NT-1 and CT-1 domains, and T-cell responses were characterized. We previously reported the identification of two T helper (Th) cell epitopes in B. bigemina RAP-1alpha1 protein (I. Hötzel, W. C. Brown, T. F. McElwain, S. D. Rodriguez, and G. H. Palmer, Mol. Biochem. Parasitol. 81:89-99, 1996). One epitope mapped to the constant domain of RAP-1 (amino acids [aa] 144 to 187), and one mapped to the CT-1 variable domain (aa 386 to 480). Th1-like clones responding to these epitopes proliferated differentially to different strains of B. bigemina, raising the possibilities that the T-cell epitopes may vary antigenically and that CT-1 may be differentially expressed with respect to the other RAP-1 CT domains in the different strains. In this report, we definitively map the T-cell epitope identified in the constant domain of RAP-1 to aa 159 to 187 (FVVSLLKKNVVRDPESNDVENFASQYFYM) and show that the predicted amino acid sequence is completely conserved among seven strains. The T-cell epitope in the CT-1 domain was mapped to aa 436 to 465 (VNSEKVDADDAGNAETQQLPDAENEVRADD), which is also completely conserved among eight strains of B. bigemina. We further show that the RAP-1alpha1-immunized cattle were protected against homologous B. bigemina challenge, thus suggesting an association between protective immunity and the helper T-cell response against the two epitopes. The immunogenic and highly conserved nature of these T-cell epitopes and their ability to stimulate functionally relevant Th cells that express gamma interferon support their inclusion in a vaccine.

Amino Acid Sequence↗

PubMed1998

Stimulation of nitric oxide production in macrophages by Babesia bovis.

Gamma interferon (IFN-gamma)-activated macrophages are believed to play a key role in resistance to Babesia bovis through parasite suppression by macrophage secretory products. However, relatively little is known about interactions between this intraerythrocytic parasite and the macrophages of its bovine host. In this study, we examined the in vitro effect of intact and fractionated B. bovis merozoites on bovine macrophage nitric oxide (NO) production. In the presence of IFN-gamma, B. bovis merozoites stimulated NO production, as indicated by the presence of increased L-arginine-dependent nitrite (NO2-) levels in culture supernatants of macrophages isolated from several cattle. The merozoite crude membrane (CM) fraction stimulated greater production of NO, in a dose-dependent manner, than did the merozoite homogenate or the soluble, cytosolic high-speed supernatant fraction. Stimulation of NO production by CM was enhanced by as little as 1 U of IFN-gamma per ml of culture medium. Upregulation of inducible NO synthase mRNA in bovine macrophages by either B. bovis-parasitized erythrocytes and IFN-gamma or CM was also observed. B. bovis-specific T-helper lymphocyte culture supernatants, all of which contained IFN-gamma, were also found to induce L-arginine-dependent NO2- production. Supernatants that induced the highest levels of NO also contained biologically active TNF. These results show that B. bovis merozoites and antigen-stimulated B. bovis-immune T cells can induce the production of NO, a molecule implicated in both protection and pathologic changes associated with hemoprotozoan parasite infections.

Animals↗

PubMed1998

B-lymphocyte proliferation during bovine leukemia virus-induced persistent lymphocytosis is enhanced by T-lymphocyte-derived interleukin-2.

Bovine leukemia virus (BLV)-induced persistent lymphocytosis is characterized by a polyclonal expansion of CD5+ B lymphocytes. To examine the role of the cytokine microenvironment in this virus-induced B-lymphocyte expansion, the expression of interleukin-2 (IL-2), IL-4, IL-10, and gamma interferon (IFN-gamma) mRNA, was measured in stimulated peripheral blood mononuclear cells from persistently lymphocytotic BLV-infected cows, nonlymphocytotic BLV-infected cows, and uninfected cows. IL-2 and IL-10 mRNA expression and IL-2 functional activity were significantly increased when peripheral blood mononuclear cells from persistently lymphocytotic cows were stimulated with concanavalin A (ConA). Additionally, during persistent lymphocytosis, peak IL-2 and IL-10 mRNA expression was delayed, and elevated expression was prolonged. To determine the potential biologic importance of increased IL-2 and IL-10 expression, the response of isolated B lymphocytes from persistently lymphocytotic cows to human recombinant cytokines and to cytokine-containing supernatants from isolated T lymphocytes was examined. While recombinant human IL-10 (rhIL-10) did not consistently induce detectable changes, rhIL-2 increased viral protein (p24) and IL-2 receptor expression in isolated B lymphocytes from persistently lymphocytotic cows. Additionally, rhIL-2 and supernatant from ConA-stimulated T lymphocytes enhanced B-lymphocyte proliferation. The stimulatory activity of the T-lymphocyte supernatant could be completely inhibited with a polyclonal anti-rhIL-2 antibody. Finally, polyclonal anti-rhIL-2 antibody, as well as anti-BLV antibody, inhibited spontaneous proliferation of peripheral blood mononuclear cells from persistently lymphocytotic cows, demonstrating that the spontaneous lymphoproliferation characteristic of BLV-induced persistent lymphocytosis is IL-2 dependent and antigen dependent. Collectively, these findings strongly suggest that increased T-lymphocyte expression of IL-2 in BLV-infected cows contributes to development and/or maintenance of persistent B lymphocytosis.

Animals↗

PubMed1998

Love styles among Guatemalans in a local village.

81 participants in a village in Guatemala were administered a translated version of the 1986 Love Attitudes Scale of Hendrick and Hendrick. Analysis showed that the men scored significantly higher on the Ludus love style than the women. Research among other Latinos outside the United States is suggested.

Adolescent↗