A new transmissible AIDS-like disease in mice induced by alloimmune stimuli.

The search for a suitable and reliable animal model for human AIDS that is easy to use on a large scale continues. Here we describe a new condition in mice that closely resembles human AIDS, namely, chronic lymphoproliferation with dramatic depletion of CD4-positive cells, progressive impairment of the immune responses, and Kaposi's sarcoma-like tumors or terminal B-lymphomas. The AIDS-like disease was primarily induced by mating BALB/c female mice to C57BL/6 males during a 1-year period (7-10 allogeneic pregnancies) followed by immunization with paternal lymphocytes. The disease is sexually and vertically transmissible, transferrable by cell-free plasma and is associated with autoimmune reactions to major histocompatibility complex antigens and CD4 cells. We hope that this becomes a model for studying the mechanisms of AIDS immunopathogenesis and immune-based treatment approaches.

Suppression of the humoral antibody response in natural retrovirus infections.

The feline leukemia virus (FeLV) frequently causes death by predisposing the host to acute infections by other pathogens rather than by inducing leukemia. In a previous study, cats infected with FeLV were found to have prolonged homograft rejection responses but there was no evidence that the humoral immune response was impaired. In the present study, the humoral response to the synthetic multichain polypeptide (L-tyrosine-L-glutamic acid)-poly-DL-alanine-poly-L-lysine, denoted (T.G)AL, was found to be significantly depressed in healthy cats that were naturally infected with FeLV compared to uninfected controls. In cats with persistent FeLV viremia the major antibody response to (T.G)AL, normally seen at days 9 to 14 after immunization, was both delayed and greatly reduced.

Ceruloplasmin as a marker of neoplastic activity in rabbits bearing the VX-2 carcinoma.

The establishment of an experimental model with rabbits in which VX-2 carcinoma was implanted in the gastrocnemius muscle and subsequently successfully cured by a second tumor cell inoculation has been reported previously. Tumor growth and regression could be followed by manula palpation. The changes in serum ceruloplasmin (CP, EC 1.10.3.2) levels of individual rabbits during tumor development and regression were followed. CP levels increased 4- to 8-fold of normal during the progression of the malignant process, often before tumors could be detected by palpation. With tumor regression CP levels returned to normal. When metastasis developed, the CP levels remained high. This phenomenon seems to be related to the VX-2 carcinoma, since CP levels in rabbits challenged with various antigens and suffering from induced multiple s.c. abscesses did not change significantly, while in pregnant rabbits CP levels increased up to at most 3-fold. It is concluded that serum CP level can serve as a reliable biochemical marker of the activity of this malignant process. The practical application of this finding lies in the follow-up of malignant processes in humans and is now under investigation.

Levels and role of cytokines in bovine leukemia virus (BLV) infection.

Bovine leukemia virus belongs to a small subfamily of exogenous retroviruses that includes the human retroviruses HTLV-1, HTLV-II and the simian virus, STLV-1. Like other retroviruses, infection with BLV results in deregulation of the host immune system at both humoral and cellular levels. An approach which might help in the elucidation of some immune impairment phenomena is the investigation of the role that cytokines play in the pathogenesis and immune response of BLV infected animals. Here we describe our findings on IL-6 and TNF. We have found that the levels of IL-6 in the sera of BLV infected cows which show persistent lymphocytosis (BLV+ PL+) were significantly higher than those of BLV infected with no lymphocytosis (BLV+ PL-) or BLV negative cows (BLV-). The same results were obtained by measuring the spontaneous production of IL-6 in peripheral blood mononuclear cells (PBMC). Furthermore, PBMC derived from BLV+PL+ cows secrete higher levels of IL-6 and TNF alpha than those derived from BLV+PL- and BLV- ones following in vitro exposure to the BLV gp51 antigen, bacterial endotoxins (LPS) and ConA. Similar results were obtained when supernatants from stimulated adherent (monocytes, macrophages) and non-adherent cells (B and T lymphocytes) were tested. When exogenous IL-6 and TNF alpha were added to BLV infected cells in vitro, the expression of viral antigens was strongly suppressed. Thus, the possibility exists that the elevated production of IL-6 and even more than that of TNF alpha play a role as contributing factors to the latency of the clinical expression in BLV infection.

A new retroviral AIDS-like disease and/or acute leukemia induced in mice by alloimmune stimuli.

We have shown a new phenomenon demonstrating that BALB/c female mice mated to C57BL/6 males during a year (7-10 pregnancies) develop AIDS-like disease or acute leukemia after an additional immunization with fixed ConA activated paternal (C57BL/6) lymphocytes. The AIDS-like disease is sexually and vertically transmissible and easily transferable to intact BALB/c and C57BL/6 mice by filtered plasma of affected animals.

Somatic cell hybridization between bovine leukemia virus-infected lymphocytes and murine plasma cell tumors: cell fusion studies with bovine cells.

Hybridization of peripheral blood lymphocytes from bovine leukemia virus-infected cows with murine myeloma cells resulted in the generation of hybrid cells secreting immunoglobulins composed of various combinations of heavy and light chains of both bovine and murine Ig origin. Some hybrid cells derived from the light-chain producer, but non-secretor murine myeloma NSI cell line, secreted IgM molecules composed of bovine mu-chain linked to bovine and/or murine light chains. Other hybridomas secreted mouse and bovine light-chain dimers and/or monomers, or failed to secrete any Ig polypeptide chain whatsoever. Immunoglobulins secreted by hybridomas obtained upon hybridization of bovine cells with the IgG-secreting murine myeloma P2X63 cell line, contained bovine mu-chain in one of the seven hybridomas obtained, and bovine light chain in two of them. All the cell lines secreted murine light- and gamma-chains.

A specific enzyme-linked immunosorbent assay for determination of bovine IgM: use in screening hybridoma cell lines.

A highly specific enzyme-linked immunosorbent assay (ELISA) is described for determination of bovine IgM and use in screening IgM-secreting hybridoma cell lines. The advantages of this assay over the method of radioactive labelling of cells followed by immune precipitation, SDS-PAGE, fluorography and autofluorography are demonstrated.

Cellular immune response cytokine expression during the initial stage of bovine leukemia virus (BLV) infection determines the disease progression to persistent lymphocytosis.

We have established experimental models of bovine leukemia virus (BLV) infection followed by progression to persistent lymphocytosis (PL) positive (BLV+PL+) or PL negative (BLV+PL-) stages of infection. Two out of six BLV infected animals developed PL+ 4 weeks after BLV infection. One other animal became PL+ late in the course of infection and three infected animals stayed PL-. These animals (PL-) exhibited transient lymphocytosis 3-4 weeks after infection and sustained PL- lymphocyte counts up to 24 weeks after infection. Competitive RT-PCR analysis of IFN-gamma mRNA expression revealed that peripheral blood mononuclear cells (PBMC) of animals with PL+ status developed by 4 weeks after infection had augmented IFN-gamma mRNA expression 3-4 weeks after BLV infection. However PBMC of animals that sustained a long-termed PL- lymphocyte count had elevated IFN-gamma mRNA expression 1-24 weeks after infection. Competitive RT-PCR analysis of IL-2 mRNA expression showed an increase in the levels of IL-2 mRNA in PL animals. Interleukin-10 (IL-10) mRNAs expression were elevated both in PL+ and PL- animals from 3 and 12 weeks after infection respectively. We suggest that early and extended expression of cellular response cytokines may delay the progression to PL+ in enzootic bovine leukemia.

Effect of bovine lactoferrin on a transmissible AIDS-like disease in mice.

The effect of bovine lactoferrin (bLF) was examined on an AIDS-like disease (ALD) in mice. Induction of disease was achieved by inoculation with infected cell-free plasma from diseased mice to uninfected ones. The effect of treatment with bLF was investigated when administered simultaneously with the virus, 20 days prior to infection, or 20 days after infection. Animals underwent clinical surveillance and enumeration of white blood cells (WBC) and lymphocytes, as well as fluorescent staining of CD4 and CD8 bearing cells. Simultaneous administration of bLF and virus did not affect the pattern of ALD progress along the course of the experiment. Pretreatment with bLF prior to virus inoculation abolished on day 21 the detrimental effect of viral infection that lasted for two months. An opposite outcome was observed when bLF was administered 20 days after the virus. It seems that bLF had played a preventive role for a restricted period of time. However, an adverse response was elicited when bLF was administered 20 days after viral infection.

Reactions of peripheral blood mononuclear cells (PBMC) of camels with monoclonal antibodies against ruminant leukocytes.

The particular immune system of the camel has been but little investigated. In this work circulating camel peripheral blood mononuclear cells (PBMC) were studied by flow cytometry. Monoclonal antibodies (mAbs) raised against ruminant leukocytes were used for the detection of cell surface antigens. Monoclonals to T-cell markers, CD4 (CACT138A) and CD8 (CACT80C), exhibited no reactivity towards camel PBMC in contrast to their reactivity to PBMC of other ruminant species and those of cattle in particular. A relatively high percentage (29.1+/-8.9%) of camel PBMC reacted with a non-immunoglobulin cell surface marker, B-B2, comparable to the reactivity of bovine PBMC. The B-B7 cell marker revealed 22.4+/-10.0% of reactive camel PBMC while the CD45 leukocyte common antigen was identified only on 19.4+/-3.1% of camel PBMC as compared to 74.7+/-4.9% for bovine PBMC. IgM (PIg45A) was detected on 9.1+/-1.4% of camel PBMC and on 46.6+/-19.5% of the bovine PBMC. Double fluorescent labeling with two B-cell markers and an anti-ruminant lambda light-chain mAb revealed 7-9% of cells bearing both B and lambda L-chain markers. Light chain reactivity was also assessed using an anti-goat F(ab')(2) antiserum. The values obtained, 14.3+/-5.8% for the camel and 47.8+/-2.7% for the cattle, are close to the values observed for surface IgM. These data suggest that camels, like other ruminants, possess L-chain bearing cells of the B-cell lineage. However, in the camel, Igs are different in that in addition to regular four chain Igs, about 65% of them possess two heavy chain Igs devoid of light chains. Because different sets of V(H) gene segments are used by four and two chain Igs, it is possible that there might be two lineages of B-cells each secreting a different form of antibodies.

BLV-infected lymphocytes exhibit two patterns of expression as determined by Ig and CD5 markers.

Lymphocytes were defined by their cell surface markers, Ig and CD5 in three groups of cows naturally infected with bovine leucosis virus (BLV). Lymphocytes were enumerated and groups were designated BLV seropositive with persistent lymphocytosis (BLV + PL +), BLV seropositive without persistent lymphocytosis (BLV + PL-) and BLV negative. The competence of peripheral blood mononuclear cells (PBMC) from the tested cows to express these two markers was determined by the double staining immunofluorescence procedure. Cows which developed persistent lymphocytosis (PL) as a result of BLV infection consequently underwent massive proliferation of B lymphocytes which express both Ig and CD5 antigens. In contrast, cows which were defined as BLV positive and PL negative showed a remarkable decrease of CD5 + Ig-, CD5- Ig+ and CD5+ Ig+ cells and also in the total number of lymphocytes. We suggest that BLV infection affects bovine lymphocytes through two different pathways of expression which might be related to the genetic properties of the target cells.

Staphylococcus aureus vaccine against mastitis in dairy cows, composition and evaluation of its immunogenicity in a mouse model.

Bovine mastitis caused by Staphylococcus aureus (S. aureus) is a most important infection disease that affects both the quality and the quantity of milk production. Antibiotic therapies formulated for intramammary use are generally unsuccessful in eliminating existing S. aureus infections. Vaccination is a logical approach to the control of S. aureus udder infections. However, to date commercially available S. aureus vaccine have shown limited efficacy under field conditions, mainly due to the paucity of information regarding relevant antigens which will induce a broad spectrum immunization. In the present paper the attempt to develop a new vaccine designated MASTIVAC I is described. MASTIVAC I is composed of three strains of S. aureus namely: VLVL8407; ZO3984 and BS449 which were isolated from clinical and sub-clinical cases of bovine mastitis. A mouse model was used to evaluate the S. aureus specific antibody production and protection of mice against virulent S. aureus strains. The results obtained showed that this vaccine exhibits a broad spectrum of antigenic and immunogenic properties that protects mice from homologues and hetrologous S. aureus challenge.

Circulating immune complexes in bovine leukemia virus (BLV)-infected cattle.

Circulating immune complexes (ICs) were detected in the sera of bovine leukemia virus (BLV)-seropositive cattle. Immune complexes were precipitated in 2.5% polyethylene glycol (PEG) and further dissociated. Bovine leukemia virus antigens, IgG and IgM molecules were detected after solubilization in the presence of sodium dodecyl sulphate, and quantitated by enzyme-linked immunosorbent assay (ELISA) assays. Mean values of IgG and IgM in BLV-containing ICs did not significantly differ from those obtained from ICs originating from BLV-seronegative animals. However, differences were found in the composition of ICs from older BLV-positive animals as compared to those obtained from young animals. The ratio of IgG/IgM was 5.02 in animals aged 5-10 years, while this ratio was 11.66 in animals of less than 5 years of age and 10.19 in controls. This might indicate a possible increase in the contribution of IgM molecules to the structural composition of ICs in BLV-infected cattle as related to age or stage of infection.

The implication of BLV infection in the productivity, reproductive capacity and survival rate of a dairy cow.

The objective of the present study was to find out whether BLV infection, known to cause immunodisturbances in cows, might also bring about decreased productivity, reproductive rate and a shorter life span. More than 100 pairs of dairy cows, and a whole population of 3000 milch cows, were studied for this report. The findings revealed that a BLV-positive cow had a shorter life span than both its seronegative counterpart and the entire milch cow population. It also produced a total of 3.5% less milk and had a mean of 48 more days open than did the BLV-negative cow. The differences in survival rate were highly significant, while, at a level of 5%, those of productivity and reproductive rate were not. The implications of these findings are discussed. A highly significant correlation was also shown between BLV infection and the persistence of Trichophyton verrucosum infection in cows. The presented data indicate that BLV infection might affect the immune system of a cow to such a degree that it ceases to be productive enough to be kept within a herd. Thus it is usually culled before any severe symptoms of disease emerge.

Effect of permeabilization on peripheral blood lymphocytes of bovine leukemia virus (BLV)-infected cattle.

Cell surface proteins serve as markers for immunophenotypic characterization of lymphocyte subsets by appropriate monoclonal antibodies and fluorescence-activated cell sorter (FACS) analysis. By the same method, internal antigens or those that are only partially expressed on the cell surface can be determined after permeabilization of the cells. Peripheral blood lymphocytes obtained from bovine leukemia virus (BLV)-infected cattle and from BLV-free cattle were permeabilized and several lymphocyte populations were examined. BoCD4, BoCD8 and three CD4 CD8-T-cell subsets retained their original frequencies after permeabilization in both groups of animals. The recognition of the B-B2 lymphocyte molecule was only partially expressed on the cell surface of intact lymphocytes and was further revealed on permeabilization. The frequency of permeabilized, but not intact, cells stained with this mAb was significantly higher for BLV-infected cattle than for BLV-free animals (P = 0.006). Reactivities of an anti-heat shock protein (Hsp) 70 were measured before and after permeabilization of PBLs. Similar increased cell frequencies were obtained for both groups of bovines. These data indicate that flow cytometry studies should be conducted on both permeabilized and intact cells for a better assessment of protein expression on the cell surface, as well as in the cytoplasm.

Detrimental effect of bovine leukemia virus (BLV) on the immunological state of cattle.

Bovine leukemia virus (BLV) is a retrovirus which seems to affect both the humoral and the cellular immune response. Cows affected by enzootic bovine leukemia (EBL) showed a reduction of IgM-producing cells in the spleen and lymph nodes. Experimentally infected calves had lower levels of secretory IgM and a decrease in T lymphocytes in the peripheral blood. The reduction in the amount of T cells was noticed mainly in cells bearing the CD4 markers. BLV-infected animals showed diminished responsiveness to newly encountered antigens. Cows naturally infected by BLV produced Igs with impaired structural or biological reactivity. The primary immune response was shown to be deficient in BLV-infected cows following vaccination with synthetic antigen. A marked shift in the proportion of PBL, especially of the CD5+ subset, was noticed. Peripheral blood mononuclear cells from BLV-infected cows secrete elevated levels of certain cytokines and contain increased levels of cytokine mRNA. High levels of cytokines are also found in the sera of BLV-infected cows compared to non-infected animals. A correlation was found between BLV infection and lack of spontaneous recovery from Trichophyton verrucosum infection. Moreover, some studies ascertained a significant association between the herd BLV infection status and disease incidence. The culling rate was higher and milk production lower in BLV-infected vs. BLV-free herds. It seems that BLV infection affects the immune system of a cow to such an extent that it ceases to be productive enough to be kept and, in most cases, the animal is culled before any symptoms of illness associated with persistent immunodeficiency become apparent.

gamma delta T-lymphocytes and anti-heat shock protein reactivity in bovine leukemia virus infected cattle.

Bovine leukemia virus (BLV) induces a chronic infection in cattle that may result in persistent lymphocytosis (PL) and, sometimes, enzootic bovine leukosis. The cellular and humoral immune responses of the host following infection have been extensively investigated but little is known about the involvement of gamma delta T-cells in BLV pathogenesis. The affluence of these cells in cattle, and particularly in the peripheral blood of young ruminants, may suggest a particular role for them in defense mechanisms. In this study we have examined circulating gamma delta lymphocytes that express workshop clusters 1 (WC1) and 2 (WC2). In healthy cattle the WC1 cell count tends to decrease with age and adult cattle blood has statistically lower numbers (19.0 +/- 6.6%) than that of young animals (40.1 +/- 7.2%). However, in the blood of BLV-seropositive adult cattle and mainly in BLV+ PL+ animals the population of WC1 cells is elevated compared with uninfected animals (P < 0.007). Likewise, the WC2 cells count is increased (P < 0.01) in BLV+PL+. Furthermore, we have investigated whether BLV infection up-regulates the expression of heat shock proteins (HSP) which in turn could augment the humoral response. Anti-HSP70 activity was examined in the sera of 34 BLV-infected cattle and 40 healthy controls by ELISA. Significantly higher activities (P < 0.001) were observed in BLV-infected cattle.

Short-termed expression of interleukin-12 during experimental BLV infection may direct disease progression to persistent lymphocytosis.

In this study an attempt was made to elucidate cellular response cytokine expression upon experimental bovine leukemia virus (BLV) infection in cattle. Progression of infection was monitored by BLV gp51 mRNA expression or DNA amplification by RT-PCR or PCR, respectively, to detect provirus infected cells. Antibodies to BLV were detected by an agar gel immuno-diffusion (AGID) test in 5 weeks and persistent lymphocytosis (PL+) was established in all four BLV-infected animals in 24 weeks after infection. At the initial stage of infection a strong cellular immune response was induced mediated by IL-12p40 mRNA expression. Short-termed IL-12p40 expression was observed in peripheral blood mononuclear cells (PBMC) in two out of four infected animals following 1-3 weeks after infection, while viral mRNA expression was observed 2 weeks following infection. Expression of genes coding for the pro-inflammatory TNFalpha, IL-1beta and cellular response cytokines IFNgamma and IL-2 was detected beginning with the second and third week after infection in all BLV-infected animals. However, IFNgamma expression significantly decreased in 12 weeks after infection in three animals while IL-10 message initially detected 3 weeks after infection increased by 12 weeks and persisted. The observed immediate short-termed cell mediated immune response characterized by IL-12p40 and IFNgamma expression followed by an early shift to an IL-10 induced humoral response, may change the cytokine balance and direct disease progression to the PL+ stage.

Humoral and cellular responses in calves experimentally infected with bovine leukemia virus (BLV).

In order to elucidate whether infection by Bovine Leukemia Virus (BLV) might induce an immunodeficient state, we inoculated sixteen calves with BLV. The calves were followed up for two years and were tested for humoral and cellular responses using various parameters, namely the appearance of antibodies to the BLV antigens, the changes in the numbers of lymphocytes involved, and the ratio between the two main populations of lymphocytes. Antibodies to the BLV antigens were of both the IgG and the IgM classes of immunoglobulins. The levels of antibodies of the IgM class were higher than those of IgG. There was a temporary decrease of reactive antibodies to the BLV antigens, to below detectable levels, during the 14-24 weeks post infection. A significant decrease in the level of plasma IgM was found in all BLV infected calves exhibiting lymphocytosis, while the level of IgG in the plasma of all experimental calves did not diverge significantly from the initial values, throughout the experiment. BLV infection was followed by lymphocytosis of B-cells in most infected calves, which persisted for the whole course of the experiment, while a decrease in the population of T-cells in peripheral blood was observed for a period of several months in all infected calves.

Membrane IgM of bovine lymphocytes.

The membrane immunoglobulins of peripheral blood lymphocytes (PBL) obtained from four bovine-leukemia-virus infected calves and from a normal cow were isolated and characterized. They were found to consist of an IgM exhibiting a mu-chain of an apparent molecular weight (AMW) of 95,000 daltons, which is 10,000 daltons more than found for the mu-chain of serum IgM. It thus seems that this is a property of membrane-bound IgM of bovine origin.