Streptococcal products and leukocyte activities.

Various streptococcal species are directly responsible for udder infections which should normally be countered by polymorphonuclear neutrophils (PMNs). In order to detect a putative inhibition of streptococcal products on the activities of bovine PMNs, we used a combination of four tests which permits an adequate evaluation of PMNs functions, e.g. PMN adherence on endothelial cells, chemotactic assay, phagocytosis of bacteria labelled with fluorescein isothiocyanate (FITC) and measurement of anion superoxide production. The conclusion is that neither of the two pathogenic streptococcal species isolated from mastitis appeared to produce in vitro factors affecting PMN activities.

FACS analysis of bovine leukemia virus (BLV)-infected cell lines with monoclonal antibodies (mAbs) to B cells and to monocytes/macrophages.

The eighteen monoclonal antibodies (mAbs) to B cells and the fourteen mAbs to accessory cells submitted to the workshop were analysed by FACS on three established, bovine leukemia virus (BLV)-infected bovine cell lines. Several mAbs of previously defined specificity were run in parallel. This analysis allowed us to gain further insight on the precise phenotype of those peculiar cells and to cluster the submitted mAbs according to their staining patterns. The BLV-infected cell lines seemed to belong to the B cell type though some of them lack detectable surface immunoglobulins. Moreover, all lines express the CD5 T cell marker and several myeloid markers.

Monoclonal antibody specific for bovine CD 5 antigen which enhances mitogen-induced blastogenesis and IL-2 production.

This study reports on the functional characteristics of a bovine T-cell differentiation antigen recognized by the monoclonal antibody (mAb) 8C11. This mAb has previously been found to react with a 67-kD molecule shared by thymocytes and peripheral blood T cells and to be undetectable on the B cells of healthy animals. This antigen is also largely expressed on the B cells from bovine leukemia virus-infected animals. Molecules with a similar cell distribution have been described in other species (mouse, human, rat and sheep), and were termed CD5 molecules. In order to confirm the CD5-like nature of the target molecule recognized by 8C11, functional T-cell assays were carried out. We report here that this mAb, like its human and murine homologues, enhances the proliferative responses of T cells to mitogens or alloantigens but does not directly stimulate T-cell division. Moreover, we have shown an enhancing effect of this 8C11 mAb on bovine interleukin-2 production by concanavalin A-stimulated bovine peripheral blood mononuclear cells.

B cells from bovine leukemia virus- (BLV) infected sheep with hematological disorders express the CD5 T cell marker.

Though peripheral blood B cells from healthy sheep were known to be devoid of the CD5 T cell marker, it appears from our study that most B cells from bovine leukemia virus- (BLV) infected sheep with hematological disorders express both the CD5 marker and surface IgM. The possible meaning of this T cell marker expression on B cells from BLV-infected sheep is briefly discussed.

Bovine leukemia virus (BLV)-infected B-cells express a marker similar to the CD5 T cell marker.

In the course of generating monoclonal antibodies to bovine thymus-dependent differentiation antigens, we were able to characterize an antibody, termed 8C11, that detects an antigen shared by a majority of thymocytes and peripheral T cells (in blood and thymus-dependent area of spleen and lymph-nodes), but undetectable on normal B cells. However, this antibody was reactive with B cells from cows infected with bovine leukemia virus (BLV). These BLV-infected B cells were found to express simultaneously high concentrations of both surface IgM and 8C11-detected antigen. The antigen recognized by this antibody was shown to be a 67.5 kDa molecule. Because similar findings have been made on mouse myelomas and on human chronic leukemia cells, where this antigen was considered to be the equivalent of mouse Ly-1 antigen and human Leu-1 or CD5 antigen, the T cell antigen detected on BLV-infected cells could be the bovine counterpart of the CD5 antigen. By another way, it has been found that the CD5 T cell antigen is also present on a minor subpopulation of B cells in the spleen but not in the blood. We suggest that in the bovine a similar B cell subpopulation should be the BLV target and expand as a consequence of viral insertion, leading to the persistent lymphocytosis observed on BLV-infected animals.

The use of amphotericin B in prevention of Trypanosoma theileri growth in bovine cell culture.

Trypanosoma theileri is an ubiquitous blood parasite of cattle. This parasite grows easily in RPMI medium and perturbs lymphocyte tests based on [3H]thymidine uptake. This paper reports that 0.5 microgram ml-1 amphotericin B in the medium is adequate to get Trypanosoma growth inhibition without alteration of either mitogenic or allogenic stimulation of bovine lymphoid cells in vitro.

Determination of N-terminal amino acid sequence of bovine class I antigens.

The amino acid sequence of the N-terminal 12 residues of papain digested BoLA class I molecules has been determined. This sequence except for its 5th amino acid, appears largely similar to those reported in man, mouse and rat.

Production and characterization of monoclonal antibodies raised against BoLA class I antigens.

Monoclonal antibodies (MoAbs) reacting with bovine leukocyte membrane antigens have been prepared by fusion of mouse myeloma cells (SP2/0.Ag.14) and spleen cells of mice immunized with various cell types. Three of these MoAbs detected membrane components showing the typical structure of class I MHC molecules; indeed, immunoprecipitation studies revealed that these components were proteins composed of two subunits of 44,000 and 12,000 daltons apparent molecular weight. The density of these antigens in the cells of various leukocyte lineages was determined by solid phase radioimmunoassay, immunogold staining and cytofluorometry. Their expression seemed similar to that of class I molecules in other species, namely heavy on the mononuclear blood cells and weaker on the neutrophils and platelets. The eosinophils appeared more positive than the neutrophils, while the erythrocytes were negative. Cross-inhibition and sequential immunoprecipitation experiments demonstrated that these MoAbs recognised different epitopes either on a single molecule or on cross-reacting molecules. One antibody appeared to be raised against the monomorphic bovine beta-2-microglobulin, while the two other antibodies detected the heavy chain of polymorphic class I-like products. The authors propose that the BoLA class I polymorphism should be studied by determination of the fixation ratio of the monomorphic anti-beta 2M versus the polymorphic anticlass I antibodies amongst the animals.

A bovine 'Ia-like' antigen detected by a xenogeneic monoclonal antibody.

A monoclonal antibody termed B2 Val 7C7, was produced by the fusion of xenoimmune mouse spleen cells with Sp2/0.Ag 14 myeloma cells. This antibody is specific for a polymorphic lymphocyte antigen; it was detected on cells from 138 out of 177 cattle by both 125I-labelled protein A (solid-phase radioimmunoassay, SPRIA) and gold-labelled protein A (immunogold). Its binding was tested on various cell types (peripheral blood lymphocytes, monocytes, polymorphonuclear cells (PMN), thymocytes) from a variety of normal bovine donors. On the one hand, B2 Val 7C7 detects a determinant present on all IgG-bearing lymphocytes, on 20% of the non-IgG-bearing lymphocytes and on the majority of the monocytes. On the other hand, no binding occurs on any PMN or thymocytes. The detected membrane antigen was isolated by immunoprecipitation from an NP 40 extract of 3H-leucine-labelled cells. On SDS-PAGE, it appears to be composed of two sub-units: a 32 000-dalton and a 27 000-dalton chain. These results show that B2 Val 7C7 recognizes an alloantigenic specificity present on an Ia-like antigen.

Adrenaline influence on the immune response. II. Its effects through action on the suppressor T-cells.

Experiments were carried out to specify the adrenaline target among the immunocompetent cells. Adrenaline administered for some hours exerted opposite effects on the natural PFC and RFC: the first were enhanced and the second significantly reduced. These paradoxical results were interpreted as a consequence of the inhibition of the suppressor T-cells in the resting status. Adrenaline appeared to act on the sensitive cells through beta-rather than through alpha-receptors. Further experiments on the adrenaline influence on the syngeneic barrier phenomenon and on the cellular balance at its termination seemed to indicate that adrenaline was directly inhibitory for the Ts but not for their precursors. These results are discussed in the light of the cellular networks regulating the immune response.

Adrenaline influence on the immune response. I. Accelerating or suppressor effects according to the time of application.

The intervention of adrenaline in the immunoregulation was investigated through the modification of the anti-SRBC PFC response of mice after its i.p. administration (4 micrograms) at various intervals before SRBC antigen. When the interval was less than 24 h, adrenaline accelerated the immune kinetics. This modification was apparent on both direct and indirect PFC, as well as on naive and immune mice. However, mice treated from 2 days showed a suppression of the response. The adrenaline effect subsisted on the adoptive response of spleen cells drug-treated either in vivo or in vitro. The mitogenic response after in vitro PHA or LPS stimulation of spleen cells from adrenaline-treated mice indicated that the T-cells were the drug target. The physiological role of the adrenaline and immunological influences of acute stress are discussed in the paper.

Interference between two immune responses. II.--Conferrability by the serum of the influences exerted by a response to horse RBC on the subsequent response to sheep RBC in the mouse.

Serum taken from mice responding to HoRBC and given to syngeneic mice influences the kinetics of a subsequent anti-SRBC response. The serum taken at the 36th of the anti-HoRBC response exerts after transfer an accelerating effect on the anti-SRBC response while this of the 7th day has an inhibitory effect. Both effects are especially demonstrative on mice irradiated and restored with spleen cells from a single donor because of the low variation coefficient of their anti-SRBC response.

Interference between two immune responses. I.--Modification of the kinetics of the mouse response to SRBC by responses to heterologous RBC.

Influence of a preceding immunization with HoRBC on the immune response to SRBC was studied in Swiss and CBA inbred mice. In Swiss mice, a short interval (1day) between the two immunizations produced an acceleration of anti-SRBC response, while a longer interval (4-7 days) inhibited this response. This was interpreted as manifestations of nonspecific helper and suppressor activities triggered by the previous challenge. At any time of a response, there would exist a variable ratio of these two opposite activities. The helper activity was radiation-resistant while the suppressor one was radiation-sensitive at the time of its appearance and was effective on the IgG as well as on the IgM response. CBA mice response to SRBC was only influenced by the suppressor and not by the helper component of the preceding challenge with HoRBC. These CBA mice were very good responders to this antigen, and this may be the cause of their strain particularities. If we consider that each individual is permanently submitted to natural immunizations, the ratio of the resulting non-specific activities must be esentially variable with time; it can partly account for the individual amplitude variation of an engaged immune response.