Treatment of murine cytomegalovirus salivary-gland infection by combined therapy with ganciclovir and thymic humoral factor gamma 2.

An optimal therapeutic regimen against primary CMV salivary-gland infection has not yet been developed. We used a murine CMV (MCMV) model system to assess the ability of combined thymic humoral factor THF-gamma 2 immunotherapy and ganciclovir (GCV) antiviral chemotherapy to eliminate detectable viral DNA from salivary glands of infected animals. Mice in different experimental groups were inoculated intraperitoneally with MCMV, treated, and then sacrificed either 2 weeks or 3 months later. To amplify and detect MCMV DNA in infected salivary-gland tissue, we developed a sensitive polymerase chain reaction (PCR) using a glycoprotein B gene primer pair that amplifies a 356 bp segment. During the acute phase of the infection, the detection of high titers of infectious virus in the salivary glands correlated with a strong PCR amplification signal. Although active virions could not be recovered from untreated animals 3 months after viral inoculation, the PCR assay detected a latent MCMV genome. Treatment with either GCV alone or THF-gamma 2 alone had little or no effect on the presence of MCMV DNA. By contrast, combined treatment with THF-gamma 2 and GCV significantly reduced the amount of salivary-gland MCMV DNA to below the limit of PCR detection. The results presented here, and experimental data from previous MCMV research in our laboratories, imply that elimination of the virus from the salivary glands could be due in part to THF-gamma 2 restoration of the various MCMV-suppressed, cell mediated immune-responses. Combining THF-gamma 2 immunotherapy and GCV antiviral chemotherapy may be an important step toward an effective therapeutic regimen that has the potential to prevent the establishment of viral latency ensuing from primary MCMV salivary-gland infection.

Tyrosine phosphorylation of measles virus P-phosphoprotein in persistently infected neuroblastoma cells.

Replication and encapsidation of measles virus (MV) requires the interaction between the nuclear protein (N) and the phosphoprotein (P). It is known that both proteins are phosphorylated on serine and threonine residues. Recently we have shown that N is phosphorylated on tyrosine in persistently-infected mouse neuroblastoma cells (NS20Y/MS). Here, we show that P in NS20Y/MS is also phosphorylated on tyrosine. To investigate whether cellular tyrosine kinases can bind and phosphorylate P, a solid phase kinase assay was employed. We show that bacterially-expressed MV P fragments, were phosphorylated on tyrosine by purified mouse c-Src protein-tyrosine kinase and when mixed with uninfected neuroblastoma cell (NS20Y) extracts, these P fragments were phosphorylated on tyrosine in addition to serine and threonine. These results imply that MV P is a substrate for tyrosine phosphorylation by cellular tyrosine kinase(s).

Novel therapeutic strategies against cytomegalovirus infection.

Murine CMV (MCMV) presents a model for the study of the role of the immune system in the pathogenesis of human CMV (HCMV) infection. MCMV causes T cell immune impairment in the infected mice, manifested by suppressed responses to T cell mitogens and a profound reduction of Con A induced IL-2 production. Thymic humoral factor (THF-gamma 2) is an octapeptide which was first isolated from calf thymus, characterized and chemically synthesized. This peptide has been shown to have immunoregulatory effects in various systems. Systemic treatment of MCMV-infected mice with THF-gamma 2 resulted in the enhancement of protective efficacy of MCMV immune spleen cells and the reconstitution of mitogenic responses and IL-2 secretion.

Restoration of murine cytomegalovirus (MCMV) induced myelosuppression by AS101.

Infection with cytomegalovirus (CMV) continues to be one of the most common complications following allogeneic bone marrow transplantation. The gravest danger for the host occurs when the virus is reactivated as a result of immunosuppression. In this report we studied the effects of sublethal murine cytomegalovirus (MCMV) infection on the hemopoietic system including bone marrow (BM) cellularity, production of colony stimulating factor (CSF) and interleukin-6 (IL-6) and the development of granulocyte-macrophage colony forming units (CFU-GM), and BM stromal cell viability. Our findings show that the virus infection led to a significant decrease in the number of BM cells and in the production levels of CSF and IL-6. There was also a decrease in the number of stromal cells, as reflected by the number of colony forming unit fibroblasts (CFU-F), and in the relative number of CFU-GM progenitors. Treatment of MCMV infected mice with the immunomodulator AS101 [ammonium trichloro (dioxyethylene 0-0')tellurate] restored significantly CSF and IL-6 production by BM cells to levels of uninfected control mice as well as the number of CFU-F and stromal cell elements which consequently led to the restoration of the total number of BM cells. Results presented here indicate that AS101 may have immunomodulatory effects on MCMV mediated myelosuppression. Administration of AS101 to patients with CMV associated BM damage may improve the restoration of their BM function.

Thymic humoral factor, THF-gamma 2, enhances immunotherapy of murine cytomegalovirus (MCMV) infection by both CD4+ and CD8+ immune T cells.

Infection of mice with murine cytomegalovirus (CMV) presents a model for the study of the role of the immune system in the pathogenesis of human CMV. The contribution of the different spleen cell subsets in conferring curative immunocytotherapy to fatally MCMV-infected immunosuppressed mice was assessed using adoptive immunotherapy. It was found that the efficacy of passively transferred immune spleen cells is dose dependent and that the therapeutic effect can be enhanced considerably by treating donor mice with thymic humoral factor (THF-gamma 2). Polymerase chain reaction (PCR) of the donor spleen population was negative, indicating that no MCMV-DNA was transferred with the immune cells. Analysis of the donor mice after THF-gamma 2 treatment showed increased levels of CMV-neutralizing antibodies, while enhancement of natural killer (NK) activity was transient and lasted only during the early phase of the infection. FACS analysis demonstrated that treatment with THF-gamma 2 restored the size of both cell subsets CD4+ and CD8+ that were decreased following MCMV infection. It is shown that both CD4+ and CD8+ T-cell subsets participate in controlling the development of the fatal disease in MCMV-infected immunosuppressed recipients. It is suggested that the enhancement of the immunocompetence of both populations of spleen cells from treated donors is mediated in part by the restoration of Interleukin-2 (IL-2) production by THF-gamma 2.