Biomedical subjects

R Gimeno

Publications and source records attributed to R Gimeno.

8 recordsLinked to original sources

PubMed2000

Distinct requirements for IFNs and STAT1 in NK cell function.

NK cell functions were examined in mice with a targeted mutation of the STAT1 gene, an essential mediator of IFN signaling. Mice deficient in STAT1 displayed impaired basal NK cytolytic activity in vitro and were unable to reject transplanted tumors in vivo, despite the presence of normal numbers of NK cells. IL-12 enhanced NK-mediated cytolysis, but poly(I:C) did not, and a similar phenotype occurred in mice lacking IFNalpha receptors. Molecules involved in activation and lytic function of NK cells (granzyme A, granzyme B, perforin, DAP10, and DAP12) were expressed at comparable levels in both wild-type and STAT1(-/-) mice, and serine esterase activity necessary for CTL function was normal, showing that the lytic machinery was intact. NK cells with normal cytolytic activity could be derived from STAT1(-/-) bone marrow progenitors in response to IL-15 in vitro, and enhanced NK lytic activity and normal levels of IFN-gamma were produced in response to IL-12 treatment in vivo. Despite these normal responses to cytokines, STAT1(-/-) mice could not reject the NK-sensitive tumor RMA-S, even following IL-12 treatment in vivo. Whereas in vitro NK cytolysis was also reduced in mice lacking both type I and type II IFN receptors, these mice resisted tumor challenge. These results demonstrate that both IFN-alpha and IFN-gamma are required to maintain NK cell function and define a STAT1-dependent but partially IFN-independent pathway required for NK-mediated antitumor activity.

Adaptor Proteins, Signal Transducing↗

PubMed2000

STAT1 affects lymphocyte survival and proliferation partially independent of its role downstream of IFN-gamma.

Lymphocytes derived from mice deficient in STAT1 showed reduced apoptosis and enhanced proliferation in vitro. To understand the involvement of STAT1 in the observed reduction in apoptosis, we examined the levels of caspase and bcl-2 family genes that are involved in cell survival and/or apoptosis. The levels of caspase 1 and 11, two enzymes involved in both cytokine protein processing and induction of apoptosis, were reduced in STAT1-/- cells compared with wild-type. However, the levels of bcl-2 genes were comparable in both mice. STAT1-/- cells also displayed an enhanced proliferation following TCR stimulation. This hyperproliferation could not be ascribed completely to the loss of IFN-gamma-mediated antiproliferation. First, similar phenotypes were also observed in fibroblasts and pre-B cells derived from STAT1-/- mice, which do not produce IFN-gamma. Second, comparisons with cells lacking the gene for IFN-gamma or with cells treated with neutralizing Abs to IFN-gamma only partially mimicked the STAT1-/- phenotype. Interestingly, the kinetics of degradation of p27kip1, a CDK inhibitor, following TCR ligation were faster, and, concomitantly, the up-regulation of CDK2 kinase activity and protein levels were increased in stimulated T cells of STAT1-/- mice relative to those of wild-type mice. Furthermore, STAT1-/- animals were more susceptible to carcinogen-induced thymic tumors, a possible consequence of altered T cell growth and/or survival. These results demonstrate an essential role for STAT1 for lymphocyte survival and proliferation that is only partially dependent on IFN-gamma signaling.

Animals↗

PubMed1999

Differential regulation of constitutive major histocompatibility complex class I expression in T and B lymphocytes.

Major histocompatibility complex (MHC) class I antigens are constitutively expressed yet highly induced by interferon (IFN) during inflammation. We found that not only IFN-induced but also normal basal expression of MHC I required IFN receptors and signal transducer and activator of transcription (STAT)1, providing genetic evidence for continuous IFN signaling. Surprisingly, an IFN-independent requirement for STAT1 was also found, specifically in T lymphocytes, where MHC class I expression was not fully accounted for by IFN signaling. This IFN-independent pathway maintained tyrosine phosphorylation of STAT1 in T but not B lymphocytes even in the absence of IFN receptors. Interestingly, interleukin (IL)-7 selectively activated STAT1 and induced MHC class I in mature T but not B cells. These loss of function studies demonstrate an essential role of endogenous IFN and activated STAT1 for constitutive MHC class I expression in normal mice and define IL-7-dependent but IFN-independent regulation of STAT1 restricted to T lymphocytes.

Animals↗

PubMed1999

LST1 is a SEC24 homologue used for selective export of the plasma membrane ATPase from the endoplasmic reticulum.

In Saccharomyces cerevisiae, vesicles that carry proteins from the ER to the Golgi compartment are encapsulated by COPII coat proteins. We identified mutations in ten genes, designated LST (lethal with sec-thirteen), that were lethal in combination with the COPII mutation sec13-1. LST1 showed synthetic-lethal interactions with the complete set of COPII genes, indicating that LST1 encodes a new COPII function. LST1 codes for a protein similar in sequence to the COPII subunit Sec24p. Like Sec24p, Lst1p is a peripheral ER membrane protein that binds to the COPII subunit Sec23p. Chromosomal deletion of LST1 is not lethal, but inhibits transport of the plasma membrane proton-ATPase (Pma1p) to the cell surface, causing poor growth on media of low pH. Localization by both immunofluorescence microscopy and cell fractionation shows that the export of Pma1p from the ER is impaired in lst1Delta mutants. Transport of other proteins from the ER was not affected by lst1Delta, nor was Pma1p transport found to be particularly sensitive to other COPII defects. Together, these findings suggest that a specialized form of the COPII coat subunit, with Lst1p in place of Sec24p, is used for the efficient packaging of Pma1p into vesicles derived from the ER.

Amino Acid Sequence↗

PubMed1997

Signal transducer and activator of transcription 1 in human muscle: implications in inflammatory myopathies.

Polymyositis (PM) and dermatomyositis (DM) are two major and distinct inflammatory myopathies. Cytokines, implicated in the immune process, have been recognized in the muscle tissue from PM and DM patients, but their functional in situ role has not been identified. We analyzed the expression of the signal transducer and activator of transcription 1 (STAT1), a molecule whose up-regulation indicates the interaction of cytokines, or growth factors, with their target receptors in muscle fibers and inflammatory infiltrates in PM and DM. An immunohistochemical analysis was performed using monoclonal antibodies to STAT1 in 57 muscle biopsies from 10 patients with DM, 10 with PM, and 37 controls. The profile of STAT1 up-regulation was also investigated in cultured muscle stimulated by interferon-gamma, epidermal growth factor, platelet-derived growth factor, and interleukin-2, using semiquantitative polymerase chain reaction and Western blot. High STAT1 expression was observed in many perifascicular atrophic muscle fibers from DM patients in 10/10 biopsies. In contrast, only a few muscle fibers undergoing necrosis were STAT1 positive in 2/10 patients with PM and in 2/37 controls. STAT1 reactivity was noted in most cells of the infiltrates in DM, PM, and controls. In vitro, STAT1 was stimulated by interferon-gamma but not by the other molecules studied. These results suggest that in DM, but not in PM, there is distinctive functional local cytokine activity able to increase STAT1 expression in muscle fibers. As interferon-gamma specifically activates STAT1 in vitro, this cytokine in conjunction with ischemia is probably involved in perifascicular muscle fiber pathology in DM.

Blotting, Western↗

PubMed1996

The two isoforms of the 90-kDalton nucleolus organizer region autoantigen (upstream binding factor) bind with different avidity to DNA modified by the antitumor drug cisplatin.

It has been previously described that some proteins containing HMG boxes are able to bind more strongly to DNA modified with cis-diamminedichloroplatinum (II) (cisplatin) than to unmodified DNA. In the present study, we analyzed the interaction of cisplatin-modified DNA with the human autoantigen NOR-90 (UBF), a transcription factor that contains several HMG boxes. Using autoantibodies against NOR-90 to perform ELISA and immunoprecipitation, it was confirmed that NOR-90 (UBF) was able to bind cisplatin-modified DNA more avidly than unmodified DNA or trans-diamminedichloroplatinum(II) (transplatin) modified DNA. Moreover, by Southwestern, we observed that the 97 kDalton isoform of NOR-90 (UBF1) was able to bind cisplatin-modified DNA more strongly than the 94 kDalton isoform (UBF2); binding of unmodified DNA or transplatin-modified DNA was not detected with either isoform. Sera containing autoantibodies against NOR-90 did not inhibit, but increased the binding of NOR-90 to cisplatin-modified DNA.

Autoantigens↗

PubMed1996

Stat1 implication in the immune response to superantigens in vivo.

We analyzed the activation and changes in the protein level of STAT1 as a consequence of in vivo treatment with superantigens. Ninety minutes after i.p. injection of the staphylococcal enterotoxin B (SEB), a complex containing STAT1 that was able to specifically bind to DNA containing GAS-like sequences was activated in mouse splenocytes. This complex had the same characteristics as that induced by IFN-gamma in several in vitro systems. Activation of the complex was inhibited by cyclosporin A, and Abs against IFN-gamma severely decreased the amount of complex detected. When splenocytes were analyzed 24 h after SEB treatment, a high increase in the amount of the STAT1 isoforms, STAT91 and STAT84, was observed by Western analysis, but binding to GAS-like sequences was clearly decreased when compared with analysis at 90 min. Nevertheless, when SEB was injected a second time 24 h after the first injection, the binding of STAT1 to GAS-like sequences had risen again. This approach corroborates the implication of IFN-gamma in the response to superantigens in vivo and shows the relevance of analysis of transcription factors in defining the molecular events involved in the immune response.

Animals↗

PubMed1985

Long-term behavior of bovine collagen membrane used as vascular substitute. Experimental study in rats.

The aim of the present study was to evaluate the sequence of the immediate and the mid/long-term organization of the blood interface of a collagenous membrane used as vascular substitute in rats. The implants were prepared from calf skin type I insoluble collagen, obtained after acidic dispersion, in absence of chemical or tanning treatment. They were used to patch an aortic defect by means of microsurgical techniques. The animals were sequentially sacrificed for immediate hemocompatibility studies at 10 s, 30 s, 10 min, 3 h, and 6 h, for long-term analyses of the organization of the blood material interface at the 7th, 15th, 45th, 60th, 90th day following the surgery and each month until 14 months after aortic replacement. The superficial immediate events at the blood patch interface demonstrated erythrocytes heavily engulfed in a thin but dense fibrin mesh both at the patch and at the adjacent aortic wall surfaces. Neither adherent platelet nor platelet aggregate were detectable on the collagen patch surface. This fibrinoerythrocytic membrane covered the patch completely at 60 s and at 3 h the deposit was limited to 5-6 erythrocyte layers as confirmed by histology. It did not further develop on the 7th day. At the blood-collagen interface there progressively developed a tissue composed of active myofibroblasts, collagen bundles, and elastic fibers. After 4 months, nests of fibroendothelial cells were present, and between 6 and 14 months surface cell differentiation, although complete on the adjacent aorta was still incomplete on the bovine collagen patch, amorphous fibers, and fibroendothelial cells coexisting. Heterologous patch debris were still present 14 months after implantation and were associated with macroscopic and ultrastructural calcification, which need further investigations concerning the exact nature and mechanism of mineralization of vascular substitutes of biological nature.

Animals↗