Intravenous delivery of HIV-based lentiviral vectors preferentially transduces F4/80+ and Ly-6C+ cells in spleen, important target cells in autoimmune arthritis

Contains fulltext : 118684.pdf (publisher's version ) (Open Access)Antigen presenting cells (APCs) play an important role in arthritis and APC specific gene therapeutic targeting will enable intracellular modulation of cell activity. Viral mediated overexpression is a potent approach to achieve adequate transgene expression levels and lentivirus (LV) is useful for sustained expression in target cells. Therefore, we studied the feasibility of lentiviral mediated targeting of APCs in experimental arthritis. Third generation VSV-G pseudotyped self-inactivating (SIN)-LV were injected intravenously and spleen cells were analyzed with flow cytometry for green fluorescent protein (GFP) transgene expression and cell surface markers. Collagen-induced arthritis (CIA) was induced by immunization with bovine collagen type II in complete Freund's adjuvant. Effect on inflammation was monitored macroscopically and T-cell subsets in spleen were analyzed by flow cytometry. Synovium from arthritic knee joints were analyzed for proinflammatory cytokine expression. Lentiviruses injected via the tail vein preferentially infected the spleen and transduction peaks at day 10. A dose escalating study showed that 8% of all spleen cells were targeted and further analysis showed that predominantly Ly6C+ and F4/80+ cells in spleen were targeted by the LV. To study the feasibility of blocking TAK1-dependent pathways by this approach, a catalytically inactive mutant of TAK1 (TAK1-K63W) was overexpressed during CIA. LV-TAK1-K63W significantly reduced incidence and arthritis severity macroscopically. Further histological analysis showed a significant decrease in bone erosion in LV-TAK1-K63W treated animals. Moreover, systemic Th17 levels were decreased by LV-TAK1-K63W treatment in addition to diminished IL-6 and KC production in inflamed synovium. In conclusion, systemically delivered LV efficiently targets monocytes and macrophages in spleen that are involved in autoimmune arthritis. Moreover, this study confirms efficacy of TAK1 targeting in arthritis. This approach may provide a valuable tool in targeting splenic APCs, to unravel their role in autoimmune arthritis and to identify and validate APC specific therapeutic targets

Myeloid cell expression of the RNA-binding protein HuR protects mice from pathologic inflammation and colorectal carcinogenesis

The innate immune response involves a variety of inflammatory reactions that can result in inflammatory disease and cancer if they are not resolved and instead are allowed to persist. The effective activation and resolution of innate immune responses relies on the production and posttranscriptional regulation of mRNAs encoding inflammatory effector proteins. The RNA-binding protein HuR binds to and regulates such mRNAs, but its exact role in inflammation remains unclear. Here we show that HuR maintains inflammatory homeostasis by controlling macrophage plasticity and migration. Mice lacking HuR in myeloid-lineage cells, which include many of the cells of the innate immune system, displayed enhanced sensitivity to endotoxemia, rapid progression of chemical-induced colitis, and severe susceptibility to colitis-associated cancer. The myeloid cell-specific HuR-deficient mice had an exacerbated inflammatory cytokine profile and showed enhanced CCR2-mediated macrophage chemotaxis. At the molecular level, activated macrophages from these mice showed enhancements in the use of inflammatory mRNAs (including Tnf, Tgfb, Il10, Ccr2, and Ccl2) due to a lack of inhibitory effects on their inducible translation and/or stability. Conversely, myeloid overexpression of HuR induced posttranscriptional silencing, reduced inflammatory profiles, and protected mice from colitis and cancer. Our results highlight the role of HuR as a homeostatic coordinator of mRNAs that encode molecules that guide innate inflammatory effects and demonstrate the potential of harnessing the effects of HuR for clinical benefit against pathologic inflammation and cancer

Identification of high frequency of Y chromosome deletions in patients with sex chromosome mosaicism and correlation with the clinical phenotype and Y-chromosome instability

A mosaic karyotype consisting of a 45,X cell line and a second cell line containing a normal or an abnormal Y chromosome is relatively common and is associated with a wide spectrum of clinical phenotypes. The aim of this study was to investigate patients with such a mosaic karyotype for Y chromosome material loss and then study the possible association of the absence of these regions with the phenotype, diagnosis, and Y-chromosome instability. We studied 17 clinically well-characterized mosaic patients whose karyotype consisted of a 45,X cell line and a second cell line containing a normal or an abnormal Y chromosome. The presence of the Y chromosome centromere was verified by fluorescence in situ hybridization (FISH) and was then characterized by 44 Y-chromosome specific-sequence tagged site (STS) markers. This study identifies a high frequency of Yq chromosome deletions (47%). The deletions extend from interval 5 to 7 sharing a common deleted interval (6F), which overlaps with the azoospermia factor region (AZF) region. This study finds no association between Y-chromosome loci hosting genes other than SRY, and the phenotypic sex, the diagnosis, and the phenotype of the patients. Furthermore, this study shows a possible association of these deletions with Y-chromosome instability. (c) 2005 Wiley-Liss, Inc

Generation and characterization of p38beta (MAPK11) gene-targeted mice

p38 mitogen-activated protein kinases (MAPKs) are activated primarily in response to inflammatory cytokines and cellular stress, and inhibitors which target the p38α and p38β MAPKs have shown potential for the treatment of inflammatory disease. Here we report the generation and initial characterization of a knockout of the p38β (MAPK11) gene. p38β(−/−) mice were viable and exhibited no apparent health problems. The expression and activation of p38α, ERK1/2, and JNK in response to cellular stress was normal in embryonic fibroblasts from p38β(−/−) mice, as was the activation of p38-activated kinases MAPKAP-K2 and MSK1. The transcription of p38-dependent immediate-early genes was also not affected by the knockout of p38β, suggesting that p38α is the predominant isoform involved in these processes. The p38β(−/−) mice also showed normal T-cell development. Lipopolysaccharide-induced cytokine production was also normal in the p38β(−/−) mice. As p38 is activated by tumor necrosis factor, the p38β(−/−) mice were crossed onto a TNFΔARE mouse line. These mice overexpress tumor necrosis factor, which results in development symptoms similar to rheumatoid arthritis and inflammatory bowel disease. The progression of these diseases was not however moderated by knockout of p38β. Together these results suggest that p38α, and not p38β, is the major p38 isoform involved in the immune response and that it would not be necessary to retain activity against p38β during the development of p38 inhibitors

Candidate gene association study of insulin signaling genes and Alzheimer's disease: Evidence for SOS2, PCK1, and PPAR gamma as susceptibility loci

Epidemiological evidence supports the existence of a possible link between type II diabetes mellitus (T2DM) and late-onset Alzheimer's disease (LOAD). Polymorphisms from candidate genes for T2DM were genotyped in a two-stage approach to identify novel risk factors for LOAD. One hundred fifty-two polymorphisms were initially genotyped in a case:control cohort: nine SNPs showed individual association with disease status under at least one genetic model, while an additional two SNPs showed a haplotype association. In a replication study, we confirmed significant association of SNPs within three genes-PPAR gamma, SOS2, and PCK1-with Alzheimer's disease. In particular, our data suggest that the effect of variants within these genes might be influenced by gender. (c) 2007 Wiley-Liss, In