Human interleukin-12 upregulates proliferation and interferon-gamma production by parasite antigen-stimulated Th cell clones and gamma/delta T cells of cattle.
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Animals↗
Biomedical subjects
W C Davis
Publications and source records attributed to W C Davis.
Malignant catarrhal fever virus. Characterization of a United States isolate and development of diagnostic assays.
Malignant catarrhal fever (MCF) is a severe lymphoproliferative disease of certain domestic and wild ruminants. Two distinct but closely related viruses cause clinically indistinguishable syndromes in susceptible ruminant species: wildebeest-associated MCF virus (WA-MCFV) and sheep-associated MCF virus (SA-MCFV). Neither the pathogenesis nor the epidemiology of SA-MCF is understood, primarily because of a lack of adequate detection methods for the etiologic agent or antibody against that agent. Work designed to develop these tests has been under way in our laboratory. To obtain basic information about the virus, the in vitro growth properties of a US isolate of MCF virus were studied and its major viral proteins identified and characterized by a panel of monoclonal antibodies generated against the isolate. A monoclonal antibody to a broadly conserved epitope of MCF virus was identified, and a competitive-inhibition ELISA (CI-ELISA) was developed for detection of anti-MCF antibody in sheep and other ruminants. The monoclonal antibody (15-A) reacted with an epitope located on a glycoprotein complex, which was present in all isolates of MCF virus examined. Antibody from a wide variety of ruminants infected with MCF virus of both sheep and wildebeest origin competed with the monoclonal antibody 15-A for the epitope, which was not present on 14 other common ruminant viruses. The assay detected antibody in inapparently infected sheep, and in cattle, deer, and bison with clinical MCF. A PCR assay for DNA of the sheep-associated virus was developed, based on previously reported primers. Comparative studies demonstrated that the CI-ELISA was specific for MCFV antibody and that the PCR was more reliable for diagnosis of clinical MCF.
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Identification of gamma delta T lymphocyte subsets that populate calf ileal mucosa after birth.
Ileal intraepithelial and lamina propria lymphocytes from newborn, 1.5-week-old, and 3-week-old calves were compared to determine to what extent the mucosa becomes populated after birth. Single and dual fluorescence flow cytometry were used with monoclonal antibodies to bovine (Bo) CD molecules to identify lymphocyte subpopulations. Few ileal mucosal lymphocytes were present in calves at birth. However, by 1.5 weeks of age, the villi were populated with large numbers of lymphocytes, and by 3 weeks of age, the numbers had increased further. These included a prominent subpopulation of gamma delta T cells. Several subsets of gamma delta T cells populated ileal mucosa after birth. The predominant subset coexpressed BoCD2, and a smaller subset coexpressed BoCD8. WC1+ gamma delta T cells comprised the smallest subset. All gamma delta T cell subsets coexpressed ACT2, a molecule expressed on activated WC1+ and WC1- gamma delta T cells from cattle.
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Cloning, sequencing and expression of the bovine CD3 epsilon and TCR-zeta chains, two invariant components of the T-cell receptor complex.
CD3 epsilon and the zeta-chain of the bovine T-cell receptor (TCR) are two invariant molecules with an important role in signal transduction via the TCR/CD3 complex. The nucleotide sequence of a bovine CD3 epsilon cDNA clone containing the complete coding sequence was determined and the deduced amino acid (aa) sequence compared to that of other species. The cytoplasmic domains of the different CD3 epsilon clearly show a higher degree of conservation than the extracellular domains. Bovine CD3 epsilon produced in Escherichia coli using different bacterial expression vectors was recognised by antibodies (Ab) directed against the intracytoplasmic domain of human CD3 epsilon. A partial bovine TCR zeta-chain cDNA was generated by the polymerase chain reaction (PCR) using primers that were based on sequences that are conserved between different species; 3' and 5' RACE-PCR were carried out to obtain the complete TCR zeta-chain cDNA sequence. A comparison of the predicted TCR zeta-chain aa sequence reveals that the GDP/GTP-binding motif, which is conserved in other species, shows marked differences in the bovine and ovine TCR zeta-chains. In contrast to CD3 epsilon, the short extracellular domain of the TCR zeta-chain is 100% conserved between the different species and the transmembrane domain also shows a high degree of identity. Ab were raised against the TCR zeta-chain, produced as a glutathione S-transferase fusion protein in E. coli, and were used in Western blot analysis to further characterise TCR zeta-chain expression in T-cells. The regents provide valuable tools for the study of signal transduction pathways in normal and transformed bovine T-cells.
Amino Acid Sequence↗
Effect of dietary restriction on cell-mediated immune responses in cattle infected with Mycobacterium bovis.
Nine M. bovis-infected cattle on a diet deficient in both protein and energy for 133 days lost approximately 17% of their original body weight. However, dietary restriction did not result in any significant reduction in skin sensitivity to PPD, in vitro production of IFN-gamma or lymphocyte blastogenesis. The number of circulating BoCD4+ cells and B cells were similar in both the malnourished and the control cattle. However, significantly lower numbers (P < 0.01) of circulating BoCD2+ cells, BoCD8+ cells, WC1+ gamma delta T cells and ACT2+ cells were found in the malnourished cattle. With the exception of inorganic phosphate, the changes in plasma biochemical parameters were unremarkable.
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Lymphocyte subpopulations in the mammary gland of the goat.
Mammary glands of pregnant, lactating and resting goats were studied by immunohistochemistry for lymphocyte subpopulations using a panel of monoclonal antibodies. All T lymphocyte subpopulations that may have a role in the immune response, CD2+, CD4+, CD8+ and gamma delta T cells and subsets, were present in the mammary gland and were noted to increase in number progressively during pregnancy, decrease significantly during lactation, and then moderately increase during the resting period. CD4+ cells, the predominant cell type in the mammary gland, were located mainly in the connective tissue, whereas CD2+, CD8+ and TcR1-N24+ cells were predominant in the intraepithelial areas. TcR1-N6+ cells were detected almost exclusively during pregnancy, being localized mainly in the connective tissue. Their proportion decreased markedly following parturition. Very few WC1-N3+ and -N4+ cells were detected in the mammary gland. It is suggested that the majority of gamma delta T lymphocytes in the mammary gland of the goat are CD2+ CD8+ WCl-, a distinctive subset from that of the WCl+ subset in peripheral blood.
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Ruminant cluster CD71.
Two monoclonal antibodies (mAbs) belong to this cluster: IL-A77 and IL-A165. They clustered closely in PC16c. The first mAb, an IgM, was raised against bovine activated B cells, the second was an IgG1 raised against bovine bone marrow cells.
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Analysis of monoclonal antibodies specific for the gamma delta TcR.
gamma delta T cells in ruminants can be subdivided in two or more subpopulations on the basis of the expression of surface antigen WC1, which can exist in different isoforms. In this study, 18 monoclonal antibodies (mAbs) submitted to the Third International Workshop that were predicted to react with gamma delta TcR molecules were analysed and expression of their antigens was investigated on the different gamma delta T cell subpopulations. A set of control mAbs positive for TcR1 (86D), BoCD3 (MM1A), WC1 (B7A1, BAQ4A, CACTB32A, and BAQ89A) was included for comparative studies. Previous investigations demonstrated eight of the mAbs immunoprecipitated peptides with apparent M(r)s of 37 and/or 47 kDa, indicating they recognized determinants on the T cell receptor, TcR1. Two color flow cytometric analyses in the present study demonstrated the mAbs formed three groups; group 1, a set of mAbs that recognize TcR1 determinants expressed on all gamma delta T cells and groups 2 and 3, sets of mAbs that recognize TcR1 determinants on some gamma delta T cells: TcR1-N6 and TcR1-N7 respectively. mAbs from the latter groups define families of TcR1 molecules that express either one or both of the determinants. These antigenically distinct forms of TcR1 are expressed in equal proportion on the two gamma delta T cell populations that express one of the mutually exclusive isoforms of WC1, WC1-N3 and WC1-N4. The data indicate usage of the mAb-defined families of the gamma delta TcR is primarily restricted to the WC1+ subpopulation of gamma delta T cells. However, a small subpopulation of CD2+, WC1- gamma delta T cells expresses a form of TcR1 positive for the determinant TcR1-N6.
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Analysis of monoclonal antibodies reactive with molecules upregulated or expressed only on activated lymphocytes.
Monoclonal antibodies potentially specific for antigens expressed or upregulated on activated leukocytes were selected for further analysis from the panel submitted to the third international workshop on ruminant leukocyte antigens. The kinetics of expression of these activation antigens on resting peripheral mononuclear cells (PBMC) and PBMC stimulated with concanavalin A or staphylococcal superantigen SECI for 4, 24 or 96 h were compared, as well as their appearance on various subsets of cells. For some of them, a molecular mass could be determined after immunoprecipitation from radio-labeled, lectin-stimulated cells. Based on the results from the clustering, kinetic studies and biochemical data, evidence was gathered for assigning two additional mAbs to cluster BoCD25 (IL-2 receptor) and two mAbs to cluster BoCD71 (transferrin receptor). Four mAbs recognized an early activation antigen predominantly expressed on gamma delta T cells in short-term cultures. A number of other activation antigens were further characterized.
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Immunohistological study on bovine lymph node using monoclonal antibodies to interleukin-2 receptor.
Three monoclonal antibodies to IL-2R alpha (LCTB32, CACT116 and LCTB2) were used for immunohistologic staining of lymph nodes from normal young cattle and a calf suffering from phlegmon. In the normal cattle, a few IL-2R alpha positive cells were detected in the paracortex region. In the calf, IL-2R alpha was mostly expressed on follicle cells.
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Changes in the distribution of cells expressing tumour-associated antigen in lymph nodes during the progression of enzootic bovine leukosis.
A monoclonal antibody (MAb), c143, that recognizes a tumour-associated antigen that is "upregulated" on neoplastic B cells in cattle with enzootic bovine leukosis (EBL), was used as a marker to study disease progression. An immunohistochemical examination of neoplastic tissue and superficial cervical lymph nodes from 14 animals with EBL revealed three morphologically definable stages of change in the structure of lymph nodes, associated with the distribution of c143-positive cells: (1) the presence of c143-positive cells at the marginal sinus with no apparent changes in lymph node structure; (2) the presence of positive cells extending into and distorting the architecture of the lymph node, with clear evidence of proliferation before overt changes (enlargement of lymph nodes) were evident; and (3) the presence of positive cells throughout the lymph node with total disruption of lymph node structure when clinical signs of lymph node enlargement were evident. The results indicated that the bovine leukaemia virus-transformed lymphocytes or neoplastic cells in peripheral blood accumulate in the marginal sinus area at the earliest stages, and subsequently proliferate and infiltrate into follicles, leading to the development of clinical signs of lymphosarcoma.
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Effect on intraerythrocytic Anaplasma marginale of soluble factors from infected calf blood mononuclear cells.
Blood mononuclear cells (lymphocytes and monocytes) were isolated from infected calves during in vivo control of acute anaplasmosis and cultured with Anaplasma marginale organisms. Supernatants from the cultures reduced the proportion of erythrocytes containing viable A. marginale in vitro, indicating that an antibody-independent mechanism of rickettsemia control might occur during acute anaplasmosis.
Acute Disease↗
Comparative effects of bluetongue virus infection of ovine and bovine endothelial cells.
Bluetongue virus (BTV) infection results in disparate clinical syndromes among ruminant species. An in vitro model system of BTV/target cell interaction was developed using umbilical vein endothelial cells (EC)from fetal lambs and calves. These cells had microscopic, ultrastructural, and immunocytochemical features typical of EC. BTV infection in these cells was examined using virus binding assays, plaque assays, a whole-cell enzyme-linked immunosorbent assay, flow cytometry, electron microscopy, and a bioassay for interferon activity. EC from both species supported cytopathic BTV infections. Ovine EC bound more BTV initially and produced more virus over time, whereas bovine EC underwent more rapid lysis subsequent to infection. An ultrastructural comparison of BTV-infected ovine and bovine EC, grown as differentiated capillary-like cords on a laminin-rich matrix or as monolayers, revealed no significant interspecies differences in viral morphogenesis between 1 minute and 24 hours after infection. The intracellular distribution of BTV nonstructural protein 1, which localized to virus inclusion bodies and tubules, was identical for ovine and bovine endothelial cells. Ovine and bovine EC produced a soluble mediator of interferon activity in response to BTV infection; however, ovine EC produced higher levels of interferon activity at lower levels of infection. These findings indicate differences in BTV-EC interaction that may contribute to the pathogenesis of the severe inflammatory disease that is characteristic of clinical bluetongue disease in sheep.
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The proto-oncogene c-myb is expressed in sporadic bovine lymphoma, but not in enzootic bovine leukosis.
We examined bovine c-myb gene expression in six samples of sporadic bovine lymphomas (two calf, three thymic and one intermediate) and five of enzootic bovine leukosis. Tumor cells of the sporadic bovine lymphomas were of immature cell lineage (one B lymphoma and five T lymphomas). The c-myb mRNA was expressed in almost all the sporadic bovine lymphomas (except for one thymic form) including a BoCD8 single positive T lymphoma. On the contrary, c-myb was not expressed in mature B lymphomas of enzootic bovine leukosis. The results suggest that c-myb expression is closely associated with tumor cell differentiation of bovine lymphomas.
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A sequential study of the bovine tuberculin reaction.
The sequential histopathological and immunocytochemical changes that characterize the tuberculin reaction were studied in 13 cattle experimentally sensitized to Mycobacterium bovis, and 14 cattle naturally infected with M. bovis. There were two distinct, temporally related patterns of morphological change that were similar for both groups of cattle. The first phase, between 6 hr and 24 hr after the intradermal injection of purified protein derivative (PPD), was characterized by a perivascular aggregation of WC1+ gamma delta T cells and neutrophils and the presence of leucocytoclastic vasculitis within the papillary dermis. The second phase of the reaction was characterized by increased numbers of infiltrating BoCD4+ cells, BoCD8+ cells and macrophages, as well as an increase in expression of the interleukin-2 (IL-2) receptor and the ACT2 antigen. Macrophages were the most numerous infiltrating leucocytes between 24 hr and 72 hr after the intradermal injection of PPD. At 72 hr, the reaction was characterized by intense perivascular cuffing with BoCD4+ cells, BoCD8+ cells and macrophages; gamma delta T cells and neutrophils were a minor component of the reaction and leucocytoclastic vasculitis was no longer observed. No B cells were detected in the dermis throughout the period of study. The increase in skin thickness was primarily because of inflammatory oedema that was contained within the area by a meshwork of fibrin deposited around the collagen bundles of the reticular dermis.
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Identification of monoclonal antibodies for immunohistochemical staining of feline B lymphocytes in frozen and formalin-fixed paraffin-embedded tissues.
Commercially-available monoclonal antibodies to B lymphocytes were evaluated for immunohistochemical staining of feline B lymphocytes in frozen and formalin-fixed, paraffin-embedded tissues using an avidin biotin complex immunoperoxidase immunohistochemical technique. Three monoclonal antibodies: F46A and F72A raised to "carnivore" B lymphocytes and RA3.6B2 raised to murine B lymphocytes, stained B lymphocyte-dependent areas of frozen feline lymphoid tissue. In addition, antibody RA3.6B2 stained B lymphocyte dependent areas in formalin-fixed, paraffin-embedded feline tissues. There was no staining of T lymphocyte-dependent areas in either frozen or formalin-fixed tissues. Dual parameter flow cytometry, using an anti-pan-T lymphocyte antibody, revealed that greater than 99% of the cells stained by RA3.6B2 are a population distinct from T lymphocytes. F46A was shown to stain a sub-population of those cells stained with RA3.6B2. These antibodies may be useful in the identification of feline B lymphocytes using immunohistochemistry and flow cytometry and thereby provide additional tools to study B lymphocyte ontogeny and the significance of lymphocyte phenotype in lymphoid neoplasia in cats.
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Elevated pim-1 and c-myc proto-oncogene induction in B lymphocytes from BLV-infected cows with persistent B lymphocytosis.
Bovine leukemia virus (BLV) induces a non-malignant, polyclonal, persistent lymphocytosis (PL) of circulating, CD5 B lymphocytes in cattle, with variable progression to CD5 B cell leukemia or lymphoma. We analyzed the expression of two proto-oncogenes, pim-1 and c-myc, proto-oncogenes deregulated in some human B cell leukemias and lymphomas, in peripheral blood mononuclear leukocytes (PBML) from BLV-infected PL cows. Results demonstrate that pim-1 and c-myc mRNA levels are elevated in unfractionated stimulated PBML from a sample of PL cows naturally infected with BLV. Results confirm that pim-1 is constitutively expressed, but not inducible in normal bovine peripheral blood B lymphocytes, but can be induced in the predominantly CD5 B lymphocytes from BLV-infected PL cows. Results further demonstrate that c-myc is inducible in bovine B and T lymphocytes regardless of BLV status, but the amount of induction is greater in B lymphocytes from BLV-infected PL cows than in B lymphocytes from noninfected control cows. These results suggest that pim-1 and c-myc are upregulated in B lymphocytes from BLV-infected PL cows and that deregulation of proto-oncogene expression is not limited to completely transformed cells, but can also characterize a naturally occurring, pre-neoplastic lymphocytic state.
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Expression of an anti-H58A monoclonal antibody recognized molecule in rabbit tonsillar epithelium.
The H58A monoclonal antibody (mAb) recognizes a highly conserved determinant on MHC class I molecules in various species. This mAb was used in immunohistochemical studies to determine the presence of this molecule in rabbit tonsils. The molecule is strongly expressed in all layers but the germinal one, of the stratified epithelium (SSE) of the oral cavity and tonsillar crypt. The expression of this molecule is completely abolished in the lymphoepithelial regions of the crypt epithelium. In cortison-induced immunosuppressed animals, the lymphoepithelium is depleted and gradually transforms to SSE. Consequently, the expression of the H58A mAb recognized molecule reappears. Further studies are needed to determine if this molecule plays any kind of functional role in the formation of lymphoepithelial tissue.
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