Biomedical subjects

Elizabeth D Fixman

Publications and source records attributed to Elizabeth D Fixman.

7 recordsLinked to original sources

PubMed2006

Enhanced transduction of antigen-stimulated T lymphocytes with recombinant retroviruses concentrated by centrifugal filtration.

Retroviral gene transduction of antigen-specific T cells and reintroduction of the gene-modified T cells into animals or human subjects is attractive for experimental disease-modeling applications and gene therapy approaches for autoimmune or allergic diseases. However, retrovirus titers are often a limiting factor for the efficient gene transfer of mature T cells, which have proven to be relatively refractory to gene transduction. Retrovirus-containing supernatants with titers sufficient for effective transduction of immortalized T cell lines may fail to transduce peripheral T cells. The use of high-titer retroviruses pseudotyped with vesicular stomatitis virus G protein and concentrated by ultracentrifugation is limited by the loss of specific tropism, lower lymphocyte transduction efficiency on infectious particle basis and pseudotransduction. Herein, we present a simple method to concentrate retroviruses by centrifugal filtration at low g force. We compared the ability of unconcentrated and concentrated retroviruses to transduce immortalized fibroblasts as well as primary rat splenocytes activated with antigen and we evaluated transduction efficiency and mean fluorescence intensity of transgene expression in transduced cells. Our data demonstrate that, with this technique, retrovirus titers were increased nearly 10-fold without significant loss of infectious particles. Compared to unconcentrated retroviral preparations, the concentrated retrovirus supernatants more effectively transduced antigen-stimulated, primary rat T cells. This simple method of concentrating retroviruses may be exploited to generate gene-modified T cells for gene therapy applications in animal models of human autoimmune or allergic disease and may also be applicable for T lymphocyte-based gene therapy approaches in humans.

Animals↗

PubMed2006

Endothelin-1 regulates proliferative responses, both alone and synergistically with PDGF, in rat tracheal smooth muscle cells.

The peptide, endothelin-1 (ET-1) regulates proliferative responses in numerous cell types. Recently, a dual ET receptor antagonist was shown to prevent the increase in airway smooth muscle cell (SMC) proliferation that accompanies airway smooth muscle remodeling in a rat model of experimental asthma. Thus, we used [(3)H]-thymidine incorporation assays and western immunoblotting to identify signaling pathways that regulate proliferative responses in cultured rat tracheal SMC. Our data indicate that ET-1 activation of the ET A receptor subtype induced [(3)H]-thymidine incorporation and activation of ERK 1/2 in primary rat tracheal SMC. ET-1-induced [(3)H]-thymidine incorporation and activation of ERK 1/2 were inhibited by pretreatment of SMC with pertussis toxin or down regulation of phorbol ester responsive isoforms of PKC. While ET- 1-induced ERK 1/2 activation was unaffected following inhibition of Rho kinase, ET-1-induced [(3)H]-thymidine incorporation was abrogated. ET-1 also potentiated [(3)H]-thymidine incorporation as well as cell proliferation of SMC stimulated with PDGF-BB and this response did not appear to be regulated by ERK1/ 2. These data demonstrate that ET-1 induces activation of multiple G proteins that regulate rat tracheal SMC proliferative responses, likely through signaling pathways downstream of ERK1/2 and Rho kinase.

Animals↗

PubMed2005

Antigen-specific CD4+ T cells drive airway smooth muscle remodeling in experimental asthma.

Airway smooth muscle (ASM) growth contributes to the mechanism of airway hyperresponsiveness in asthma. Here we demonstrate that CD4+ T cells, central to chronic airway inflammation, drive ASM remodeling in experimental asthma. Adoptive transfer of CD4+ T cells from sensitized rats induced an increase in proliferation and inhibition of apoptosis of airway myocytes in naive recipients upon repeated antigen challenge, which resulted in an increase in ASM mass. Genetically modified CD4+ T cells expressing enhanced GFP (EGFP) were localized by confocal microscopy in juxtaposition to ASM cells, which suggests that CD4+ T cells may modulate ASM cell function through direct cell-cell interaction in vivo. Coculture of antigen-stimulated CD4+ T cells with cell cycle-arrested ASM cells induced myocyte proliferation, dependent on T cell activation and direct T cell-myocyte contact. Reciprocally, direct cell contact prevented postactivation T cell apoptosis, which suggests receptor-mediated T cell-myocyte crosstalk. Overall, our data demonstrate that activated CD4+ T cells drive ASM remodeling in experimental asthma and suggest that a direct cell-cell interaction participates in CD4+ T cell regulation of myocyte turnover and induction of remodeling.

Animals↗

PubMed2005

The Epstein-Barr virus replication protein BBLF2/3 provides an origin-tethering function through interaction with the zinc finger DNA binding protein ZBRK1 and the KAP-1 corepressor.

Herpesviruses encode a set of core proteins essential for lytic replication of their genomes. Three of these proteins form a tripartite helix-primase complex that, in the case of Epstein-Barr virus (EBV), consists of the helicase BBLF4, the primase BSLF1, and the linker protein BBLF2/3. BBLF2/3 and its homologs in the other herpesviruses remain relatively poorly characterized. To better understand the contribution to replication made by BBLF2/3, a yeast two-hybrid screen was performed with BBLF2/3 as the bait protein. This screen identified as interactors a number of cell replication-related proteins such as DNA polymerase beta and subunits of DNA polymerase delta along with the EBV-encoded DNase BGLF5. The screen also identified the DNA binding zinc finger protein ZBRK1 and the ZBRK1 corepressor KAP-1 as BBLF2/3 interactors. Interaction between BBLF2/3 and ZBRK1 and KAP-1 was confirmed in coimmunoprecipitation assays. A binding site for ZBRK1 in the EBV oriLyt enhancer was identified by electrophoretic mobility shift assay. ZBRK1, KAP-1, and the ZBRK1 binding protein BRCA1 were shown by indirect immunofluorescence to be present in replication compartments in lytically induced D98-HR1 cells, and additionally, chromatin immunoprecipitation assays determined that these proteins associated with oriLyt DNA. Replication of an oriLyt plasmid and a variant oriLyt (DeltaZBRK1) plasmid was examined in lytically induced D98-HR1 cells. Exogenous ZBRK1, KAP-1, or BRCA1 increased the efficiency of oriLyt replication, while deletion of the ZBRK1 binding site impaired replication. These experiments identify ZBRK1 as another cell protein that, through BBLF2/3, provides a tethering point on oriLyt for the EBV replication complex. The data also suggest that BBLF2/3 may serve as a contact interface for cell proteins involved in replication of EBV oriLyt.

Animals↗

PubMed2004

Proliferative aspects of airway smooth muscle.

Increased airway smooth muscle (ASM) mass is perhaps the most important component of the airway wall remodeling process in asthma. Known mediators of ASM proliferation in cell culture models fall into 2 categories: those that activate receptors with intrinsic receptor tyrosine kinase activity and those that have their effects through receptors linked to heterotrimeric guanosine triphosphate-binding proteins. The major candidate signaling pathways activated by ASM mitogens are those dependent on extracellular signal-regulated kinase and phosphoinositide 3'-kinase. Increases in ASM mass may also involve ASM migration, and in culture, the key signaling mechanisms have been identified as the p38 mitogen-activated protein kinase and the p21-activated kinase 1 pathways. New evidence from an in vivo rat model indicates that primed CD4(+) T cells are sufficient to trigger ASM and epithelial remodeling after allergen challenge. Hyperplasia has been observed in an equine model of asthma and may account for the increase in ASM mass. Reduction in the rate of apoptosis may also play a role. beta(2)-Adrenergic receptor agonists and glucocorticoids have antiproliferative activity against a broad spectrum of mitogens, although it has become apparent that mitogens are differentially sensitive. Culture of ASM on collagen type I has been shown to enhance proliferative activity and prevent the inhibitory effect of glucocorticoids, whereas beta(2)-agonists are minimally affected. There is no evidence that long-acting beta(2)-agonists are more effective than short-acting agonists, but persistent stimulation of the beta(2)-adrenergic receptor probably helps suppress growth responses. The maximum response of fluticasone propionate against thrombin-induced proliferation is increased when it is combined with salmeterol.

Adrenergic beta-Agonists↗

PubMed2002

Suppression of IL-4- and CD40-induced B-lymphocyte activation by intravenous immunoglobulin is not mediated through the inhibitory IgG receptor FcgammaRIIb.

BACKGROUND: Intravenous immunoglobulin (IVIG) has been used extensively in the treatment of autoimmune and allergic diseases, but the precise mechanism behind its efficacy remains unclear. Ligation of the low-affinity IgG Fc receptor FcgammaRIIb can inhibit B-lymphocyte activation. Our laboratory has shown that IVIG suppresses proliferation and IgE production by human B cells stimulated with IL-4 and anti-CD40 antibodies. OBJECTIVE: We sought to determine whether the regulatory action of IVIG is mediated through binding FcgammaRIIb, phosphorylation of the receptor, and induction of phosphatases, including SH2-containing inositol-5'-phosphatase. METHODS: All experiments were performed on human tonsillar B cells. Phenotyping was performed by means of flow cytometry. Cells were cultured with IL-4 and anti-CD40 antibodies with or without IVIG (10 mg/mL), and FCgammaRIIb receptor activation and phosphorylation were measured by means of Western blot analysis. RESULTS: FcgammaRIIb was the predominant isoform of Fcgamma receptor expressed on tonsillar B cells, and preincubation with IVIG failed to block binding of FcgammaRIIb antibody. Anti-FcgammaRIIb antibodies did not reverse inhibition of B-cell proliferation or IgE production by IVIG. Treatment of stimulated B lymphocytes with IVIG for 1 to 60 minutes did not change the global protein tyrosine phosphorylation pattern, except for tyrosine phosphorylation of an unidentified 30-kd protein. We directly examined tyrosine phosphorylation of FcgammaRIIb and its downstream-associated phosphatase, SH2-containing inositol-5'-phosphatase. Both remained unchanged after IVIG treatment, as did other related phosphatases. CONCLUSION: These data argue against the involvement of FcgammaRIIb in the inhibition of CD40/IL-4-induced B-cell activation by IVIG.

Antigens, CD↗

PubMed2002

Src modulates serotonin-induced calcium signaling by regulating phosphatidylinositol 4,5-bisphosphate.

We tested the hypothesis that, in airway smooth muscle cells, stimulation of G-protein-coupled receptors by contractile agonists activates Src kinase and that this kinase modulates cell contractility and Ca(2+) signaling by affecting the levels of the phospholipase C substrate phosphatidylinositol 4,5-bisphosphate (PIP(2)). Stimulation of cultured rat tracheal smooth muscle cells with serotonin (5-HT) induced an increase in Src activity, Ca(2+) mobilization, and contraction (decrease in cell area). 5-HT-evoked cell contraction was reduced by a specific inhibitor of Src family kinases, 4-amino-5(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP1). Peak Ca(2+) responses to 5-HT were attenuated by PP1 and an anti-Src-blocking antibody and augmented by expression of constitutively activated Y529F Src. Sustained phases of Ca(2+) responses to 5-HT and Ca(2+) influx resulting from emptying of Ca(2+) stores in the endoplasmic reticulum by thapsigargin were also decreased after PP1 treatment. PP1 significantly reduced the turnover of inositol phosphates produced on 5-HT stimulation and the amount of PIP(2) in the Triton X-100-insoluble lipid fraction. Overall, these data demonstrate that, in rat tracheal smooth muscle cells, Src kinase modulates 5-HT-evoked cell contractility and Ca(2+) signaling by regulating PIP(2) levels and Ca(2+) influx.

Animals↗