A-770041, a novel and selective small-molecule inhibitor of Lck, prevents heart allograft rejection.

Lck, one of eight members of the Src family of tyrosine kinases, is activated after T cell stimulation and is required for T-cell proliferation and interleukin (IL)-2 production. Inhibition of Lck has been a target to prevent lymphocyte activation and acute rejection. Here, we report the pharmacologic characterization of 1-methyl-1H-indole-2-carboxylic acid (4-{1-[4-(4-acetyl-piperazin-l-yl)-cyclohexyl]-4-amino-1H-pyrazolo[3,4-d]pyrimidin-3-yl}-2-methoxy-phenyl)-amide (A-770041), an orally bioavailable pyrazolo[3,4-d]pyrimidine with increased selectivity for Lck compared with previously reported compounds.

A-420983: a potent, orally active inhibitor of lck with efficacy in a model of transplant rejection.

We have identified the pyrazolo[3,4-d]pyrimidine A-420983 (compound 7) as a potent inhibitor of lck. A-420983 exhibits oral efficacy in animal models of delayed-type hypersensitivity and organ transplant rejection.

Lck inhibitors as a therapeutic approach to autoimmune disease and transplant rejection.

T-cells play an important role in the pathogenesis of many diseases. These include diseases with large commercial markets and also with significant unmet medical needs, such as rheumatoid arthritis and asthma in addition to those with smaller markets such as organ transplantation, multiple sclerosis, inflammatory bowel diseases, type 1 diabetes, systemic lupus erythematosus and psoriasis. The use of currently available immunomodulatory agents is often limited by the appearance of dose-limiting side effects that result from the actions of these agents on non-lymphoid tissues.

Pyrrolo[2,3-d]pyrimidines containing an extended 5-substituent as potent and selective inhibitors of lck I.

Pyrrolo[2,3-d]pyrimidines containing a 5-(4-phenoxyphenyl) substituent are potent and selective inhibitors of Ick in vitro; some compounds are selective for lck over src. Data are shown for two compounds demonstrating that they are potent and selective inhibitors of IL2 production in cells.

SHP2-interacting transmembrane adaptor protein (SIT), a novel disulfide-linked dimer regulating human T cell activation.

T lymphocytes express several low molecular weight transmembrane adaptor proteins that recruit src homology (SH)2 domain-containing intracellular molecules to the cell membrane via tyrosine-based signaling motifs. We describe here a novel molecule of this group termed SIT (SHP2 interacting transmembrane adaptor protein). SIT is a disulfide-linked homodimeric glycoprotein that is expressed in lymphocytes.

Proteolytic activation of protein kinase C delta by an ICE/CED 3-like protease induces characteristics of apoptosis.

Recent studies have shown that protein kinase C (PKC) delta is proteolytically activated at the onset of apoptosis induced by DNA-damaging agents, tumor necrosis factor, and anti-Fas antibody. However, the relationship of PKC delta cleavage to induction of apoptosis is unknown. The present studies demonstrate that full-length PKC delta is cleaved at DMQD330N to a catalytically active fragment by the cysteine protease CPP32.

LPAP, a novel 32-kDa phosphoprotein that interacts with CD45 in human lymphocytes.

CD45, a leukocyte-specific protein tyrosine phosphatase involved in signal transduction, has previously been shown to associate with a 32-kDa phosphoprotein in human T-lymphocytes and T-lymphoma cell lines. The 32-kDa protein was purified and its coding cDNA cloned. Since expression of the protein was found to be restricted to B- and T-lymphocytes it was termed LPAP (lymphocyte phosphatase-associated phosphoprotein).

Identification of a novel dimeric phosphoprotein (PP29/30) associated with signaling receptors in human T lymphocytes and natural killer cells.

Two-dimensional gel electrophoresis of in vitro phosphorylated proteins coprecipitated by CD2 monoclonal antibody (mAb) from Brij58 lysates of resting human T lymphocytes and natural killer (NK) cells resulted in the identification of a novel 29/30-kD disulfide-linked dimer (pp29/30). Comparative two-dimensional analysis of CD2, CD3, CD4, CD5, and CD8 immunoprecipitates revealed that pp29/30 associates with these signaling receptor complexes but not with CD18, CD27, and CD29 in human T lymphocytes. Analysis of CD2 immunoprecipitates prepared from T cell antigen receptor/CD3-modulated T lymphocytes indicated that pp29/30 preferentially associates and comodulates with the human T cell antigen receptor (TCR).

SH2 domains recognize specific phosphopeptide sequences.

A phosphopeptide library was used to determine the sequence specificity of the peptide-binding sites of SH2 domains. One group of SH2 domains (Src, Fyn, Lck, Fgr, Abl, Crk, and Nck) preferred sequences with the general motif pTyr-hydrophilic-hydrophilic-Ile/Pro while another group (SH2 domains of p85, phospholipase C-gamma, and SHPTP2) selected the general motif pTyr-hydrophobic-X-hydrophobic. Individual members of these groups selected unique sequences, except the Src subfamily (Src, Fyn, Lck, and Fgr), which all selected the sequence pTyr-Glu-Glu-Ile.

The cytoplasmic domain of CD4 is required for stable association with the lymphocyte-specific tyrosine protein kinase p56lck.

The CD4 T cell surface molecule binds MHC class II determinants expressed on antigen-presenting cells. CD4 is thought to enhance T cell activation by serving as an adhesion molecule as well as possibly by transducing an independent intracellular signal during the process of antigen stimulation. The recent observation that CD4 is physically associated with the Src-related tyrosine protein kinase p56lck suggests that tyrosine phosphorylation might be involved in these CD4 "signaling" events.

Direct evidence for binding of CD8 to HLA class I antigens.

Adhesion of T lymphocytes is an essential step for antigen recognition and lymphocyte activation. mAbs to T cell surface proteins have been used to define the receptor-ligand proteins that appear to be involved in adhesion. Since most assays measure the effects of mAbs on T lymphocyte function, it is not known whether mAb-mediated blocking is due to a disruption of receptor-ligand interactions or results in inhibition of some aspect of receptor-mediated triggering.

Expression and function of CD8 in a murine T cell hybridoma.

In general, the human CD8 molecule is expressed on T cells specific for HLA class I molecules. Studies designed to delineate the function and to define the ligand of the CD8 molecule have been complicated by the fact that the presumptive ligand for CD8 is on the HLA class I molecule, the same molecule encoding the ligand for the antigen-specific T cell receptor. The ability to express genes in cells other than their natural host has produced a new technology with which to approach CD8 functional studies.

Excretory urography in current practice: evidence against overutilization.

Excretory urography could be performed less frequently if some combinations of genitourinary signs and symptoms were found to be predictive of either a specific disease or normality. To explore this possibility, the authors conducted a prospective study involving more than 3,000 patients at three institutions (a teaching hospital, a community hospital, and a health maintenance organization). Predictive algorithms were obtained by application of a polychotomous logistic regression model but did poorly at differentiating normal from abnormal patients or arriving at a specific diagnosis.

Selection criteria for upper gastrointestinal examinations: attempts at improvement.

An attempt was made to improve upon selection criteria for the performance of upper gastrointestinal (UGI) series in three settings: a teaching hospital, a community hospital, and a health maintenance organization. Two statistical techniques, the polychotomous logistic model (to develop predictive algorithms for the identification of specific diseases) and the maximum attainable discrimination technique, were used to show the relationship between the percentage of patients with any disease detected and the percentage of UGI examinations performed. Results showed that neither technique improved significantly upon selection criteria for identifying patients with abnormal UGI series.

In vitro generation of helper T cells and suppressor T cells that regulate the cytolytic T lymphocyte response to trinitrophenyl-modified syngeneic cells.

Helper T cells and suppressor T cells have been generated in vitro that regulate the cytolytic T lymphocyte (CTL) response to trinitrophenyl (TNP)-modified syngeneic cells. B6D2F1 helper cells generated to TNP-modified parental (P1) cells augment the CTL response to those P1-TNP-modified antigens but not to P2-TNP-modified antigens. The generation of these helper T cells requires the presence of splenic adherent cells and these helper T cells are radioresistant.

Antigen-specific suppression of cytotoxic T cell responses: an idiotype-bearing factor regulates the cytotoxic T cell response to azobenzenearsonate-coupled cells.

When A/J mice are injected subcutaneously with azobenzenearsonate- (ABA) coupled spleen cells, their splenocytes contain primed ABA-specific cytotoxic T lymphocyte (CTL) precursors. Animals that are not primed in vivo do not develop vigorous CTL activity when assessed after in vitro culture with ABA-coupled stimulators. Suppressor molecules derived from ligand-induced first-order ABA-specific suppressor T cells were evaluated for their ability to limit cytolytic T cell development.

T lymphocyte-mediated suppression of myeloma function in vitro. II. Evidence for regulation of hapten-binding myelomas by syngeneic hapten-specific cytolytic T lymphocytes.

BALB/c splenocytes stimulated in vitro with trinitrophenyl (TNP)-modified syngeneic cells inhibit the secretion of antibody by the TNP-binding BALB/c myeloma MOPC 315 in the presence of soluble TNP-Keyhole limpet hemocyanin (KLH). The effector cells are hapten-specific, H-2-restricted, Thy-1.2-bearing, Ly-2-positive T lymphocytes whose precursors are resistant to pretreatment with cyclophosphamide.

Mouse cytolytic T lymphocytes induced by xenogeneic rat stimulator cells exhibit specificity for H-2 complex alloantigens.

When mouse spleen cells were stimulated with irradiated xenogeneic, allogeneic, or trinitrophenyl-modified syngeneic lymphoid cells, the strongest cytolytic response was induced by alloantigens. Mouse cytolytic T lymphocytes generated to rat lymphoid cells demonstrated specificity for the immunizing rat strain, but extensive lysis of allogeneic target cells from certain mouse strains was also observed. Cold target inhibition studies indicated that separate clones of xenoantigen-induced cytolytic T lymphocytes lysed each of the allogeneic murine targets.