Altered apoptosis and proliferation in milk cells and PBMc from BLV-infected bovines with different proviral loads: Possible role of the BCL-2 family proteins, TNF-alpha, and receptors.

Bovines infected by bovine leukemia virus (BLV) are characterized by presenting low proviral load (LPL) or high proviral load (HPL). It is reported that animals with HPL in peripheral blood mononuclear cells (PBMCs) present a decrease in apoptosis, an increase in viability and the proliferation rate, while animals that maintain an LPL have an intrinsic ability to control the infection, presenting an increased apoptosis rate of their PBMCs. However, there is little information on the effect of BLV on these mechanisms when the virus infects somatic milk cells (SC). This study investigates the mechanisms underlying apoptosis in milk and blood from BLV-infected animals with HPL and LPL. Relative levels of mRNA of tumor necrosis factor-α (TNF-α), TNF receptor 1 (TNF-RI), TNF receptor 2 (TNF-RII), anti-apoptotic B-cell lymphoma 2 protein (Bcl-2), and pro-apoptotic Bcl-2-like protein 4 (Bax) were measured in SC and PBMCs using quantitative reverse transcription-polymerase chain reaction (RT-qPCR) assay. A significant decrease in the expression of TNF-α in SC from HPL animals vs non-infected bovines was observed, but the infection in SC with BLV did not show a modulation on the expression of TNF receptors. A significant increase in TNF-RI expression in PBMCs from HPL bovines compared to LPL bovines was observed. No significant differences in PBMCs between HPL and LPL compared to non-infected animals concerning TNF-α, TNF-RI, and TNF-RII expression were found. There was a significant increase of both Bcl-2 and Bax in SC from LPL compared to non-infected bovines, but the Bcl-2/Bax ratio showed an anti-apoptotic profile in LPL and HPL bovines compared to non-infected ones. Reduced mRNA expression levels of Bax were determined in the PBMCs from HPL compared to LPL subjects. In contrast, BLV-infected bovines did not differ significantly in the mRNA expression of Bax compared to non-infected bovines. Our data suggest that the increased mRNA expression of Bax corresponds to the late lactation state of bovine evaluated and the exacerbated increase of mRNA expression of Bcl-2 may be one of the mechanisms for the negative apoptosis regulation in the mammary gland induced by BLV infection. These results provide new insights into the mechanism of mammary cell death in HPL and LPL BLV-infected bovine mammary gland cells during lactation.

Modulation of cathelicidins, IFNß and TNFα by bovine alpha-herpesviruses is dependent on the stage of the infectious cycle.

Production of antimicrobial peptides cathelicidins, interferons and cytokines is an important feature in airway epithelial host defense. The innate immune response to alpha-herpesvirus infection at the sites of primary replication has not been fully studied. Thus, the aim of this study was to determine the expression of innate immune components, cathelicidins, IFNß, TNFα and TNF receptors (TNFRI and TNFRII) during acute infection and reactivation of bovine herpesvirus type 1 (BoHV-1) and 5 (BoHV-5) in the respiratory tract and lymphoid tissue of their natural host. We found that BoHV infection modulates mainly the expression of BMAP28, a key cathelicidin in cattle. It was downregulated by both viruses in retropharyngeal lymph nodes of acutely infected-calves, and it was accompanied by a lower expression of IFNß, TNFα and TNFRI. BoHV-5 showed a pronounced role in the downregulation of BMAP28, even in nasal mucosa and lung. However, during reactivation, BoHV-5 upregulated both BMAP28 and IFNß in retropharyngeal lymph nodes. Acute replication induced also TNFα mRNA and protein synthesis, and expression of TNFRI and II was positively regulated during both acute infection and reactivation, particularly in the trachea. Moreover, BMAP27 was detected during BoHV-1 reactivation suggesting a potential role at this stage. Thus, cathelicidins are implicated in alpha-herpesvirus infections of the bovine respiratory system and the response is distinct during BoHV-1 and BoHV-5 acute infection and reactivation. This demonstrates that these viruses modulate differentially the components of innate immune response, possibly influencing their pathogenesis. This study provides an initial pilot analysis of factors that might be implicated in alpha-herpesvirus infection of the bovine respiratory system.

Rapamycin increases the cellular concentration of the BCL-2 protein and exerts an anti-apoptotic effect.

The immunosuppressant rapamycin, an immunophilin-binding antibiotic, has been studied in follicular B-cell lymphoma lines that express the highest level of the BCL-2 protein. The growth rate of human follicular B-cell lymphoma lines was slowed more efficiently than that of other human B-cell lines or non-B-cell lines. This effect was dependent on the arrest of cells in the G(1) phase; the number of apoptotic cells was not increased. Rapamycin inhibited apoptosis or caspase activation induced by cytotoxic drugs, whereas caspase activation by doxorubicin was not inhibited. The increase in the cellular concentration of BCL-2 protein was related to its concentration in the steady state and was unrelated to the amount of bcl-2 mRNA. The increase of BCL-2 level in the cells rather than its level in the steady state may be important for drug resistance. The biochemical target of rapamycin, the mTOR kinase, may be a candidate sensitising agent for chemotherapy. This effect of rapamycin shows that G(1) arrest and protection from apoptosis are combined events susceptible to regulation by pharmacological means.

BCL-2 regulation targeting the AU-rich domain.

In an effort to identify potential upregulators of bcl-2 activity in t(14;18) follicular B lymphoma cells, we detected a hybrid bcl-2/IgH RNA transcribed in antisense orientation. This antisense transcript may contribute to upregulation of bcl-2 expression in t(14;18) cells, overlapping AU-rich motifs present in the 3'-untranslated region of bcl-2 mRNA. We have studied the enzymatic efficiency of a ribozyme directed towards the bcl-2 AU-rich region in a cell-free system determining its kinetic parameters.