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Biomedical subjects

S A McCarthy

Publications and source records attributed to S A McCarthy.

At least 37 records · Page 2Linked to original sources
PubMed1997

Rapid phosphorylation of Ets-2 accompanies mitogen-activated protein kinase activation and the induction of heparin-binding epidermal growth factor gene expression by oncogenic Raf-1.

Heparin-binding epidermal growth factor (HB-EGF) gene transcription is rapidly activated in NIH 3T3 cells transformed by oncogenic Ras and Raf and mediates the autocrine activation of the c-Jun N-terminal kinases (JNKs) observed in these cells. A 1.7-kb fragment of the promoter of the murine HB-EGF gene linked to a luciferase reporter was strongly induced following activation of deltaRaf-1:ER, a conditionally active form of oncogenic human Raf-1. Promoter activation by deltaRaf-1:ER required a composite AP-1/Ets transcription factor binding site located between bp -974 and -988 upstream of the translation initiation site. In vivo genomic footprinting indicated that the basal level of occupancy of this composite AP-1/Ets element increased following deltaRaf-1:ER activation. Cotransfection of Ets-2 and p44 mitogen-activated protein (MAP) kinase expression vectors strongly potentiated HB-EGF promoter activation in response to deltaRaf-1:ER. Potentiated activation required both p44 MAP kinase catalytic activity and threonine 72 in the Pointed domain of Ets-2. Biochemical assays demonstrated the ability of the p42 and p44 MAP kinases to phosphorylate Ets-2 on threonine 72. Importantly, in intact cells, the kinetics of phosphorylation of Ets-2 on this residue closely mirror the activation of the p42 and p44 MAP kinases and the observed onset of HB-EGF gene transcription following deltaRaf-1:ER activation. These data firmly establish Ets-2 as a direct target of the Raf-MEK-MAP kinase signaling pathway and strongly implicate Ets-2 in the regulation of HB-EGF gene expression.

3T3 Cells↗
PubMed1996

Bystander injury of host lymphoid tissue during murine graft-verus-host disease is mediated by nitric oxide.

The suppressed lymphocyte proliferative responses characteristic of graft-versus-host disease (GVHD) are due, in part, to production of nitric oxide (NO). In order to more fully elucidate the role of NO during GVHD, an NO synthesis inhibitor, aminoguanidine (AG), was administered to unirradiated (C57BL/6J X DBA/2J)F1 mice injected with 5 X 10(7) C57BL/6J splenocytes. Administration of AG resulTed in abrogation of the elevation in serum NO2- + NO3- levels characteristic of GVHD. A significantly increased percentage of splenocytes of host phenotype (H2b/d, B220+, and THY1.2+) and a significantly higher hematocrit value were detected in GVHD animals receiving AG therapy. Additionally, the Con A-induced proliferative response of splenocytes obtained from GVHD mice receiving AG therapy was increased compared with the responses of splenocytes from animals that did not receive AG therapy. Parameters not affected by AG therapy included NO synthesis by recovered peritoneal macrophages, donor antihost cytolytic activity in splenocyte populations, serum GM-CSF levels and long-term engraftment of donor cells. These data indicate that NO may play a role in the destruction of both lymphoid and erythroid host tissue as well as the reduced lymphoproliferative responses associated with the acute phase of GVHD.

Animals↗
PubMed1996

Making sense of the combined effect of interleukin-2 and interleukin-4 on lymphocytes using a mathematical model.

The cytokines are the information superhighway of the immune system. They are an important component of the integrated behavior of the system. In order to be able to have a good understanding of the immune system, we must be able to model the effect of cytokines and their combined effect. This work is a step in that direction. We study the combined effect of two cytokines: interleukin-2 (IL-2) and interleukin-4 (IL-4) on some cells of the immune system. Interleukin-2 and interleukin-4 are important growth and differentiation factors for B and T cells. Interleukin-4 antagonizes the effect of interleukin-2 on B cells and some T cells while it synergizes with interleukin-2 on other T cells. We build a mathematical model of the interaction of both cytokines on T and B cells as a building block toward a model of the Th1/Th2 cross-regulation. The response of a given cell to the combination of interleukin-2 and interleukin-4 is shown to involve competing dynamical effects which can lead to either antagonistic or synergistic combined effect.

Animals↗
PubMed1996

Functional changes of intestinal intraepithelial lymphocytes during acute graft versus host disease: correlation with phenotype.

While the pathological characteristics of acute graft versus host disease (GvHD) have been defined for many target organs, little attention has been paid to the functional changes to lymphocytes in target organs such as the liver and small intestine. We have shown previously, utilizing a nonlethal parent (C57BL/6J) into the F1 (C57BL/6J x DBA2J F1) GvHD model, that the intestinal mucosa is infiltrated exclusively by donor T cells with a CD8+ phenotype during the first 3 weeks post-GvHD induction. The present study investigated the functional changes associated with the phenotypic changes of intestinal intraepithelial lymphocytes (IEL) during the acute phase of GvHD. IEL displayed specific anti-host cytolytic activity during the second and third week after GvHD induction. In addition, enhanced cytolytic activity, detected in the presence of anti-CD3 antibody, was apparent during the second through fourth week, with a peak in the third week after GvHD induction. The IEL also showed enhanced proliferative responses to immobilized anti-CD3 during the first 2 weeks of acute GvHD, while profound inhibition of proliferation was observed in the splenocyte population of the same animals. During weeks 2 and 3 post-GvHd induction, the V beta distribution of host IEL remained unchanged while the V beta distribution of infiltrating donor CD8+ cells resembled that of the donor IEL population. In the small intestine, the increased cytolytic and proliferative activity of IEL during the course of the disease may provide a rationale for the involvement of this organ in the pathology associated with GvHD.

Acute Disease↗
PubMed1996

Raf, but not MEK or ERK, is sufficient for differentiation of hippocampal neuronal cells.

To elucidate signal transduction pathways leading to neuronal differentiation, we have investigated a conditionally immortalized cell line from rat hippocampal neurons (H19-7) that express a temperature sensitive simian virus 40 large T antigen. Treatment of H19-7 cells with the differentiating agent basic fibroblast growth factor at 39 degrees C, the nonpermissive temperature for T function, resulted in the activation of c-Raf-1, MEK, and mitogen-activated protein (MAP) kinases (ERK1 and -2). To evaluate the role of Raf-1 in neuronal cell differentiation, we stably transfected H19-7 cells with v-raf or an oncogenic human Raf-1-estrogen receptor fusion gene (deltaRaf-1:ER). deltaRaf-1:ER transfectants in the presence of estradiol for 1 to 2 days expressed a differentiation phenotype only at the nonpermissive temperature. However, extended exposure of the deltaRaf-1:ER transfectants to estradiol or stable expression of the v-raf construct yielded cells that extended processes at the permissive as well as the nonpermissive temperature, suggesting that cells expressing the large T antigen are capable of responding to the Raf differentiation signal. deltaRaf-1:ER, MEK, and MAP kinase activities in the deltaRaf-1:ER cells were elevated constitutively for up to 36 h of estradiol treatment at the permissive temperature. At the nonpermissive temperature, MEK and ERKs were activated to a significantly lesser extent, suggesting that prolonged MAP kinase activation may not be sufficient for differentiation. To test this possibility, H19-7 cells were transfected or microinjected with constitutively activated MEK. The results indicate that prolonged activation of MEK or MAP kinases (ERK1 and -2) is not sufficient for differentiation of H19-7 neuronal cells and raise the possibility that an alternative signaling pathway is required for differentiation of H19-7 cells by Raf.

Animals↗
PubMed1995

Rapid induction of heparin-binding epidermal growth factor/diphtheria toxin receptor expression by Raf and Ras oncogenes.

We have used differential display PCR to search for mRNAs induced by delta Raf-1:ER, an estradiol-dependent form of Raf-1 kinase. Through this approach the gene encoding heparin-binding epidermal growth factor (HB-EGF) was identified as an immediate-early transcriptional target of oncogenic Raf kinases. Activation of delta Raf-1:ER and a conditional oncogenic form of B-Raf, delta B-RAF:ER, resulted in rapid and sustained induction of HB-EGF mRNA expression and secretion of mature HB-EGF from cells. Neutralizing anti-HB-EGF antisera prevented the delayed activation of the c-Jun amino-terminal kinases that is observed in cells transformed by delta Raf-1:ER. These results demonstrate that distinct signaling pathways can cross talk via the secretion of polypeptide growth factors. Furthermore, cells transformed by oncogenic Ras, which also induced HB-EGF expression, demonstrated a marked increase in sensitivity to the cytotoxic action of diphtheria toxin, for which the membrane anchored HB-EGF precursor acts as a cell-surface receptor.

3T3 Cells↗
PubMed1995

Phenotypic analysis of donor cells infiltrating the small intestinal epithelium and spleen during graft-versus-host disease.

One of the principal target organs during graft-versus-host disease (GvHD) is the intestinal epithelium, although the reasons for the preferential involvement of particular organs in this disease are not known. This study analyzed the subset distribution of donor and host lymphocytes in the small intestinal epithelium and the spleen during GvHD in a parent (C57BL/6J) into F1 (C57BL/6JxDBA2/J F1) model. While the donor cell population in the spleen consisted of B and T cells, the donor cell population in the intestine contained only T cells during the course of GvHD. These infiltrating donor cells resembled the host intraepithelial lymphocytes (IELs), which are predominantly CD8+ T cells. This subset distribution of donor cells in the intestinal epithelium was remarkable since they originated from a donor splenocyte population containing few CD8+ lymphocytes. In addition, although the injected donor splenic T cells were virtually all alpha/beta TCR+, several months after GvHD induction more than 30% of the donor cells in the intestine were gamma/delta TCR+, thereby resembling the host IELs not only in their expression of CD4 and CD8, but also in their TCR expression. In contrast, no gamma/delta TCR+ donor cells were detectable in the spleen of GvHD mice. The subset distribution of donor and host IELs remained constant throughout the disease, while in the spleen a decrease of both donor and host B cells and a temporary increase of both donor and host CD8+ cells was observed. These findings demonstrate that in a given target organ during GvHD the disease process affects both donor and host lymphoid populations. In addition the different tissue microenvironments eventually lead to donor cell repopulation with a subset distribution similar to the host natural lymphoid population of the particular target organ.

Animals↗
PubMed1994

Regulation of apoptosis in transgenic mice by simian virus 40 T antigen-mediated inactivation of p53.

Several proteins encoded by DNA tumor viruses are thought to disrupt cellular growth control by interacting with key cellular proteins, such as p53 and pRB, that normally function to regulate cell growth. However, the biological consequences of intracellular complexing between the viral oncoproteins and cellular proteins have remained unclear. Such complexes could either facilitate functional inactivation of the cellular proteins, leading to a loss-of-function phenotype, or could activate new functions, leading to a gain-of-function phenotype. Here we demonstrate that the simian virus 40 large tumor (T) antigen produces a loss-of-p53-function phenotype when introduced into the thymocytes of transgenic mice. Like thymocytes from the recently characterized p53-null mice, thymocytes from transgenic mice expressing a T-antigen variant capable of binding to p53 are resistant to irradiation-induced apoptosis. Thymocytes from transgenic mice expressing a mutant T antigen that is unable to complex p53, but retains the ability to complex the pRB and p107 proteins, retain sensitivity to irradiation. We further demonstrate that although irradiation-induced apoptosis is impaired by T antigen, clonal deletion of autoreactive thymocytes via p53-independent apoptosis is not perturbed by T antigen. These results provide convincing evidence that T antigen inactivates p53 in thymocytes in vivo and suggest a mechanism by which T antigen predisposes thymocytes to tumorigenesis in T antigen-transgenic mice.

Animals↗
PubMed1994

Activin-A binds to a heterotrimeric receptor complex on the vascular endothelial cell surface. Evidence for a type 3 activin receptor.

The effect of transfection of the type 2 activin receptor, ACTR2, on binding of 125I-activin-A to the surface of bovine aortic endothelial cells (BAEC) was investigated. BAEC transfected either with full-length ACTR2 or with a truncated form of ACTR2 lacking the intracellular kinase domain (ACTR2T) displayed two classes of 125I-activin-A binding sites, one of high affinity (Kd = 250-254 pM) and one of low affinity (Kd = 6.5-16 nM). Affinity labeling of ACTR2-transfected BAEC with 125I-activin-A revealed labeled species of 55, 95, 100, and 160 kDa, all four of which were immunoprecipitated by an anti-ACTR2 monoclonal antibody. Only the 95- and 100-kDa species, however, were immunoprecipitated following denaturation of the affinity-labeled cell lysate with SDS. BAEC transfected with an epitope-tagged form of ACTR2T (ACTR2TMyc) displayed intense 55- and 70-kDa affinity-labeled forms of the truncated receptor, together with a 160-kDa species. As with the full-length receptor, the 160-kDa species associated non-covalently with ACTR2TMyc. These data indicate that, in vascular endothelial cells, ACTR2 forms a high affinity heterotrimeric receptor complex with activin-binding proteins characteristic of type 1 and type 3 activin receptors, and that formation of the complex does not require the kinase domain of ACTR2.

Activin Receptors↗
PubMed1994

Granulocyte-macrophage colony-stimulating factor augmentation of T-cell receptor-dependent and T-cell receptor-independent thymocyte proliferation.

The effects of granulocyte-macrophage colony-stimulating factor (GM-CSF) are not confined to cells of the myeloid lineage. GM-CSF has been shown to have effects on mature T cells and both mature and immature T-cell lines. We therefore examined the GM-CSF responsiveness of murine thymocytes to investigate whether GM-CSF also affected normal immature T lymphocytes. The studies presented here indicate that GM-CSF augments accessory cell (AC)-dependent T-cell receptor (TCR)-mediated proliferation of unseparated thymocyte populations. To identify the GM-CSF responsive cell type, thymic AC and T cells were examined for GM-CSF responsiveness. We found that GM-CSF augmentation of TCR-induced thymocyte proliferation appears to be mediated via augmentation of AC function, and not via direct effects on mature single-positive (SP) thymocytes. Enriched double-negative (DN) thymocytes were also tested for GM-CSF responsiveness. GM-CSF induced the proliferation of adult and fetal DN thymocytes in an AC-independent and TCR-independent single-cell assay. Thus, in contrast to the SP thymocytes, a DN thymocyte population was directly responsive to GM-CSF. GM-CSF therefore may play a direct role in the expansion of DN thymocytes and an indirect role in the expansion of SP thymocytes.

Animals↗
PubMed1994

Macrophage synthesis of nitric oxide in the mouse mixed leucocyte reaction.

The production of nitric oxide (.N = O) in the splenocyte mixed leucocyte reaction (MLR) results in inhibition of allospecific lymphocyte effector function. In order to more clearly define the circumstances which promote .N = O synthesis in the MLR, responder accessory cell depleted spleen cells (ACDSC) were co-cultured with allogeneic macrophage cell lines or peritoneal macrophages. .N = O synthesis and C57BL/6 (H-2b) ACDSC proliferation were concurrently monitored in cultures comparing RAW 264.7 (H-2d, a high .N = O producer), P388D1 (H-2d, a low .N = O and BALB/c (H-2d) peritoneal macrophages as allogeneic antigen presenting cells (APC). A concentration-dependent increase in lymphocyte proliferation was observed in the presence of 1 x 10(4) to 1 x 10(5) P388D1. In contrast, addition of NG-monomethyl-L-arginine (NMA), a competitive inhibitor of .N = O synthase, was necessary in order to observe lymphocyte proliferation in the presence of increasing numbers of RAW 264.7 and BALB/c peritoneal macrophages. The addition of both anti-IL-2 and anti-IFN gamma (interferon-gamma) monoclonal antibodies inhibited .N = O synthesis in alloantigen-stimulated cultures. The IFN gamma induced expression of class II antigen, as well as the constitutive expression of class I antigen, on RAW 264.7 was similar in the presence or absence of NMA, indicating that induction of .N = O synthesis by IFN gamma does not inhibit H-2 antigen expression. Thus, cytokines produced as a result of alloimmune interaction initiate macrophage .N = O synthesis. However, allogeneic APC function, as assessed by H-2 antigen expression and subsequent stimulatory capacity of MLR, is not affected by initiation of the .N = O pathway.

Animals↗
PubMed1994

Monoclonal antibodies that recognize the type-2 activin receptor, ACTR2.

Monoclonal antibodies (MAbs) were raised in mice against a bacterial fusion protein composed of the intracellular serine/threonine kinase domain of the type-2 activin receptor, ACTR2, fused to glutathione S-transferase. Three MAbs with high affinity toward the ACTR2 kinase domain were isolated, one of which recognized specifically ACTR2 expressed transiently in vascular endothelial cells. These reagents should be of use in the elucidation of mechanisms of transmembrane signaling by this member of the emerging receptor serine threonine kinase family.

Activin Receptors↗
PubMed1994

Characterization of an antibiotic produced by Alteromonas luteoviolacea Gauthier 1982, 85 isolated from Kinko Bay, Japan.

An antibiotic produced by Alteromonas luteoviolacea strain 9K-V10 was recovered after cold acetone precipitation of culture supernatant fluids or lysates that had been frozen and thawed. The precipitate obtained from cell-free lysates was fractioned by DEAE ion-exchange chromatography. Further purification by gel-filtration chromatography yielded a single peak of antibiotic activity that corresponded to a protein peak with a molecular mass of approximately 100 kDa. After non-denaturing polyacrylamide gel electrophoresis, antibiotic activity co-migrated with a protein band. The isoelectric point of the antibiotic was estimated to be 7.7. Treatment of the concentrated active fraction with proteinase K or heating at 70 degrees C for 10 min resulted in total loss of antibiotic activity. These results show that the antibiotic produced by Alt. luteoviolacea 9K-V10 is of a proteinaceous nature.

Anti-Bacterial Agents↗
PubMed1994

International dissemination of epidemic Vibrio cholerae by cargo ship ballast and other nonpotable waters.

In 1991 and 1992, toxigenic Vibrio cholerae O1, serotype Inaba, biotype El Tor, was recovered from nonpotable (ballast, bilge, and sewage) water from five cargo ships docked in ports of the U.S. Gulf of Mexico. Four of these ships had taken on ballast water in cholera-infected countries; the fifth took on ballast in a noninfected country. Isolates examined by pulsed-field gel electrophoresis were indistinguishable from the Latin American epidemic strain, C6707; however, they differed significantly from the endemic Gulf Coast strain (VRL 1984), the sixth-pandemic strain (569-B), and a V. cholerae non-O1 strain isolated from a ship arriving from a foreign port. On the basis of our findings, the Food and Drug Administration recommended that the U.S. Coast Guard issue an advisory to shipping agents and captains requesting that ballast waters be exchanged on the high seas before entry of ships into U.S. ports.

Cholera↗
PubMed1993

Inhibition of vascular endothelial cell growth by activin-A.

The demonstration of type 2 activin receptor expression in human umbilical vein endothelial cells prompted an investigation of the effects of the activin/inhibin family of hormones on vascular endothelial cell growth. Recombinant activin-A inhibited [3H]methylthymidine uptake and growth of a panel of endothelial cell types; recombinant inhibin-A was without effect. Affinity cross-linking studies demonstrated the presence of type 1 and type 2 activin receptors on the surface of bovine aortic endothelial cells, while detailed analysis of type 2 activin receptor expression revealed both type 2 and type 2B activin receptor mRNA in all endothelial cell types analyzed. In addition, capillary endothelial cells were found to express activin-beta A subunit mRNA and protein, the levels of which were increased in response to transforming growth factor (TGF)-beta. Furthermore, activin-A and TGF-beta caused additive inhibition of capillary endothelial cell [3H]methylthymidine uptake. These findings implicate the activins in the regulation of endothelial cell function, and suggest that TGF-beta and activin may interact to inhibit capillary endothelial cell growth.

Activin Receptors↗
PubMed1993

Characterization of the immunosuppressive effects of nitric oxide in graft vs host disease.

The generation of nitric oxide (.N = O) during in vitro assays involving lymphocyte-macrophage interaction can result in profound inhibition of lymphocyte proliferation. The present study examined whether .N = O synthesis plays a role in the suppression observed in immune function assays during graft vs host disease (GvHD). By using a parent to F1 model to induce GvHD (C57BL/6J to C57BL/6J x DBA 2J F1), a mild but transient increase in serum NO2- plus NO3- levels was observed on day 12 after inoculation. Resident peritoneal macrophages obtained from mice with GvHD demonstrated enhanced .N = O synthesis in response to LPS, compared with control F1 peritoneal macrophages. Similarly, when splenocytes from GvHD mice were cultured with Con A or LPS enhanced supernatant NO2- levels were observed, compared with control F1 mice. Addition of NG-monomethyl-L-arginine (NMA), a competitive inhibitor of .N = O synthesis, resulted in decreased NO2- levels and greatly enhanced proliferation in response to Con A. Addition of NMA to LPS-stimulated cultures did not enhance proliferation, perhaps as the result of the paucity of B cells in the GvHD population. LPS-induced .N = O synthesis by GvHD splenocytes was blocked by anti-IFN-gamma mAb, whereas Con A-induced .N = O synthesis was relatively unaffected by similar concentrations of anti-IFN-gamma mAb, suggesting different mechanisms of induction of .N = O synthesis. A proliferative response of splenocytes from mice with GvHD to third-party alloantigen was not detectable, even in the presence of NMA. The suppression observed when splenocytes from GvHD animals were added to control TNP-modified self cultures was partially reversed in the presence of NMA. These results demonstrate that .N = O synthesis in both splenocyte and peritoneal macrophage populations from GvHD mice is enhanced, revealing that in vivo priming of macrophages for .N = O synthesis occurs during GvHD. Some, but not all, in vitro tests of immune function by using GvHD splenocytes are suppressed by the generation of .N = O.

Animals↗
PubMed1993

Effect of granulocyte-macrophage colony-stimulating factor on lymphokine-activated killer cell induction.

The treatment of cancer with lymphokine-activated killer (LAK) cells in conjunction with high-dose interleukin-2 (IL-2) has been limited by the toxicity of IL-2 and the narrow range of tumors that respond to therapy. Cytokines that are capable of augmenting lower doses of IL-2 are, therefore, a major focus of research. We report here that granulocyte-macrophage colony-stimulating factor (GM-CSF) can augment low-dose IL-2 LAK induction from murine splenocytes. Anti-tumor necrosis factor alpha (anti-TNF alpha) or anti-interferon gamma (anti-IFN gamma) monoclonal antibodies did not inhibit (IL-2 + GM-CSF)-induced LAK generation, indicating that GM-CSF augmentation does not require TNF alpha or IFN gamma activity. Depletion of natural killer cells before culture did not inhibit low-dose IL-2-induced LAK generation or the ability of GM-CSF to augment LAK generation. In contrast, depletion of both CD4+ and CD8+ T cells before culture inhibited the generation of LAK activity. However, depletion of only CD4+ T cells, or only CD8+ T cells, did not inhibit the generation of IL-2 or (IL-2 + GM-CSF) LAK activity. These results suggest that LAK precursors are present in both the CD4+ and CD8+ T-cell populations and suggest that the addition of GM-CSF to low-dose IL-2 may result in the generation of T-derived LAK cells.

Animals↗