Naphthyl ketones: a new class of Janus kinase 3 inhibitors.

Potent inhibition of Janus kinase 3 was found for a series of naphthyl(beta-aminoethyl)ketones (e.g. 7, pIC50 = 7.1+/-0.3). Further studies indicated that these compounds fragment in less than 1 h by retro-Michael reaction in the Jak3 in vitro ELISA assay procedure. The breakdown product of 7, 2-naphthylvinyl ketone (22, pIC50 = 6.8+/-0.3) showed very similar inhibitory activity to 7. Compounds 7 (in neutral buffer) and 22 will be useful pharmacological tools for the investigation of the Janus tyrosine kinase Jak3.

A novel series of non-nucleoside inhibitors of inosine 5'-monophosphate dehydrogenase with immunosuppressive activity.

Inhibitors of inosine 5'-monophosphate dehydrogenase (IMPDH, EC 1.1.1.205) are effective immunosuppressive drugs that may also have additional potential applications as antitumour and antimicrobial agents. The clinical value of the most potent and specific inhibitor of IMPDH, mycophenolic acid, is limited by its rapid metabolism in vivo to an inactive glucuronide derivative. There is, therefore, a considerable incentive to develop structurally novel, preferably non-nucleoside, inhibitors with greater metabolic stability than mycophenolic acid. Here, we describe a high throughput screen for inhibitors of IMPDH, which facilitated the discovery of a single novel non-nucleoside inhibitor from a collection of approximately 80,000 compounds. The inhibitor is a pyridazine, which, like mycophenolic acid, exerts uncompetitive inhibition of IMPDH. Analysis of the enzyme kinetics suggests that the inhibitory action of the pyridazine is similar to that of mycophenolic acid, which involves trapping of a covalent intermediate formed during the conversion of IMP to xanthosine monophosphate. Chemical modification of the lead compound resulted in pyridazine derivatives with enhanced potency against IMPDH and guanine nucleotide synthesis in cultured cells in vitro and also against guanine nucleotide synthesis in the mouse spleen in vivo. One of the compounds was available in sufficient quantity to demonstrate highly effective immunosuppressive activity in a model of delayed type hypersensitivity in mice. To our knowledge, the novel pyridazines described in this report represent the first non-nucleoside uncompetitive inhibitors of IMPDH with immunosuppressive activity since the discovery of the inhibitory activity of mycophenolic acid and its derivatives thirty years ago.

Antigenicity of rat erythrocyte glycophorins.

The relationship between rat red blood cell (RBC) glycophorins and the antigens recognised by anti-rat RBC antibodies was examined. Initially, murine monoclonal antibodies specific for surface epitopes on whole rat RBCs were tested for their reactivity with RBC membranes on Western blots and two were found which reacted with blotted antigens. These antibodies recognised two bands corresponding to the major PAS-stainable bands of rat RBC membranes (i.e., the glycophorins) and a number of minor bands, thus demonstrating that the bands are antigenically related. This band-pattern was remarkably similar to that obtained with mouse anti-rat RBC serum. Digestion with neuraminidase altered the electrophoretic mobility of most of the bands, providing additional evidence that they are sialoglycoproteins, although sialic acid was shown not to contribute to their antigenicity. The glycophorin nature of the major antigens was verified by reelectrophoresis and blotting of bands excised from SDS gels, which showed that they were interconvertible monomeric and dimeric forms of the same polypeptide chain. It is suggested that rat RBC glycophorins are a related family of sialoglycoproteins with the high molecular weight members being formed by dimerization of five lower molecular weight polypeptide chains in various combinations.

Identification of an erythrocyte autoantigen using monoclonal autoantibodies induced by immunization of mice with rat erythrocyte.

An erythrocyte autoantigen has been identified by means of monoclonal autoantibodies raised by immunizing mice with rat red blood cells (RBC). The autoantibodies reacted with intact rat and mouse RBC as judged by a cellular radioimmunoassay, and with a 52K band on western blots of rat and mouse RBC. They did not react with intact sheep RBC or blotted sheep erythrocyte membranes. Although anti-rat erythrocyte antibodies did react with bands in the molecular weight region of 50-55 K (on blots of rat erythrocyte membranes), these bands were susceptible to neuraminidase digestion, thus distinguishing them from the 52 K band recognized by monoclonal autoantibodies. The implications of the above results for the known autoantibody specificity of suppression is discussed, and it is suggested that they favour the existence of autoantigen-specific suppressor cells.

A phenotypically dominant regulatory mechanism suppresses major histocompatibility complex class II gene expression in a murine plasmacytoma.

The expression of major histocompatibility complex (MHC) class II antigens is down-regulated when B cells differentiate into plasma cells. We have studied the mechanism of down-regulation of MHC class II expression in a BALB/c strain-derived murine plasmacytoma cell line, NS1. NS1 cells express MHC class I antigens but not MHC class II antigens. We tested 20 uncloned hybrid cell lines obtained from the fusion of NS1 cells with MHC class II-expressing splenic B cells prepared from CBA, SJL or BALB/c mice. All the hybrid cell lines expressed MHC class I antigens of either or both parental haplotypes but did not express MHC class II. One NS1 X splenic B cell hybrid clone, K3, was used to further validate these results; K3 cells expressed MHC class I but not MHC class II antigens. K3 was fused to the MHC class II-expressing B lymphoma A20, and the seven resulting hybrid cell lines were again found to express MHC class I but not MHC class II antigens. Since NS1 is a subclone of the P3-X63Ag8 murine plasmacytoma, we also tested one P3-X63Ag8 x splenic B cell hybrid, Sp2/0, and two Sp2/0 x splenic B cell hybrids. All were found to express the appropriate MHC class I antigens but did not express MHC class II. Thus, our results suggest that the NS1 plasmacytoma suppresses MHC class II expression by a phenotypically dominant regulatory mechanism. We found that NS1 cells express correctly sized mRNA for the MHC class II genes A alpha, E alpha and the invariant chain. The co-expression of MHC class I protein and I-A and I-E region gene transcripts provides strong evidence that the MHC gene cluster is structurally intact, and that lack of class II expression is due to a genetic regulatory mechanism. The amounts of class II mRNA expressed by NS1 cells were at least equivalent to those found in splenic lymphocytes. Therefore, this regulation must operate post-transcriptionally.

Identification of rat erythrocyte antigens with a new non-radioactive immunoprecipitation technique.

In order to examine how rat erythrocytes stimulate erythrocyte autoantibody production at the molecular level, we have identified rat erythrocyte antigens by immunoprecipitation and western blotting using monoclonal antibodies and antisera. A novel non-radioactive immunoprecipitation technique was used, which employed biotin as a label and a luminescent detection system. The new method was validated by comparison with conventional immunoprecipitation using 125I. Glycophorins of relative molecular mass (Mr) 81,000 and 38,000 were found to be the major antigenic components of rat erythrocytes, while band 3 (the most abundant erythrocyte membrane protein) was not recognized by rat-specific antibodies. The same surface antigens were recognized by sera from mice producing erythrocyte autoantibodies and by sera from mice in which autoantibody production was suppressed. Nine other minor rat-specific antigens were identified by blotting, ranging in Mr from 23,000 to 147,000. Analysis of the integral membrane proteins of rat and mouse erythrocytes by sodium dodecyl sulphate (SDS) electrophoresis followed by silver or periodic acid-Schiff (PAS) stains revealed differences between the glycophorins, but not between rat and mouse band 3. Thus, the major antigenic differences correspond to discernible biochemical differences between rat and mouse erythrocyte sialoglycoproteins.

The relationship between secreted and cell surface antigen-binding molecules synthesized by T cells. I. Function-related isotypic determinants on T cells defined by antisera to antigen-specific helper and suppressor factors.

Rabbit antisera have been used to define 'constant region' markers which distinguish between mouse T cell-derived helper and suppressor factors, regardless of their antigen-specificity or strain of origin. These antisera have also been shown to bind to functional T-cell lines. After several absorption steps, rabbit anti-helper factor serum bound specifically to mouse helper cell lines, whereas rabbit anti-suppressor factor serum bound specifically to suppressor cell lines. Neither antiserum bound to cytotoxic T cell lines. The 'isotypic' determinants defined by these antisera were demonstrated to be present on distinct subpopulations of non-transformed T cell populations, such as splenic T cells, cortisone-resistant thymocytes and Con A blasts, but were not found on Thy-1- spleen cells, bone marrow, brain, heart, liver, kidney or heart tissue. The antisera did not stain significant numbers of normal thymocytes, and so expression was restricted to mature T cells. T cells reactive with rabbit anti-helper factor serum were found in the Lyt 2- population of cortisone-resistant thymocytes, and constituted a major subpopulation of in vitro induced helper cells, while rabbit anti-suppressor factor serum stained cells found in the Lyt 2+ population of cortisone-resistant thymocytes, as well as the majority of in vitro induced suppressor cells. Thus, these antisera are potentially of great value in the definition and isolation of functionally-distinct T cell subpopulations.

A monoclonal antibody against antigen-specific helper factor augments T-cell help.

Antigen-specific molecules, commonly termed 'factors', have been shown to be released from helper and suppressor T cells. These factors mimic the activity of the cells that secrete them and there is much speculation about the relationship of antigen-specific factors to T-cell receptors for antigen. We have raised a variety of antisera in rabbits which were shown to react against conserved 'constant' determinants on either helper or suppressor factors independently of antigenic specificity or mouse strain of origin of the factor. In contrast, syngeneic mouse antisera were found to react with 'variable' factor determinants in an antigen-specific and mouse strain-dependent manner. These antisera thus define two regions on factor molecules, one 'variable' (related to antigen specificity) and the other 'constant' (related to function). However, potential contaminants in these antisera have limited their usefulness. Thus, we are now generating monoclonal antibodies against T-cell factors and report here the properties of a monoclonal antibody (AF3.44.4) which reacts with antigen-specific helper factors. This antibody also binds to helper T cells and, in the presence of antigen, augments helper cell induction in vitro, which, in turn, leads to enhanced antibody production in vitro. These characteristics suggest that AF3.44.4 recognizes a determinant shared by helper factor and the antigen receptor on helper T cells.

Lymphoid cell lines as indicators of lymphokine generation.

We investigated the migration characteristics of the cells of four human lymphoid lines, normal peripheral blood leucocytes (PBL), and normal mouse splenocytes (MSC). Two lines (QIMR-WIL and Namalwa) were actively migratory, as were the PBL and MSC. Migration was inhibited at low temperatures and reduced by inhibition of glycolysis, oxidative phosphorylation and RNA synthesis. The migration of QIMR-WIL cells, PBL and MSC but not Namalwa was inhibited by lymphokine-containing supernatants from phytohaemagglutinin-pulsed MSC or MSC stimulated by allogenic cells. The inhibitor of migration in the supernatants had properties similar to those of human leucocyte inhibition factor but distinct from those of macrophage inhibition factor. QIMR-WIL and normal human PBL were compared as indicators in an indirect leucocyte migration assay. QIMR-WIL cells were the more sensitive responder cells and are an abundant, stable, and uniform cell population for lymphokine detection.

T-cell hybridomas producing hapten-specific suppressor factors.

We have made several T-T hybridomas which secrete soluble factors capable of suppressing an in vitro antibody response to nitrophenol (NP), but not other unrelated antigens. These factors bind specifically to NP, and express determinants coded for in the I-J region of the mouse major histocompatibility complex. No determinants that cross-react with the constant regions of mouse immunoglobulins are present on the factors. Three sub-clones originating from the same initial culture well of hybridoma cells secrete factors which carry I-J determinants of different haplotypes. One clone expresses I-J determinants derived from the suppressor cell parent, another expresses I-J determinants derived from the tumour cell parent, and a third expresses both. This correlates exactly with I-J determinants expressed on the cell membrane, and suggests the participation of at least two genes in the determination of suppressor-factor structure.