Increased ratio of bcl-2/bax expression is associated with bovine leukemia virus-induced leukemogenesis in cattle

To further investigate the molecular basis underlying the dysregulation of B cell homeostasis associated with bovine leukemia virus disease progression in cattle, bovine bax was cDNA cloned and sequenced. The predicted amino acid sequence of bovine Bax revealed a 192-amino-acid protein having extensive identity with the human (97%), murine (93%), and rat (94%) homologues. Because the ratio of Bcl-2 to Bax is believed to predetermine the susceptibility to a given apoptotic stimulus, the relative expression of the genes encoding these oncoproteins was evaluated in cattle naturally infected with BLV. In BLV-infected cattle an increase in the ratios of bcl-2/bax mRNA and protein expression correlated with advancing stages of disease. These findings suggest that in addition to the maintenance of BLV-associated hematopoietic malignancies, the reciprocal expression of Bcl-2/Bax may modulate the induction of B cell expansion typical of BLV disease progression.     

Function and conformation of wild-type p53 protein are influenced by mutations in bovine leukemia virus-induced B-cell lymphosarcoma

The mutations of the p53 gene previously represented one of several genetic changes involved in the development of bovine leukemia virus (BLV)-induced lymphosarcoma, while the effects of these mutations on the function of p53 are unknown. We identified four mutations of p53 gene in BLV-infected cattle with lymphosarcoma and demonstrated clearly the existence of two functionally distinct groups of mutants: (i) the mutant forms with substitutions at codons 241 and 242, which were mapped within an evolutionally conserved region and corresponded to the human "hot-spot" mutations, had completely lost the capacities for transactivation and growth suppression and gained transdominant repression activity in p53-null SAOS-2 cells; and (ii) the mutations at codons 206 and 207 were located outside the evolutionally conserved regions. These mutants partially retained the capacity for transactivation and growth suppression and failed to inhibit the transactivation activity of coexpressed wild-type p53, instead showing an enhancement of this activity. In addition, protein analysis using an antibody specific for the mutant form revealed that the mutations at codons 206 and 242 induced a "mutant" conformation of the bovine p53 proteins. Collectively, these results show that mutations of p53 gene in BLV-infected cattle with lymphosarcoma can potentially alter its physiological function and may play an important role in BLV-induced leukemogenesis.     

Macrophages infected with bovine leukaemia virus (BLV) induce humoral response in rabbits

BLV is a lymphotropic retrovirus which infects mainly B-cells. However, the possible infection of cells of the monocyte/macrophage lineage (M/M) might explain some aspects of the disease such as latency or disease progression. We infected sheep M/M with BLV either by culturing M/M with supernatant containing virus, or coculturing M/M with persistently infected cell lines. These BLV-infected M/M were inoculated into rabbits and the serological response was followed for two years. ELISA results using adsorbed sera showed a persistent production of specific antibodies from as early as the first week post inoculation. Two tests were used to detect the response against envelope glycoprotein gp51: Agar gel immunodiffusion (AGID) and a virus neutralization test read as syncytia inhibition (SI). Sera were positive by AGID after the second or third inoculation. Neutralizing titres (SI) were higher than those seen in control rabbits inoculated with persistently infected cell lines, suggesting that the virus may be expressed better in M/M. Gag-related proteins were analyzed by Western Blot (WB). Sera from rabbits inoculated with BLV-infected M/M recognized as many viral proteins as sera from BLV immunized control rabbits or infected cows, and this profile did not change with repeated inoculations. All these results suggest that BLV may infect M/M, where viral proteins are actively expressed to the point that they induce a humoral immune response in animals, and that animals get persistently infected.     

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.     

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.     

Infectious, linear, unintegrated DNA of Moloney murine leukemia virus

A closed circular, double-stranded infectious DNA of Moloney leukemia virus has been described previously. The present report characterizes a second type of infectious, unintegrated viral DNA which is linear, largely double stranded, and of mass comparable to that of the closed circular viral DNA. The linear form is of nonpermuted sequence, and SalI endonuclease cleaves at one site 45% from one end.     

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.     

Lymphocyte activators elicit bovine leukemia virus expression differently as asymptomatic infection progresses

Since bovine leukemia virus (BLV) replicates in B-lymphocytes, viral expression and production should respond to agents activating these cells. We asked whether synthesis of BLV capsid (CA) protein and production of infectious virus would increase when peripheral blood mononuclear cells (PBMCs) from infected sheep were stimulated in short-term culture with lipopolysaccharide (LPS), a polyclonal activator of B-cells, and compared its effects with those of phytohemagglutinin (PHA), a lymphocyte activator known to increase BLV expression. LPS treatment of PBMCs from asymptomatic sheep that had been infected for 1-4 years increased the number of cells synthesizing CA protein, the amount of CA protein per cell, and the number of PBMCs acting as infectious centers. LPS derived from several different microbes was effective. During the ensuing 4 years of asymptomatic infection, the number of PBMCs expressing virus under minimal stimulation increased for each animal. The ability of LPS to recruit additional cells to express CA protein remained constant or decreased in magnitude, yet at the same time, lower concentrations of LPS were required to elicit a maximal effect. This suggests that the cellular pathways affected by LPS are endogenously more activated as infection progresses. PHA initially stimulated fewer cells to synthesize BLV CA protein than LPS did although the amount of CA protein synthesized per cell was greater with PHA. As infection progressed, PHA surpassed LPS in the numbers of PBMCs induced to express CA protein. This suggests that the cellular pathways affected by PHA become more responsive to its effects as infection progresses. LPS increased CA expression early and transiently during culture whereas the PHA-mediated increase continued to develop for several days. Thus, LPS increases BLV expression but does so differently than PHA. Moreover, these longitudinal results show that the activation state of BLV-infected cells changes as asymptomatic infection progresses.     

Esophageal intramural pseudodiverticulosis

Cows that develop a persistent lymphocytosis (PL) as a result of bovine leukemia virus (BLV) infection develop massive proliferation of B-lymphocytes expressing both IgM and CD5 markers. The association of these two markers on peripheral blood mononuclear cells (PBMC) derived from BLV-infected cows and also expressing BLV-gp51 antigen marker on these cells was determined by three-color cytometric analysis. After in vitro cultivation of PBMC in the presence of PHA for 24 h, the mean percentages of marker-reactive cells of five PL+ cows were as follows; 43% +/- 4.5 of the PBMC expressed BLV-gp51 antigen; 90% +/- 1.6 of these cells expressed both IgM and CD5 at the same time, whereas but 7.5% +/- 1.9 expressed only IgM and 2.9% +/- 0.4 expressed only CD5. The PBMC, IgM positive cells accounted for 77.8% +/- 6.8, while both CD5 and BLV-gp51 were detected simultaneously on 52.0% +/- 2.4 of the IgM+ cells, while the CD5 marker and BLV-gp51 antigen were detected independently on 35.0% +/- 1.9 and in 9.0% +/- 3.1, respectively of the IgM+ cells. Of the CD5+ cells (equivalent to 75.5% +/- 9.0 of the PBMC), 54.7% +/- 4.7 expressed simultaneously IgM and BLV-gp51, while BLV-gp51 and IgM were expressed separately by 3.0% +/- 0.5 and 37.8% +/- 3.3, respectively. An association between the B-cell phenotype and BLV tropism might exist. It is also possible that cells bearing both IgM and CD5 markers are the main target cells for BLV infection.     

The morbidity of insomnia uncomplicated by psychiatric disorders

Efficient superinfection of H9HTLVIIIB cell line (persistently infected with HIVHXB2 strain) with HIVMN strain is reported. The superinfecting viral DNA was found in the chromosomic and extrachromosomic fractions at early stages, but at 48 hours post superinfection, it remained mainly unintegrated. Interestingly, superinfected cells only produced HIVHXB2 in the supernatant and no increase of viral yield of this persistent virus was observed. Remarkably, virions of both strains. HIVHXB2 and HIVMN, were recovered after cocultivating superinfected cells with MT2 cell line. In the extrachromosomic fractions of seven different superinfected subclons of H9HTLVIIIB, viral DNA of the superinfecting HIVMN strain predominated while in the chromosomic fraction, the proportion of superinfecting viral DNA differed. The study of the presence of different integrated and unintegrated genomes in a single cell could be crucial in the understanding of HIV biology.     

Antitumor effect of diphtheria toxin A-chain gene-containing cationic liposomes conjugated with monoclonal antibody directed to tumor-associated antigen of bovine leukemia cells

Monoclonal antibody c143 against tumor-associated antigen (TAA) expressed on bovine leukemia cells was conjugated to cationic liposomes carrying a plasmid pLTR-DT which contained a gene for diphtheria toxin A-chain (DT-A) under the control of the long terminal repeat (LTR) of bovine leukemia virus (BLV) in the multicloning site of pUC-18. The specificity and antitumor effects of the conjugates were examined in vitro and in vivo using TAA-positive bovine B-cell lymphoma line as the target tumor. In vitro studies with the TAA-positive cell line indicated that luciferase gene-containing cationic liposomes associated with the c143 anti-TAA monoclonal antibody caused about 2-fold increase in luciferase activity compared with cationic liposomes having no antibody, and also that the c143-conjugated cationic liposomes containing pLTR-DT exerted selective growth-inhibitory effects on the TAA-positive B-cell line. Three injections of pLTR-DT-containing cationic liposomes coupled with c143 into tumor-bearing nude mice resulted in significant inhibition of the tumor growth. The antitumor potency of the c143-conjugated cationic liposomes containing pLTR-DT was far greater than that of normal mouse IgG-coupled cationic liposomes containing pLTR-DT as assessed in terms of tumor size. These results suggest that cationic liposomes bearing c143 are an efficient transfection reagent for BLV-infected B-cells lymphoma cells, and that the delivery of the pLTR-DT gene into BLV-infected B-cells by the use of such liposomes may become a useful technique for gene therapy of bovine leukosis.     

Viral antibodies in bovine fetuses in Argentina

In order to establish the prevalence of viral infections of the bovine fetus in Argentina, a serological survey for antibodies against viral agents currently affecting cattle in this country was conducted. Antibodies against foot-and-mouth disease virus (FMDV), bovine herpesvirus-1 (BHV-1), bovine leukaemia virus (BLV), bovine rotavirus (BRV), bovine coronavirus (BCV), bovine viral diarrhoea virus (BVDV) and parainfluenza-3 (PI-3) were investigated in a total of 315 fetal serum samples. Conventional techniques were used: indirect immunofluorescence (FMDV, BHV-1, BVDV and BCV), radial immunodiffusion (BLV), ELISA (BRV) and haemagglutination inhibition (PI-3). Antibodies against BHV-1, BVDV and PI-3 were detected in samples from fetuses in the second and third trimester of gestation, with a prevalence of 1.21 per cent (two of 165), 2.03 per cent (four of 197) and 5.08 per cent (nine of 177), respectively. Either antibodies or non-antibody factors able to bind to BRV and BCV antigens were detected with a prevalence of 2.44 per cent (five of 205) and 4.54 per cent (five of 110), respectively. In addition, 14.68 per cent of non-specific inhibitors of PI-3 mediated haemagglutination were found. No seropositives against FMDV and BLV were detected.     

Circular forms of unintegrated human immunodeficiency virus type 1 DNA and high levels of viral protein expression: association with dementia and multinucleated giant cells in the brains of patients with AIDS

Thirty-one histologically abnormal brains from patients with AIDS were studied in order to establish the relationship between multinucleated giant cells, viral protein expression, the various forms of human immunodeficiency virus type 1 (HIV-1) DNA, and clinical evidence of dementia. Unintegrated HIV-1 DNA of 2 to 8 kb was found in 22 of the 31 brains. Multinucleated giant cells without any other pathology were found in 14 cases; unintegrated 1-long terminal repeat (1-LTR) circular forms of HIV-1 DNA and strongly positive immunohistochemistry for gp41 and p24 were found in most of these brains. Most of these patients had a clinical diagnosis of HIV-1-associated dementia and cerebral atrophy. In all the other brains studied, 1-LTR circles were absent and immunohistochemistry for gp41 and p24 was usually negative. Very few of these patients had a clinical diagnosis of dementia. Sequence comparison of the LTR region from integrated HIV-1 DNA with that from unintegrated 1-LTR circular forms of HIV-1 DNA in 12 cases showed no significant differences. A further comparison of these brain-derived LTR sequences with LTR sequences derived directly from lymphoid tissue also showed strong sequence conservation. The V3 loop of the virus from the brain was sequenced in 6 cases and had a non-syncytium inducing-macrophage-tropic genotype. Our results show that (i) although unintegrated HIV-1 DNA was present in most brains from patients with AIDS, molecular evidence of high levels of viral replication was associated with the presence of multinucleated giant cells and dementia, and that (ii) the HIV-1 LTR is not a determinant of neurotropism. These observations suggest that replication of HIV-1 and not just the presence of HIV-1 DNA within giant cells makes the important contribution to central nervous system damage.