The down-regulation of CD4 by cultured monocytes has been observed by our group and by other investigators. Flow cytometric analysis was performed to elucidate factors influencing this phenomenon. The addition of LPS, GM-CSF, M-CSF or IL-10 to monocyte cultures failed to inhibit the decrease in monocyte CD4 expression following overnight culture. The down-regulation was an adherence-independent phenomenon since it was not prevented by culture in Teflon vials. The type of anticoagulant into which the peripheral blood was collected did not appear to be a factor, The presence or absence of lymphocytes within the cultures was also inconsequential. The continued culture of monocytes in a whole blood environment resulted in decreased CD4 down-regulation. Experiments in which CD4 was tagged with alpha-CD4-PE mAb prior to cell culture revealed that the down-regulation observed was the result of CD4 internalization. In an attempt to further understand monocyte CD4 function, we investigated the role of monocyte CD4 in signal transduction. Stimulation of Thp-1 monocytic cells with antibody to CD4 resulted in a Ca2+ flux, as well as in the time-dependent tyrosine phosphorylation of various proteins having molecular weights of approximately 180, 140, 120, 110, 85, 65, 55, 50 and 35 kDa. We identified the 140 and 85 kDa proteins as PLC-gamma1, and the regulatory subunit of PI3-K, respectively. Using immunoprecipitation/Western immunoblotting, we were unable, however, to show any direct association between CD4 and PLC-gamma1, PI3-K, or other signaling proteins. In an attempt to identify proteins capable of associating with the cytoplasmic tail of CD4, we generated a GST-CD4cyt fusion protein for use in far Western blots and immunoprecipitation experiments. In both types of experiments, the GST-CD4cyt fusion protein routinely associated with 45 and 55 kDa proteins. In the immunoprecipitation experiments, a 35 kDa protein was also observed. The above results suggest that the expression of monocytic CD4 is regulated during the differentiation process. Furthermore, the cytoplasmic tail of monocytic CD4 is associated with various proteins which we postulate function in signal transduction, and which may also play a role in CD4 down-regulation.

A human adenovirus type 5-rabies recombinant vaccine(Ad5RG) is a potential candidate for the oral immunization of skunks, foxes, and raccoons against rabies. Adenovirus seroprevalence studies in these species were required before using the vaccine in the field, as part of an environmental risk assessment and for determining if a barrier to immunization exists. For this purpose, two competitive enzyme-linked immunosorbent assays (cELISAs) measuring antibodies (Ab) against Ad5 and canine adenovirus type one (CAV-1) were developed and validated. Sera were examined for either Ad5 or CAV-1 neutralizing antibodies using plaque reduction neutralization tests (PRNT). The Ad5 cELISA was 80% sensitive and 75% specific when an 8% inhibition positivity cutoff was, used. The CAV-1 cELISA was 83% sensitive and 96% specific when a 17% inhibition positivity cut off was used. Contributing to the low Ad5 specificity in particular were raccoons that generally lacked Ad5 neutralizing activity but had Ab which bound purified Ad5 in the cELISA and Western blot. (Abstract shortened by UMI.)

The prevention and control of new and existing hepatitis B virus (HBV) infections remains an important global health concern. Despite current global immunization efforts, new HBV infection continues to occur. It is estimated that about 350 million people worldwide are currently afflicted by viral persistence. This body of work explores the possibility of developing improved prophylactic and possibly therapeutic vaccines against HBV using DNA technology. It has become well established that the effective control of HBV infection requires the development of strong humoral and cell mediated immunity (CMI) including the production of the pro-inflammatory cytokines interferon (IFN) gamma and tumor necrosis factor (TNF) alpha that are associated with a CMI response. In the absence of these responses infection is not resolved and persistence ensues. The ability of DNA (either in the form of a DNA vaccine or as immunostimulatory DNA sequences in combination with protein antigens) to stimulate both humoral and cellular immune responses that include the production of IFNgamma and TNFalpha, makes them attractive candidates for development of novel prophylactic and therapeutic agents in the struggle against HBV infection. This work demonstrates that immunization strategies that include DNA technology were capable of controlling HBV gene expression in a hepatitis B surface antigen transgenic mouse model as well as provide protection against infectious HBV challenge in chimpanzees. The quality of the immune responses induced in the chimpanzees suggests that the therapeutic potential of these immunization strategies observed in the mouse model may also extend to higher primates.

Infection of immune cells with human immunodeficiency virus (HIV) induces dysregulation of cytokines which may play a vital role in HIV pathogenesis. I analyzed the expression of Th1 (interferon-$\gamma,$ (IFN-$\gamma)\rbrack$ and Th2 (interleukin-4 (IL-4), IL-10) type cytokines in unstimulated and mitogen stimulated peripheral blood mononuclear cells (PBMC) from HIV seropositive (HIV$\sp+)$ patients. It was determined that IFN-$\gamma$ mRNA in unstimulated PBMC was significantly decreased and IL-10 mRNA as well as IL-10 protein was significantly increased in patients with $$400 CD4$\sp+$ T cells/mm$\sp3$ (n = 6) and normal controls (n = 16). Mitogen stimulation of PBMC revealed two groups of HIV$\sp+,$ low and normal IL-10 producers. Production of IL-4 was reduced in HIV$\sp+$ individuals with $$400 CD4$\sp+$ T cells/mm$\sp3.$ However, ability to produce IFN-y by mitogen stimulated PBMC and CD4 T$\sp+$ cells was not impaired in HIV$\sp+$ individuals. These results suggest that PBMC of HIV$\sp+$ exhibit dysregulation of Th2 type cytokines which may play a role in HIV immunopathogenesis. In the next set of experiments, the IL-10 production was correlated with the levels of proliferative responses to recall antigens. Low IL-10 producers proliferated in response to recall antigens, and demonstrated enhanced recall antigen-induced proliferation upon addition of anti-IL-10 antibodies and/or IL-12. Conversely, normal IL-10 producers had PBMC that failed to proliferate to recall antigens, and did not demonstrate enhanced recall antigen-induced proliferation upon addition of anti-IL-10 antibodies and/or IL-12. Source of the IL-10 production in PBMC of HIV$\sp+$ individuals was shown to be monocytes, while, in HIV controls, it was produced by both T cells and monocytes. The molecular mechanisms underlying the production of IL-10 are not clear. I have demonstrated that monocytes/macrophages are required for IL-10 production by normal activated T cells. IL-10 production was significantly downregulated in both T cell and monocyte depleted PBMC compared to undepleted PBMC, and IL-10 production could be restored following addition of monocyte conditioned medium (MCM), this suggested that IL-10 production by T cells is regulated by monokine(s) produced by activated monocytes. The monokine(s) responsible for IL-10 induction by T cells were further studied. Addition of IL-6 and IL-12 enhanced IL-10 production in monocyte depleted PBMC in a dose dependent and additive manner. With respect to regulation of IL-10 produced by monocytes, tumor necrosis factor $\alpha$ (TNF-$\alpha)$ was found to induce IL-10 production by resting purified monocytes. Taken together, these findings suggest that IL-10 production by human T cells and monocytes is differentially regulated. IL-12 and IL-6 induce the expression of IL-10 by PHA stimulated T cells whereas TNF-$\alpha$ induces IL-10 production by monocytes. Since IL-10 inhibits production of IL-6, IL-12 and TNF-$\alpha,$ these results may indicate a potential mechanism of negative feedback regulation of the immune system. Furthermore, mitogen stimulated PBMC from HIV$\sp+$ individuals produced significantly lower levels of IL-12 than did those from HIV-controls. A defect in IL-12 induction may partially cause IL-10 dysregulation in HIV infection.

I used the hypoxanthine guanine phosphoribosyl transferase (hprt) clonal assay to determine the in vivo frequency of mutant T cells (FMC) from the peripheral blood, synovial fluid, and synovial tissue of rheumatoid arthritis (RA) patients and controls. The results demonstrate that there is an increased FMC in the peripheral blood of RA patients compared to controls. There was also a significant elevation in the corrected FMC (cFMC), which takes in to account the cloning efficiency of the T cells, in the peripheral blood of RA patients compared to controls. There is an elevated FMC and cFMC in synovial fluid of RA patients compared to the peripheral blood of controls. However. the FMC and cFMC from the peripheral blood of unselected RA patients from the outpatient clinic is not significantly different than from the synovial fluid of RA patients suggesting that the synovial fluid does not contain the necessary mitogenic and mutagenic factors to induce T cell genetic damage. The FMC and cFMC from RA and osteoarthritis (OA) synovium is approximately ten fold greater than the FMC and cFMC from the peripheral blood of the same patients which suggests that the mitogenic and genotoxic environment of the inflamed synovium is capable of inducing T cell mutations. There was no significant difference in the cloning efficiency of T cells (CE), FMC, and cFMC from the peripheral blood of RA patients with 'active' or 'inactive' disease. No correlation between the cFMC from the peripheral blood of RA patients and clinical disease parameters and patient medication was found. (Abstract shortened by UMI.)

T cell activation and the generation of effective immune responses is critically dependent on APC-derived signalling. The relative expression of B7 isoforms on APC may be important in determining the nature and extent of the immune response, and immunoregulatory cytokines may mediate their effects through alterations in B7 isoform expression. The effects of a panel of cytokines on B7 isoform expression on resting and activated monocytes and B cells was evaluated. IL10 and IL4, which induce the development of Th2 type T cells, downregulated expression of B7-2 and modestly upregulated expression of B7-1 on unstimulated human monocytes. IFN$\gamma,$ a potent inducer of Th1 type T cells, upregulated both B7-1 and B7-2 expression. TNF$\alpha$ downregulated B7-2 expression, but did not alter B7-1 expression. Addition of anti-IL10 antibodies did not abrogate the effects of TNF$\alpha$ on B7-2 expression. LPS had effects on B7 isoform expression on purified monocytes similar to those of IL10 in PBMC, namely marked B7-2 downregulation and modest B7-1 upregulation. None of the cytokines influenced the levels of expression of B7 isoforms on either resting or activated B cells. Thus cytokines that influence development of T helper type immune responses have differential effects on expression of individual B7 isoform on monocytes but not on B cells. These findings may have important implications in activation and control of immune responses in infections, autoimmunity and malignancies.

Abstract not available.

Abstract not available.

This study shows that HSV-2 can cause down-regulation in IFN-gammaR expression on the monocyte surface in both HSV-2 seropositive and seronegative patients. The objective of this work is to examine and explain the mechanisms involved in the viral down-regulation of IFN-gammaR. The first question I tried to answer was whether humoral factors might participate in this down-regulation. Humoral factors were not involved in this phenomenon. Next, cell-to-cell interactions were examined. T, B or natural killer cell depletion experiments were conducted in peripheral blood mononuclear cells of both HSV2 seropositive and seronegative patients. The results suggest that NK cells and T cells but not B cells were involved in the IFN-gammaR downregulation in HSV-2 seropositive patients. In addition, purified monocytes also demonstrated IFN-gammaR down-regulation after HSV-2 exposure in seropositive patients. I concluded that, in HSV-2 seropositive patients, NK cells may have an inhibitory effect and T cells may have a facilitatory role in the down-regulation of IFN-gammaR. (Abstract shortened by UMI.)

CD8+ T cells secrete CD8 Antiviral Factor (CAF), an unidentified soluble factor that inhibits replication of HIV-1 by suppression of viral transcription. To date, the effect of Highly Active Antiretroviral Treatment (HAART) on CAF production in HIV+ individuals is inconclusive. Therefore, we have examined CAF production by CD8+ T cells of HIV-positive patients on various treatment regimens, using a transcription-based assay. We found that HAART does not significantly modulate CAF compared to untreated controls, but NNRTI treatment may increase CAF in some patients, as this group demonstrated a higher proportion of effective suppressors of viral gene expression. These trends were not seen at the level of viral replication, which shows that determination of CAF at the level of virus replication does not mirror results found using a transcription-based assay. These results suggest that immunological changes induced by NNRTI-based therapy may affect the ability of CD8+ T cells to produce CAF.