BACKGROUND:The CXCR3 receptor and its three interferon-inducible ligands (CXCL9, CXCL10 and CXCL11) have been implicated as playing a central role in directing a Th1 inflammatory response. Recent studies strongly support that the CXCR3 receptor is a very attractive therapeutic target for treating autoimmune diseases, such as rheumatoid arthritis, multiple sclerosis and psoriasis, and to prevent transplant rejection. We describe here the in vitro and in vivo pharmacological characterizations of a novel and potent small molecule CXCR3 antagonist, SCH 546738.RESULTS:In this study, we evaluated in vitro pharmacological properties of SCH 546738 by radioligand receptor binding and human activated T cell chemotaxis assays. In vivo efficacy of SCH 546738 was determined by mouse collagen-induced arthritis, rat and mouse experimental autoimmune encephalomyelitis, and rat cardiac transplantation models. We show that SCH 546738 binds to human CXCR3 with a high affinity of 0.4 nM. In addition, SCH 546738 displaces radiolabeled CXCL10 and CXCL11 from human CXCR3 with IC50 ranging from 0.8 to 2.2 nM in a non-competitive manner. SCH 546738 potently and specifically inhibits CXCR3-mediated chemotaxis in human activated T cells with IC90 about 10 nM. SCH 546738 attenuates the disease development in mouse collagen-induced arthritis model. SCH 546738 also significantly reduces disease severity in rat and mouse experimental autoimmune encephalomyelitis models. Furthermore, SCH 546738 alone achieves dose-dependent prolongation of rat cardiac allograft survival. Most significantly, SCH 546738 in combination with CsA supports permanent engraftment.CONCLUSIONS:SCH 546738 is a novel, potent and non-competitive small molecule CXCR3 antagonist. It is efficacious in multiple preclinical disease models. These results demonstrate that therapy with CXCR3 antagonists may serve as a new strategy for treatment of autoimmune diseases, including rheumatoid arthritis and multiple sclerosis, and to prevent transplant rejection.

Early work on the biology of the components of Cannabis sativa showed evidence for a potential influence on immune regulation. With the discovery of a peripheral cannabinoid receptor associated with immune cells, many laboratories have sought to link the immunoregulatory activities of cannabinoid compounds with this receptor, hoping that such compounds would lack the psychoactive effects of marijuana and other nonspecific cannabinoid agonists. In this report, the authors investigate the role of the cannabinoid CB2 receptor in immune regulation, with particular emphasis on compounds shown to regulate immune cell recruitment. The authors conclude by using the immune cell recruitment model to rationalise cannabinoid CB2 receptor-specific effects in modulating immune disease, particularly the increasing evidence for its role in experimental autoimmune encephalomyelitis and in influencing bone density.

Sulfatide (3'sulfogalactosylceramide) is a glycosphingolipid present within the nervous system and in the islets of Langerhans. Anti-sulfatide antibodies have been observed in both pre-diabetic and newly diagnosed type 1 diabetic patients. The aim of this study was to test in vivo, the therapeutic effect of sulfatide on the development of diabetes in the NOD mouse. In four separate experiments diabetogenic splenocytes from newly diabetic NOD mice were injected iv into 7-8 week old irradiated (700R) female NOD mice (4-10 million cells/mouse). Each experiment consisted of four treatment groups to which the mice were randomly divided: 1) sulfatide; 2) galactosylceramide (the precursor to sulfatide without sulfate); 3) GM1, a glycosphingolipid negatively charged as sulfatide but with a different sugar composition; and 4) phosphate buffered saline (PBS). The mice received 100 microg glycosphingolipid iv on the day of cell transfer and 1-3 times thereafter at four day intervals, and were screened for diabetes three times a week the next 52 days. Among all the 35 sulfatide-treated mice 54% became diabetic compared to 93 % of 43 PBS-treated animals (p < 0.00001). Correspondingly, galactosylceramide reduced diabetes incidence to 52% (25 mice, p < 0.00001). On the other hand, 86% of GM1-treated mice (n=28) became diabetic indicating that no effect was obtained by this glycosphingolipid. In two experiments in which less spleen cells were transferred (4-5 mill.) and glycosphingolipids were given 4 times, 35% of the sulfatide-treated animals (n = 17) developed diabetes compared to 85% of PBS-treated mice (n = 20, p < 0.001). A robust proliferative response to sulfatide, but none to GM1, was observed when spleen cells were rechallenged with glycosphingolipid in vitro. Thus, like insulin and GAD, sulfatide is able to prevent diabetes in NOD mice.

The 524--543 region of glutamic acid decarboxylase (GAD65), GAD65(524--543), is one of the first fragments of this islet Ag to induce proliferative T cell responses in the nonobese diabetic (NOD) mouse model of spontaneous autoimmune diabetes. Furthermore, NOD mice given tolerogenic doses of GAD65(524--543) are protected from spontaneous and cyclophosphamide-induced diabetes. In this study, we report that there are at least two I-A(g7)-restricted determinants present in the GAD65(524--543) sequence, each capable of recruiting unique T cell repertoires characterized by distinct TCR V beta gene use. CD4(+) T cells arise spontaneously in young NOD mice to an apparently dominant determinant found within the GAD65 peptide 530--543 (p530); however, T cells to the overlapping determinant 524-538 (p524) dominate the response only after immunization with GAD65(524--543). All p530-responsive T cells used the V beta 4 gene, whereas the V beta 12 gene is preferentially used to encode the TCR of p524-responsive T cell populations. T cell clones and hybridomas from both of these T cell groups were responsive to APC pulsed with GAD65(524--543) or whole rGAD65. p524-reactive cells appeared to be regulatory upon adoptive transfer into young NOD mice and could inhibit insulin-dependent diabetes mellitus development, although they were unable to produce IL-4, IL-10, or TGF beta upon antigenic challenge. Furthermore, we found that i.p. injection with p524/IFA was very effective in providing protection from cyclophosphamide-induced insulin-dependent diabetes mellitus. These data demonstrate that the regulatory T cells elicited by immunizing with GAD65(524--543) are unique and distinct from those that arise from spontaneous endogenous priming, and that T cells to this limited region of GAD65 may be either regulatory or pathogenic.

As virtually all adaptive immune responses involve the activation of CD4 T cells by specific antigen, a central question in immunology is the nature of the ligands recognized by these cells. It is now well established that CD4 T cells respond to peptide fragments of foreign proteins bound to a single peptide binding site of a class II molecule encoded in the major histocompatibility complex (MHC). Thus, determining the nature of peptides bound by MHC class II molecules is essential for an understanding of adaptive immunity. Moreover, as tolerance of CD4 T cells is crucial for maintaining self tolerance of B cells (Lin et al, 1991), CD8 cytolytic T cells (Guerder and Matzinger, 1992), and the CD4 T cells themselves, understanding the nature and distribution of self peptides bound to MHC class II molecules is also critical to understanding self tolerance. Finally, self peptides have been implicated in positive selection of T cells in the thymus for self MHC recognition capability. For all these reasons, we have sought to characterize self peptides bound to MHC class II molecules, their generation and distribution in various tissues, and T cell responses to these self peptides. In this paper, we summarize our findings and speculate about their implications for self tolerance and self MHC recognition.

Soluble extracts prepared from Babesia bigenina merozoites were tested for anti-genicity in class-specific enzyme immunoassays currently being evaluated for the differential serodiagnosis of bovine babesiosis. Intact merozoites were harvested from erythrocytes from an experimentally-infected calf by controlled hypotonic lysis and differential ultracentrifugation. The merozoites were disrupted by ultrasonication and a crude soluble extract obtained by ultracentrifugation. Fractionation of the crude extract on calibrated Sephadex G-200 column consistently produced 4 fractions with molecular weights of 600, 40, 15 and 5 k (k = 103 daltoons). Only the 100 and 15 k fractions proved to be antigenic when reacted against bovine immune sera. These fractions were incorporated into IgM- and IgG-specific enzyme immunoassays and used to determine the kinetics of the host-antibody responses to infection. The use of semi-defined antigens allowed assay standardization and good reproducibility of the results. A calf infected with a cryopreserved stabilate of B. bigemina originating from adult Boophilus microplus ticks developed a mild transient fever from 6–4 days post-infection (d.p.i.) and low parasitaemia levels from 7–16 d.p.i. IgG-antibodies first appeared at 7 d.p.i.,peaked in intensity at 12 d.p.i. and then persisted at these levels until the end of the test period at 49 d.p.i. IgM-antibodies appeared at 7 d.p.i., peaked in intensity from 12–22 d.p.i., but then declined to low levels by 28 d.p.i.. The importance of this transitory IgM-antibody response in the serodiagnosis of acute B. bigemina infections remains to be determined in clinical and field situations.

Cloned T cell lines expressing either CD4 or CD8 have been isolated from the islets of acutely diabetic NOD mice. These cloned T cell lines respond by proliferating specifically to islets from mice bearing MHC gene products of the NOD strain. Adoptive transfer of cloned T cell lines into irradiated recipients shows intense insulitis and occasional frank diabetes in recipients of both CD4+ and CD8+ cloned T cell lines, but only mild insulitis and no overt disease in recipients of either CD4 or CD8 cloned T cell lines alone. This experimental system will be used to probe three areas of insulin-dependent diabetes mellitus: first, what is the pathogenesis of ß cell destruction in IDDM. Second, what it the exact nature of the ligand recognized on ß cells by CD4+ and CD8+ cloned T cells. Third, what is the nature of the autoreactive T cell receptor, and are T cells mediating islet destruction in NOD mice homogeneous, oligoclonal, or highly variable in the expression of cell surface T cell receptors.