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.

Intracellular adaptation of Brucella abortus.

Macrophages were infected with virulent Brucella abortus strain 2308 or attenuated strain 19. Intracellular bacteria were recovered at different times after infection and their proteomes compared. The virulent strain initially reduced most biosynthesis and altered its respiration; adaptations reversed later in infection. The attenuated strain was unable to match the magnitude of the virulent strain's adjustments. The results provide insight into mechanisms utilized by Brucella to establish intracellular infections.

Inhibition of experimental allergic airways disease by local application of a cell-penetrating dominant-negative STAT-6 peptide.

Allergic airways disease is initiated and perpetuated by an aberrant Th2 inflammatory response regulated in part by the cytokines IL-4 and IL-13, each of which induces activation of the STAT-6 transcription factor. Data from murine models indicate that the clinical manifestations of acute asthma are STAT-6 dependent, and thus, STAT-6 is a target for drug development in allergic airways disease. We designed a novel chimeric peptide (STAT-6 inhibitory peptide (STAT-6-IP)) comprised of a sequence predicted to bind to and inhibit STAT-6, fused to a protein transduction domain, to facilitate cellular uptake of the STAT-6-binding peptide. Our data demonstrate that the STAT-6-IP inhibited OVA-induced production of Th2 cytokines IL-4 and IL-13 in vitro. In contrast, the STAT-6-IP did not affect production of IFN-gamma, demonstrating specificity for Th2 cytokine inhibition. Following intranasal administration, the STAT-6-IP was localized to epithelial cells in the airways. Finally, in in vivo murine models of allergic rhinitis and asthma, intranasal delivery of the STAT-6-IP inhibited OVA-induced lung inflammation and mucus production as well as accumulation of eosinophils and IL-13 in bronchoalveolar lavage fluid and OVA-dependent airway hyperresponsiveness. Together these data show that local application of cell-penetrating peptide inhibitors of STAT-6 has significant potential for the treatment of allergic rhinitis and asthma.

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.