Anti-hepatitis C virus activity and toxicity of type III phosphatidylinositol-4-kinase beta inhibitors.

Type III phosphatidylinositol-4-kinase beta (PI4KIIIß) was previously implicated in hepatitis C virus (HCV) replication by small interfering RNA (siRNA) depletion and was therefore proposed as a novel cellular target for the treatment of hepatitis C. Medicinal chemistry efforts identified highly selective PI4KIIIß inhibitors that potently inhibited the replication of genotype 1a and 1b HCV replicons and genotype 2a virus in vitro. Replicon cells required more than 5 weeks to reach low levels of 3- to 5-fold resistance, suggesting a high resistance barrier to these cellular targets. Extensive in vitro profiling of the compounds revealed a role of PI4KIIIß in lymphocyte proliferation. Previously proposed functions of PI4KIIIß in insulin secretion and the regulation of several ion channels were not perturbed with these inhibitors. Moreover, PI4KIIIß inhibitors were not generally cytotoxic as demonstrated across hundreds of cell lines and primary cells. However, an unexpected antiproliferative effect in lymphocytes precluded their further development for the treatment of hepatitis C.

Protective immune responses elicited in mice by immunization with formulations of poly(lactide-co-glycolide) microparticles.

Parenteral administration of microparticle encapsulated DNA elicits immune responses to the encoded antigens. Experiments were performed to test whether the addition of certain lipophilic agents to such formulations enhanced the activity of a beta-galactosidase (beta-gal) DNA vaccine. Addition of either taurocholic acid (TA) or monomethoxy polyethylene-glycol-distearoylphosphatidylehanolamine (PEG-DSPE) increased the efficiency of DNA encapsulation. Immunization of mice with encapsulated DNA formulations containing either compound significantly increased the number of antibody positive responders over that achieved with non-lipid containing particles. Moreover, responding animals demonstrated trends towards higher antibody titers and increased T cell responses. Tumor protection against the CT26.CL25 tumor cell line was demonstrated with lipid and non-lipid containing formulations. These results are the first demonstration of protection obtained by parenteral administration of PLG encapsulated DNA vaccines.

Biological potency of microsphere encapsulated plasmid DNA.

We evaluated the utility of three in vitro methods to monitor the biological potency of PLGA encapsulated DNA. For each assay we also determined whether the biological activity was influenced by the structural profile of DNA isomers. Collectively, the results indicate that all three methods can be used to evaluate the biological activity of DNA extracted from PLGA microspheres, but they are differentially sensitive to the structural changes of plasmid DNA that can occur during microencapsulation and microsphere storage. More specifically, mammalian cell transfection followed by an enzyme assay affords an accurate determination of DNA potency over time and is less influenced by DNA isoform than bacterial transformation. Cell-free transcription/translation systems can also be utilized, and the results of this assay are influenced by DNA isoform. Finally, bacterial transformation was found to be more sensitive to DNA isoform than the other assay methods.

Tissue distribution and persistence in mice of plasmid DNA encapsulated in a PLGA-based microsphere delivery vehicle.

Information regarding the distribution and persistence of DNA encapsulated in poly-(lactide co-glycolide) microspheres was collected to provide additional information regarding the safety of DNA vaccines and to support the clinical testing of this new delivery system for DNA. Plasmid DNA was encapsulated in poly(lactide co-glycolide) microspheres and the distribution and persistence of plasmid in murine tissues resulting from parenteral administration were examined by a sensitive PCR assay. Encapsulated DNA delivered by intramuscular or subcutaneous injection can be detected for 100 days post-injection and is distributed primarily at the site of injection and the lymphoid organs. Intravenous administration results in more widespread dissemination with long term persistence limited to the lymphoid organs and those of the reticuloendothelial system. Specific cellular uptake of DNA by professional antigen presenting cells (APCs) following injection suggests the utility of microspheres as DNA delivery agents. Distribution and persistence studies support the safety of encapsulated DNA and the specific cellular uptake of DNA by professional APCs following injection suggests the utility of microspheres as DNA delivery agents.

The immune response to soluble D10 TCR: analysis of antibody and T cell responses.

In order to evaluate the potential of TCR as vaccines for immunomodulation, the immunogenicity of soluble versions of D10 TCR has been investigated in mice. Soluble D10 TCR containing the extracellular domains were produced either as dual chain (dc) TCR lacking transmembrane and cytoplasmic regions or as a TCR-IgG1 chimeric protein. Soluble single chain (sc) D10 TCR contained only the Valpha and Vbeta segments joined by a peptide linker. Syngeneic D10 dcTCR or D10 TCR-IgG1 immunizations of AKR mice induced antibody responses to D10 clonotypic epitopes and to constant region epitopes that are not exposed on D10 cells. Only clonotypic antibodies were produced after D10 scTCR immunizations. Immunization of AKR mice with D10 dcTCR and D10 TCR-IgG1 primed I-Ak- and I-Ek-restricted CD4+ T cells recognizing constant region epitopes, but there was no detectable response to the variable region. Comparison of the in vitro proliferative responses of CD4+ T cells from D10 scTCR-primed H-2 congenic mice revealed that H-2u was a responder haplotype for the variable region. How the immunogenicity of particular regions of the TCR appears to be shaped by tolerance induction in vivo and the implications for immunotherapy with soluble TCR vaccinations are discussed.

Maternal immunization with a soluble TCR-Ig chimeric protein: long term, V beta-8 family-specific suppression of T cells by maternally transferred antibodies.

Maternal transfer of TCR clonotypic Ab protected young NOD mice against the adoptive transfer of diabetes by the BDC 2.5 T cell clone. The effect of maternal anti-TCR Vbeta-8 Ab on T cell development and function has now been investigated. SJL/J mice, which lack TCR Vbeta-8, were immunized with soluble, chimeric D10 TCR-IgG1 containing Vbeta-8.2. The (SJL/J x AKR/J) F1 offspring of immunized female SJL/J mice were severely depleted of peripheral T cells bearing Vbeta-8 until 11 to 17 wk of age. The loss of Vbeta-8 expression did not appear to be due to modulation of cell surface TCR. Since the Vbeta-8+ T cell population was unperturbed in the (AKR/J x SJL/J) F1 offspring of D10 TCR-IgG1-immunized AKR/J mothers making D10 clonotypic Ab, the effect was immunologically specific. The deletion of Vbeta-8+ T cells had functional consequences. In the in vitro response to the superantigen, staphylococcal enterotoxin B, the usually observed participation of Vbeta-8.2+ T cells was largely suppressed, whereas the recruitment of Vbeta-3+ T cells remained unaltered. In control mice, T cell responses to the 134- to 146-residue peptide of conalbumin (pCA(134-146)) were biased toward use of Valpha-2/Vbeta-8.2 TCR. In D10 TCR-IgG1 maternally immunized (SJL x AKR/J) F1 mice, the T cell responses to pCA(134-146) were suppressed, and T cell lines derived from these in vitro were devoid of Vbeta-8.2 expression. With an increased understanding of TCR V gene usage in autoimmune diseases, similar strategies for the depletion of autoreactive T cells may become feasible in humans.

Immunization with soluble BDC 2.5 T cell receptor-immunoglobulin chimeric protein:antibody specificity and protection of nonobese diabetic mice against adoptive transfer of diabetes by maternal immunization.

The BDC 2.5 T cell clone is specific for pancreatic beta-cell antigen presented by I-Ag7, and greatly accelerates diabetes when injected into 10-21-d-old nonobese diabetic (NOD) mice. The BDC 2.5 T cell receptor (TCR) has been solubilized as a TCR-IgG1 chimeric protein. All NOD mice immunized against BDC 2.5 TCR-IgG1 produced antibodies recognizing TCR C alpha/C beta epitopes that were inaccessible on the T cell surface. 56% of the mice produced antibodies against the BDC 2.5 clonotype that specifically blocked antigen activation of BDC 2.5 cells. We have used the adoptive transfer model of diabetes to demonstrate that maternal immunization with soluble TCR protects young mice from diabetes induced by the BDC 2.5 T cell clone.

Loss of RT6 message and most circulating T cells after thymectomy of diabetes prone BB rats.

T cells expressing the RT6 surface alloantigen perform important immunoregulatory functions in the rat. Diabetes prone (DP) BB rats are deficient in circulating RT6+ T cells and develop spontaneous autoimmune diabetes mellitus. Transfusions leading to engraftment of RT6+ T cells prevent the disease. Coisogenic diabetes resistant (DR) BB rats do circulate RT6+ T cells and are free of disease. We investigated the basis for the deficiency of RT6+ T cells in the DP-BB rat and made the following observations. 1. Thymectomy causes the rapid loss of most peripheral T cells in the DP-BB rat. 2. Concomitant with the loss of T cells is the total loss of mRNA encoding RT6. 3. In contrast to the effects observed in peripheral lymphoid tissues, thymectomy does not lead to a detectable loss in RT6+ protein found in the small intestine. We conclude that the deficiency of RT6+ peripheral T cells in the DP-BB rat is due either to their short life span or to their reduced proliferative capacity following release from the thymus.

Adoptive transfer of autoimmune diabetes and thyroiditis to athymic rats.

We describe the induction of autoimmune diabetes, insulitis, and thyroiditis in athymic rats following injections of major histocompatibility complex compatible spleen cells. Lymphocytes with these capabilities were found in normal rats of the YOS, WAG, PVG, and diabetes-resistant BB strains, and in diabetes-prone BB rats. Adoptive transfer was facilitated by prior in vivo depletion of RT6.1+ regulatory T cells and in vitro mitogen activation of donor spleen cells. By RT6 depleting diabetes-resistant donors and using nude recipients, transfer of diabetes and thyroiditis was accomplished by using fresh, unstimulated spleen cells. The data suggest that organ-specific autoreactive cells may be present to various degrees but suppressed to a variable extent in many rat strains. The equilibrium between autoreactive and regulatory cells appears to determine the expression of autoimmunity.

Effects of irradiation on diabetes in the BB/Wor rat.

Lymphoid irradiation is known to prevent spontaneous autoimmune diabetes in susceptible BB rats. The present studies investigated further the effects of radiation in diabetes prone (DP) and resistant (DR) BB/Wor rats, and histocompatible Yoshida (YOS) rats. Single doses of total body gamma irradiation (125-600 rads) induced diabetes within 22-44 days in 20 of 102 (20%) 30 day old DR rats, less than 1% of which develop the disease. Radiation was also associated with (1) a reduction in the ratio of W3/25+ to OX8+ peripheral blood lymphocytes within 2 weeks, and (2) a decreased percentage of lymph node cells expressing the RT6.1 surface alloantigen 3-4 weeks after treatment. Similar doses of irradiation did not alter the frequency or age at onset of diabetes in DP rats, and did not induce diabetes in YOS rats. When a single dose of 250 or 500 rads of gamma irradiation was followed by injection of mitogen activated spleen cells from acutely diabetic rats to adoptively transfer diabetes, 16 of 19 (84%) DR and 8 of 14 (57%) YOS rats became diabetic. Long term exposure to ultraviolet irradiation (UVB) did not alter the frequency or age at onset of diabetes in either DP or DR rats. We conclude that there may exist a population of regulatory cells relatively sensitive to gamma irradiation that play a role in determining the susceptibility of rats to autoimmune diabetes mellitus.

Absence of RT6+ T cells in diabetes-prone biobreeding/Worcester rats is due to genetic and cell developmental defects.

Diabetes-prone BB/Wor (DP) rats lack the RT6+ peripheral T cell subset whereas diabetes-resistant BB/Wor rats have normal numbers of RT6+ T cells. Lymphocyte transfusion experiments and in vivo depletion studies have demonstrated that RT6+ T cells have an important regulatory role in the pathogenesis of insulin-dependent diabetes mellitus in BB/Wor rats. In the present study, the results of genetic complementation studies indicate that the DP rat contains an intact RT6 gene, but fails to express the RT6.1 alloantigen in the functional absence of an accessory factor (provided by RT6+ cells). At the cellular level, irradiation chimeras demonstrate that the absence of RT6+ T cells in DP rats is due to an intrinsic defect that results in abnormal development and/or differentiation of prothymocytes into RT6+ T cells. The inability of DP prothymocytes to generate RT6+ T cells is not due to serum autoantibodies, lack of accessory cells, or to the presence of inhibitory cells. Inasmuch as DP bone marrow can transfer the susceptibility for diabetes to irradiated recipients, our present results suggest that an important predisposing factor for insulin-dependent diabetes mellitus in DP rats is the inability of DP prothymocytes to generate RT6+ T cells.

Helper and suppressor T lymphocyte function in severe alcoholic liver disease.

The immune regulatory T cell status of patients with severe alcoholic liver disease (ALD) was investigated. Using monoclonal antibodies to identify lymphocyte subsets in 22 patients, a significant decrease in the percentage of T suppressor/cytotoxic cells (P less than 0.01) and increase in the percentage T helper/inducer population (P less than 0.05) was observed when the results were compared with 20 normal controls. However, when absolute numbers of these lymphocyte subsets were calculated the patient group did not differ significantly from the controls. Further studies revealed T immunoregulatory cell function to be normal. Concanavalin A induced suppressor cells resulted in equivalent inhibition of autologous cell mitogen responsiveness in the patient and control groups. In addition, purified patient T lymphocytes were demonstrated to provide normal help to and manifest normal suppression of IgG, IgA and IgM synthesis by allogeneic B cells. When spontaneous immunoglobulin synthesis by circulating mononuclear cells was investigated, a significant increase in IgA synthesis was found in the ALD patients (P less than 0.05). These results suggest that T cell immunoregulation is normal in patients with ALD and a defect in this system is not responsible for the increased synthesis of immunoglobulin observed in ALD.

Increased concanavalin A induced suppression in treated and untreated coeliac disease.

The generation of suppression by concanavalin A in peripheral blood mononuclear cells in treated and untreated coeliac subjects using an in vitro assay was found to be significantly increased when compared with controls. The response of peripheral blood mononuclear cells to the plant mitogen concanavalin A (con A) was also significantly depressed in both groups of coeliac patients. It is proposed that the depressed cell mediated immunity found in this and other studies in coeliac patients is because of increased suppression. The possible connection between these findings and the increased incidence of malignancy also found in coeliac disease is discussed.