Novel role of toll-like receptor 3 in hepatitis C-associated glomerulonephritis

Hepatitis C virus (HCV) infection is frequently complicated by glomerulonephritis with immune complexes containing viral RNA. We examined the potential influence of Toll-like receptors (TLRs), specifically TLR3 recognition of viral dsRNA exemplified by polyriboinosinic:polyribocytidylic acid [poly(I:C) RNA]. Normal human kidney stained positive for TLR3 on mesangial cells (MCs), vascular smooth muscle cells, and collecting duct epithelium. Cultured MCs have low TLR3 mRNA levels with predominant intracellular protein localization, which was increased by tumor necrosis factor-alpha, interleukin (IL)-1beta, interferon (IFN)-gamma, and the TLR3 ligand poly(I:C) RNA. Poly(I:C) RNA stimulation of MCs increased mRNA and protein synthesis of IL-6, IL-1beta, M-CSF, IL-8/CXCL8, RANTES/CCL5, MCP-1/CCL2, and ICAM-I; it also increased anti-proliferative and proapoptotic effects, the latter of which was decreased by inhibiting caspase-8. In microdissected glomeruli of normal and non-HCV membranoproliferative glomerulonephritis biopsies, TLR3 mRNA expression was low. In contrast TLR3 mRNA expression was significantly increased in hepatitis C-positive glomerulonephritis and was associated with enhanced mRNA for RANTES/CCL5 and MCP-1/CCL2. We hypothesize that immune complexes containing viral RNA activate mesangial TLR3 during HCV infection, thereby contributing to chemokine/cytokine release and effecting proliferation and apoptosis. Thus, TLR3 expression on renal cells, and especially MCs, may establish a link between viral infections and glomerular diseases.     

The use of supplementary techniques to increase recall of sex partners in a network-based research study in Rio de Janeiro, Brazil

OBJECTIVE: To evaluate supplementary cueing as a technique to increase recall of sex partners in the year before the interview. GOAL: Recall of partners beyond those freely recalled. STUDY DESIGN: We asked volunteers at a clinic in Brazil to freely recall all regular and casual sex partners in the year before the interview. Then, we used a name generator developed by Brewer et al. in the United States, in which volunteers were prompted with 4 types of cues: location, alphabetic, social role, and network. We calculated different measures to evaluate the technique and analyzed the associations between reporting any additional partner and demographic characteristics. RESULTS: Among volunteers reporting 2 or more sexual partners (n = 590), 41 (7%) recalled 1 or more additional partners by using the supplementary technique, with 105 partners of 2090 (5%) recalled only after using the cues. For volunteers reporting 4 or more sexual partners (n = 193), 34 (18%) recalled 1 or more additional partners by using the supplementary technique, and 98 of 1177 (8%) of their sexual partners were recalled after using the cues. Men were less likely than women to report sex partners after prompting with the social role cues (OR 0.09), and overall the combined techniques were slightly less effective for older individuals (OR 0.95). CONCLUSION: The cue technique can improve sexual partners' recall in cultural contexts different than the United States, mainly for individuals already reporting several partners before the cue.     

Shotgun Optical Maps of the Whole Escherichia coli O157:H7 Genome

We have constructed NheI and XhoI optical maps of Escherichia coli O157:H7 solely from genomic DNA molecules to provide a uniquely valuable scaffold for contig closure and sequence validation. E. coli O157:H7 is a common pathogen found in contaminated food and water. Our approach obviated the need for the analysis of clones, PCR products, and hybridizations, because maps were constructed from ensembles of single DNA molecules. Shotgun sequencing of bacterial genomes remains labor-intensive, despite advances in sequencing technology. This is partly due to manual intervention required during the last stages of finishing. The applicability of optical mapping to this problem was enhanced by advances in machine vision techniques that improved mapping throughput and created a path to full automation of mapping. Comparisons were made between maps and sequence data that characterized sequence gaps and guided nascent assemblies.     

Altering glomerular epithelial function in vitro using transient and stable transfection.

Damage of the glomerular filtration barrier leads to proteinuria and progressive renal failure. Several independent lines of research have implicated the glomerular epithelial cell (GEC) as a key player in initiation and propagation of pathways leading to glomerulosclerosis. A growing number of molecules activated in this process have been identified. To further define their cellular function, manipulation of these molecules using pharmacological or genetic approaches in tissue culture systems are required. In this study, strategies for altering GEC gene expression by transient and stable transfection of fluorescence labeled proteins will be presented and discussed. The insight gained through these and comparable systems should allow a detailed dissection of the molecular pathways active in GEC function and failure.     

raf involvement in the simultaneous genetic transfer of the radioresistant and transforming phenotypes

We examined a human Alu+ mouse tertiary transformant derived from a noncancerous skin fibroblast cell line which exhibits the unique characteristic of being resistant to the killing effects of ionizing radiation. This transformed cell line was found to contain activated human c-raf-1, and demonstrated an increased level of radioresistance indicating the simultaneous transfer of both the transforming and radiation-resistant phenotypes. We have also found a relationship between the presence of activated oncogenes, specifically those with serine/threonine kinase activity and the radioresistant phenotype.     

Abnormal pattern of post-gamma-ray DNA replication in radioresistant fibroblast strains from affected members of a cancer-prone family with Li-Fraumeni syndrome.

Non-malignant dermal fibroblast strains, cultured from affected members of a Li-Fraumeni syndrome (LFS) family with diverse neoplasms associated with radiation exposure, display a unique increased resistance to the lethal effects of gamma-radiation. In the studies reported here, this radioresistance (RR) trait has been found to correlate strongly with an abnormal pattern of post-gamma-ray DNA replicative synthesis, as monitored by radiolabelled thymidine incorporation and S-phase cell autoradiography. In particular, the time interval between the gamma-ray-induced shutdown of DNA synthesis and its subsequent recovery was greater in all four RR strains examined and the post-recovery replication rate was much higher and was maintained longer than in normal and spousal controls. Alkaline sucrose sedimentation profiles of pulse-labelled cellular DNA indicated that the unusual pattern of DNA replication in irradiated RR strains may be ascribed to anomalies in both replicon initiation and DNA chain elongation processes. Moreover, the RR strain which had previously displayed the highest post-gamma-ray clonogenic survival was found to harbour a somatic (codon 234) mutation (presumably acquired during culture in vitro) in the same conserved region of the p53 tumour-suppressor gene as the germline (codon 245) mutation in the remaining three RR strains from other family members, thus coupling the RR phenotype and abnormal post-gamma-ray DNA synthesis pattern with faulty p53 expression. Significantly, these two aberrant radioresponse end points, along with documented anomalies in c-myc and c-raf-1 proto-oncogenes, are unprecedented among other LFS families carrying p53 germline mutations. We thus speculate that this peculiar cancer-prone family may possess in its germ line a second, as yet unidentified, genetic defect in addition to the p53 mutation.     

A method for predicting individual HIV infection status in the absence of clinical information

Latent structure analysis can be used to determine sensitivity and specificity rates of human immunodeficiency virus antibody assays in the absence of previous clinical or laboratory results. The technique was applied to the analysis of data obtained when a panel of serum samples, collected as part of a large-scale screening project, were subjected to four conventional bioassays (ag121, p24, gp120, and an enzyme-linked immunosorbent assay). To determine the accuracy of this statistical approach, the results of latent structure analysis were compared with the known clinical diagnoses of patients from whom the samples were taken, and nearly 100% agreement was obtained. Although a two-class latent structure model had some predictive value, a three-class model more adequately explained assay patterns. The use of the four standard assays in conjunction with the statistical methods described here would largely reduce the need for confirmatory Western blot assays in analyses of large panels of samples.