Steven H. Larsen

Hepatitis C infection and related factors in hemodialysis patients in china: systematic review and meta-analysis.

Aims: To provide a comprehensive and reliable tabulation of available data on the epidemiological characteristics and risk factors for hepatitis B virus (HBV) infection in maintenance hemodialysis (HD) patients in China and help to inform prevention programs and guide future research. Methods: A systematic review was constructed based on the computerized literature database. Confidence intervals (95% CI) of infection rates were calculated using the approximate normal distribution model. Odds ratios (OR) and 95% CI were calculated by fixed or random effects models. Hepatitis B surface antigen positivity (HBsAg (+)) was set as the sign of HBV infection. Results: Fifty studies met our inclusion criteria. The pooled prevalence of HBV infection among HD patients in China was 11.9%. Blood transfusion was correlated with an increase in HBV infection (p = 0.05). HD patients with a long-term history were more likely to be infected than those with a short-term history. The levels of alanine aminotransferase were higher in the HBsAg (+) patients (p < 0.001). Large doses of HBV vaccine (80 μg/dose) increased the seroconversion rate. The response rate of intradermal injection of HBV vaccine was higher than that of intramuscular injection. Conclusion: Hepatitis B is still one of the main complications in HD patients in China, and the frequency of blood transfusion and duration of HD were the risk factors. Large doses and intradermal injection of HBV vaccine were recommended to prevent HBV infection in HD patients. The findings of this meta-analysis have implications for optimal prevention and treatment of Hepatitis B in HD patients.

Evolutionary variants of mouse adenovirus containing cellular DNA sequences

Abstract This study was initiated to determine the amount of mouse adenovirus strain FL that is available for substitution. Evolutionary variants were isolated following serial passage at high input multiplicities. Virions containing altered genomes were detected by altered size of their DNA in agarose gels. These variants demonstrated a selective growth advantage and became the predominant species as passages were continued. Both deletions and substitutions were found; they ranged in size from a small percentage to over 90% of the AdFL genome. The ends of the viral DNA appeared to be conserved. No common DNA sequences were deleted in all variants. The DNA from one mutant was shown to contain a continuous insert of about 16 million daltons of cellular DNA that included two regions of reiterated cellular sequences.

Expression of human salivary protein genes

Human proline-rich proteins (PRPs) are polymorphic, homologous in sequence, and linked in a cluster called the human salivary protein complex (SPC). Recently this complex was localized to human chromosome band 12p13.2 (Mamulaet al., Cytogenet. Cell Genet. 39:279, 1985). We have isolated a PRP cDNA, EO27, from a human parotid gland library, identified it by DNA sequencing, and used it to study the molecular and cellular biology of PRP production. Cell-free translation and mRNA characterization with EO27 indicate that the numerous PRPs seen in saliva are produced from relatively few, large precursors, probably by posttranslational cleavage. This supports an hypothesis originally proposed by Friedman and Karn in 1977 (Am. J. Hum. Genet. 29:44A;Biochem. Genet. 15:549) and later supported by biochemical studies (Karnet al., Biochem Genet. 17:1061, 1979) and molecular studies (Mamulaet al., Fed. Proc. 43:1522, 1984; Maedaet al., J. Biol. Chem. 260:1123, 1985). EO27 was also used in this study to localize PRP mRNA production to the acinar cells of the parotid gland byin situ hybridization.

Construction of an Erythropoietin-Expressing Bioartificial Renal Tubule Assist Device

Methods: A eukaryotic expression vector pcDNA3.1-hEpo was constructed by standard cloning methods. G418 was applied to select for a stable erythropoietin (Epo) expression cell clone. Cell viability and cell growth curve were generated with MTT chromatometry. HK-2 cells were transfected with pcDNA3.1-hEpo, cultured, and then seeded into the hollow fibers of high-flux hemofilters. The expression levels of Epo in the culture supernatants were detected by enzyme-linked immunosorbent assay (ELISA). The inulin leak rate and the transport of Na+ and glucose (Glu) of the Epo-expressing bioartificial renal tubule assist device (RAD) were determined and compared with the conventional RAD. The specific inhibition of the transport by ouabain and phlorizin was determined. Results: The Epo expression plasmid pcDNA3.1-hEpo was successfully constructed and generated. Stable expression of Epo was obtained by selection of G418 after HK-2 cells were transfected. There was no obvious difference about cell growth curves between the transfected and untransfected HK-2 cells (p > 0.05). High levels of Epo expression were observed in both intra- and extraluminal culture media in the novel RAD. There was no significant difference about the inulin leak rate and the transport rate of Na+ and Glu between the novel and conventional RADs (p > 0.05 for each). And the transport function of the novel and conventional RADs was significantly inhibited by ouabain and phlorizin (p < 0.01 for each). Conclusions: A novel RAD that can express Epo was successfully constructed in vitro, in which other functions of the RAD were not affected by the transfection of pcDNA3.1-hEpo.

303. Impaired Nuclear Transport and Virus Uncoating Limit Recombinant Adeno-Associated Virus 2 Vector-Mediated Transduction of Primary Murine Hematopoietic Cells|[ast]|

The transduction efficiency of adeno-associated virus 2 (AAV) vectors varies greatly in different cells and tissues. Whereas muscle and brain cells are transduced efficiently, controversies abound regarding hematopoietic cell transduction by AAV vectors. For human hematopoietic cells, we documented this problem to be related to the extent of AAV receptor expression (J. Virol., 71: 8262–8267, 1997). Murine hematopoietic cells express the receptor, yet are transduced poorly. Using a murine fibroblast cell line, we reported that the lack of transduction was due to inefficient intracellular trafficking of AAV from cytosol into the nucleus (J. Virol., 74: 992–996, 2000), and that treatment with hydroxyurea (HU) facilitated nuclear transport of AAV in these cells (J. Virol., 75: 4080–4090, 2001). In the present studies, we extended these observations to primitive murine hematopoietic cells. Murine c-kit+, lin− hematopoietic cells were transduced with recombinant AAV-lacZ vectors. Forty-eight hrs. post-transduction, nuclear and cytoplasmic fractions were isolated and low Mr DNA samples extracted from these fractions were analyzed on Southern blots. Approximately 85% of AAV genomes were present in the cytoplasmic fraction. However, when donor mice were injected intra-peritoneally with HU at 1 mg/g body weight, 24 hrs. prior to isolation of cells, the extent of AAV genomes detected in the cytoplasmic fraction was reduced to ~40%, with a corresponding increase in the nuclear fraction, which increased to ~60%, indicating that in vivo administration of HU facilitated nuclear transport of AAV. However, when DNA samples were electrophoresed on denaturing gels and analyzed on Southern blots, it was apparent that a significant fraction of the AAV genome present in the nuclear fraction from cells obtained from HU-treated mice was single-stranded. We tested the hypothesis whether single-stranded genomes were derived from virions that failed to undergo uncoating in the nucleus. Primitive hematopoietic cells, with and without HU-administration in vivo, were transduced with recombinant AAV-lacZ vectors, and 48-hrs. post-transduction, equivalent fractions were either mock-treated, or digested exhaustively with DNase I, and analyzed on DNA slot-blots using a lacZ probe. Whereas majority of the signal, which was resistant to DNase I, was detected in the cytoplasmic fraction in cells obtained from untreated mice, as expected, a substantial fraction of the signal in the nuclear fraction in cells obtained from HU-treated mice was also resistant to DNase I, suggesting that although HU facilitated nuclear transport of AAV, most of the virions failed to undergo uncoating, thereby leading to only a partial improvement in viral second-strand DNA synthesis and transgene expression. Thus, the identification of a yet another obstacle — virus uncoating — has implications in the optimal use of recombinant AAV vectors in hematopoietic stem cell gene therapy.