Sex significantly influences transduction of murine liver by recombinant adeno-associated viral vectors through an androgen-dependent pathway.

A systematic evaluation of the influence of sex on transduction by recombinant adeno-associated viral vector (rAAV) indicated that transgene expression after liver-targeted delivery of vector particles was between 5- to 13-fold higher in male mice compared with female mice, irrespective of the proviral promoter or cDNA and mouse strain. Molecular analysis revealed that the rAAV genome was stably retained in male liver at levels that were 7-fold higher than those observed in females. Further, the sex difference in transduction was observed with AAV-2- and AAV-5-based vectors, which use distinct receptor complexes for infection. In concordance with the differences in AAV transduction, gel shift analysis with nuclear extracts derived from the liver of mice and humans revealed substantially higher binding of host nuclear protein to the rep-binding site (RBS) of AAV inverted terminal repeat (ITR) in males compared with females. Transduction efficiency and binding of nuclear protein to RBS was dramatically reduced in male mice by castration. In contrast, although oophorectomy did not significantly influence rAAV transduction, administration of 5alpha dihydrotestosterone, prior to gene transfer, increased stable hepatocyte gene transfer in females to levels observed in male mice, implying that androgens significantly influence hepatocyte gene transfer. Interestingly, sex did not have a significant effect on AAV gene transfer into nonhepatic tissue, indicating that there are distinct tissue- and sex-specific differences in the mechanisms responsible for efficient transduction with this vector. These results have significant implications for gene therapy of autosomal and acquired disorders affecting the liver

Sustained high-level expression of human factor IX (hFIX) after liver-targeted delivery of recombinant adeno-associated virus encoding the hFIX gene in rhesus macaques

The feasibility, safety, and efficacy of liver-directed gene transfer was evaluated in 5 male macaques (aged 2.5 to 6.5 years) by using a recombinant adeno-associated viral (rAAV) vector (rAAV-2 CAGG-hFIX) that had previously mediated persistent therapeutic expression of human factor IX (hFIX; 6%-10% of physiologic levels) in murine models. A dose of 4 × 1012 vector genomes (vgs)/kg of body weight was administered through the hepatic artery or portal vein. Persistence of the rAAV vgs as circular monomers and dimers and high-molecular-weight concatamers was documented in liver tissue by Southern blot analysis for periods of up to 1 year. Vector particles were present in plasma, urine, or saliva for several days after infusion (as shown by polymerase chain reaction analysis), and the vgs were detected in spleen tissue at low copy numbers. An enzyme-linked immunosorption assay capable of detecting between 1% and 25% of normal levels of hFIX in rhesus plasma was developed by using hyperimmune serum from a rhesus monkey that had received an adenoviral vector encoding hFIX. Two macaques having 3 and 40 rAAV genome equivalents/cell, respectively, in liver tissue had 4% and 8% of normal physiologic plasma levels of hFIX, respectively. A level of hFIX that was 3% of normal levels was transiently detected in one other macaque, which had a genome copy number of 25 before abrogation by a neutralizing antibody (inhibitor) to hFIX. This nonhuman-primate model will be useful in further evaluation and development of rAAV vectors for gene therapy of hemophilia B. © 2002 by The American Society of Hematology

Human anti-dsDNA antibodies induce amplification loop of TGF-β1, fibronectin and collagen type I in proximal renal tubular epithelial cells

Thursday Poster: TH-PO584BACKGROUND: Nephritis affects up to 60% of patients with systemic lupus erythematosus and is characterized by the production of anti-dsDNA antibodies and immune-mediated renal injury. Severity of tubulointerstitial fibrosis is a strong predictor of reduced renal survival. Fibronectin (FN) is amongst the matrix components showing early deposition during the process of tubulointerstitial fibrosis. This study investigates the mechanisms through which FN is induced in renal proximal tubular epithelial cells (PTEC) following stimulation with human anti-dsDNA antibodies, and its role in tubulointerstitial fibrosis of lupus nephritis. METHODS: Confluent growth-arrested PTEC were incubated with serum free medium (SFM), control IgG or human polyclonal anti-dsDNA antibodies (10µg/ml) for 24h in the presence or absence of Gö6976 or TGF-neutralizing antibody, and the synthesis of FN assessed. In separate studies, PTEC were stimulated with soluble FN to determine its effect on fibrotic processes. RESULTS: Control IgG had no effect on FN synthesis compared to SFM. Anti-dsDNA antibodies significantly increased cell-associated and soluble FN compared to SFM and control IgG (6.4- and 5.2-fold respectively for cell-associated FN; 1.85- and 1.62-fold respectively for soluble FN, P<0.01 for all). This was accompanied by significantly increased TGF-β1 secretion (P<0.05), and increased PKC-α (P<0.05) and PKC-βII (P<0.01) but not PKC-βI phosphorylation. Pre-treatment of PTEC with Gö6976 (20µM) and TGF-neutralizing antibody (100g/ml) significantly suppressed anti-dsDNA antibody-induced FN (P<0.01 for both cell-associated and soluble FN). Incubation of PTEC with soluble FN significantly increased TGF-β1 secretion and collagen type I synthesis in a dose-dependent manner (P<0.05 and P<0.01 respectively for 10µg/ml soluble FN). CONCLUSIONS: These results suggest that anti-dsDNA antibodies induce an amplification loop comprising TGF-β1, FN and collagen type I in PTEC through PKC activation.link_to_OA_fulltex

Anti-dsDNA Antibodies Bind to Ku70 Leading to Increased MCP-1 and TGF-beta1 Expression in Proximal Tubular Epithelial Cells

Poster Session: Renal Pathology, Experimental Pathology, incl. Immune and Inflammatory Mechanisms - Presentation no. MON-263Introduction: Lupus nephritis is characterized by anti-dsDNA antibody production and their deposition in the kidney parenchyma resulting in immune-mediated tissue injury. Immune deposition along the tubular basement membrane is commonly observed but its pathogenic significance remains to be determined. We investigated the binding of anti-dsDNA antibodies to proximal tubular epithelial cells (PTEC) and its impact on inflammatory and fibrotic processes. Methods: Anti-dsDNA antibodies with high binding activity to PTEC were isolated from the sera of lupus nephritis patients using affinity chromatography. Cultured PTEC were incubated with serum free medium (SFM), control IgG, or anti-dsDNA antibodies in the presence or absence of DNase I, trypsin, exogenous dsDNA, histones and nucleosomes. PTEC plasma membrane proteins were isolated and immunoprecipitated with anti-dsDNA antibodies to identify cross-reactive antigens using LC-MS/MS. Results: DNase I, dsDNA, histones and nucleosomes had no effect on anti-dsDNA antibody binding to PTEC. Limited trypsin treatment of PTEC, which removed cell surface proteins without affecting cell attachment, resulted in a significant reduction in anti-dsDNA antibody binding (P<0.01). Anti-dsDNA antibodies bound to a 70 kDa protein identified as Ku70 by LC-MS/MS. Incubation of PTEC with anti-dsDNA antibodies increased Ku70 expression in a time-dependent manner, accompanied by increased MCP-1 and TGF-beta1 expression. Gene silencing with Ku70 specific RNAi suppressed Ku70 mRNA by approximately 85.0%, accompanied by 79.4% and 33.9% reduction in MCP-1 and TGF-beta1 gene expression respectively (P<0.05, for both). Kidney biopsies from lupus nephritis patients showed a marked increase in Ku70 expression, predominantly in proximal tubular epithelial cells. Conclusions: Our data showed that anti-dsDNA antibodies bound directly to PTEC through Ku70, and this binding was accompanied by downstream pro-inflammatory and pro-fibrotic processes. These findings could have important implications on tubulo-interstitial disease in lupus nephritis

Balance performance in irradiated survivors of nasopharyngeal cancer with and without tai chi qigong training

2014-2015 > Academic research: refereed > Publication in refereed journalVersion of RecordPublishe

Identification of blood meal sources of Lutzomyia longipalpis using polymerase chain reaction-restriction fragment length polymorphism analysis of the cytochrome B gene

An analysis of the dietary content of haematophagous insects can provide important information about the transmission networks of certain zoonoses. The present study evaluated the potential of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of the mitochondrial cytochrome B (cytb)gene to differentiate between vertebrate species that were identified as possible sources of sandfly meals. The complete cytb gene sequences of 11 vertebrate species available in the National Center for Biotechnology Information database were digested with Aci I, Alu I, Hae III and Rsa I restriction enzymes in silico using Restriction Mapper software. The cytb gene fragment (358 bp) was amplified from tissue samples of vertebrate species and the dietary contents of sandflies and digested with restriction enzymes. Vertebrate species presented a restriction fragment profile that differed from that of other species, with the exception of Canis familiaris and Cerdocyon thous. The 358 bp fragment was identified in 76 sandflies. Of these, 10 were evaluated using the restriction enzymes and the food sources were predicted for four: Homo sapiens (1), Bos taurus (1) and Equus caballus (2). Thus, the PCR-RFLP technique could be a potential method for identifying the food sources of arthropods. However, some points must be clarified regarding the applicability of the method, such as the extent of DNA degradation through intestinal digestion, the potential for multiple sources of blood meals and the need for greater knowledge regarding intraspecific variations in mtDNA