Diminished transmission of drug resistant HIV-1 variants with reduced replication capacity in a human transmission model

Background

Different patterns of drug resistance are observed in treated and therapy naïve HIV-1 infected populations. Especially the NRTI-related M184I/V variants, which are among the most frequently encountered mutations in treated patients, are underrepresented in the antiretroviral naïve population. M184I/V mutations are known to have a profound effect on viral replication and tend to revert over time in the new host. However it is debated whether a diminished transmission efficacy of HIV variants with a reduced replication capacity can also contribute to the observed discrepancy in genotypic patterns.As dendritic cells (DCs) play a pivotal role in HIV-1 transmission, we used a model containing primary human Langerhans cells (LCs) and DCs to compare the transmission efficacy M184 variants (HIV-M184V/I/T) to HIV wild type (HIV-WT). As control, we used HIV harboring the NNRTI mutation K103N (HIV-K103N) which has a minor effect on replication and is found at a similar prevalence in treated and untreated individuals.

Results

In comparison to HIV-WT, the HIV-M184 variants were less efficiently transmitted to CCR5⁺ Jurkat T cells by both LCs and DCs. The transmission rate of HIV-K103N was slightly reduced to HIV-WT in LCs and even higher than HIV-WT in DCs. Replication experiments in CCR5⁺ Jurkat T cells revealed no apparent differences in replication capacity between the mutant viruses and HIV-WT. However, viral replication in LCs and DCs was in concordance with the transmission results; replication by the HIV-M184 variants was lower than replication by HIV-WT, and the level of replication of HIV-K103N was intermediate for LCs and higher than HIV-WT for DCs.

Conclusions

Our data demonstrate that drug resistant M184-variants display a reduced replication capacity in LCs and DCs which directly impairs their transmission efficacy. As such, diminished transmission efficacy may contribute to the lower prevalence of drug resistant variants in therapy naive individuals.

     

Clinical and Serological Features of Patients Referred through a Rheumatology Triage System because of Positive Antinuclear Antibodies

Background

The referral of patients with positive anti-nuclear antibody (ANA) tests has been criticized as an inappropriate use of medical resources. The utility of a positive ANA test in a central triage (CT) system was studied by determining the autoantibody profiles and clinical diagnoses of patients referred to rheumatologists through a CT system because of a positive ANA test.

Methods

Patients that met three criteria were included: (1) referred to Rheumatology CT over a three year interval; (2) reason for referral was a “positive ANA”; (3) were evaluated by a certified rheumatologist. The CT clinical database was used to obtain demographic and clinical information and a serological database was used to retrieve specific ANA and/or extractable nuclear antigen (ENA) test results. Clinical information was extracted from the consulting rheumatologist''s report.

Results

15,357 patients were referred through the CT system; 643 (4.1%) of these because of a positive ANA and of these 263 (40.9%) were evaluated by a certified rheumatologist. In 63/263 (24%) of ANA positive patients, the specialist provided a diagnosis of an ANA associated rheumatic disease (AARD) while 69 (26.2%) had no evidence of any disease; 102 (38.8%) had other rheumatologic diagnoses and 29 (11%) had conditions that did not meet AARD classification criteria. Of ANA positive archived sera, 15.1% were anti-DFS70 positive and 91.2% of these did not have an AARD.

Conclusions

This is the first study to evaluate the serological and clinical features of patients referred through a CT system because of a positive ANA. The spectrum of autoantibody specificities was wide with anti-Ro52/TRIM21 being the most common autoantibody detected. Approximately 15% of referrals had only antibodies to DFS70, the vast majority of which did not have clinical evidence for an AARD. These findings provide insight into the utility of autoantibody testing in a CT system.     

Human Health Risk of Ingested Nanoparticles That Are Added as Multifunctional Agents to Paints: an In Vitro Study

Microorganisms growing on painted surfaces are not only an aesthetic problem, but also actively contribute to the weathering and deterioration of materials. A widely used strategy to combat microbial colonization is the addition of biocides to the paint. However, ecotoxic, non-degradable biocides with a broad protection range are now prohibited in Europe, so the paint industry is considering engineered nanoparticles (ENPs) as an alternative biocide. There is concern that ENPs in paint might be released in run-off water and subsequently consumed by animals and/or humans, potentially coming into contact with cells of the gastrointestinal tract and affecting the immune system. Therefore, in the present study we evaluated the cytotoxic effects of three ENPs (nanosilver, nanotitanium dioxide and nanosilicon dioxide) that have a realistic potential for use in paints in the near future. When exposed to nanotitanium dioxide and nanosilicon dioxide in concentrations up to 243 µg/mL for 48 h, neither the gastrointestinal cells (CaCo-2) nor immune system cells (Jurkat) were significantly affected. However, when exposed to nanosilver, several cell parameters were affected, but far less than by silver ions used as a control. No differences in cytotoxicity were observed when cells were exposed to ENP-containing paint particles, compared with the same paint particles without ENPs. Paint particles containing ENPs did not affect cell morphology, the release of reactive oxygen species or cytokines, cell activity or cell death in a different manner to the same paint particles without ENPs. The results suggest that paints doped with ENPs do not pose an additional acute health hazard for humans.     

Distribution and function of actin in the developing stomatal complex of winter rye (Secale cereale cv. Puma)

Summary The dynamics of actin distribution during stomatal complex formation in leaves of winter rye was examined by means of immunofluorescence microscopy of epidermal sheets. This method results in actin localization patterns that are the same as those seen with rhodamine-phalloidin staining, but are more stable. During stomatal development MFs are extensively rearranged, and most of the time the orientation or placement of MFs is distinctly different from that of MTs, the exception being co-localization of MTs and MFs in phragmoplasts. Although MFs show an orientation similar to that of MTs in interphase guard mother cells, no banding of MFs into anything resembling the interphase MT band is observed. From prophase to telophase, a distinct, dense concentration of MFs is found in subsidiary cell mother cells (SMCs) between the nucleus and the region of the cell cortex facing the guard mother cell. Cytochalasin B treatment causes incorrect positioning of the SMC nucleus/daughter nuclei and abarrent placement and orientation of the new cell wall that forms the boundary of the subsidiary cell at cytokinesis. These results suggest that MFs are involved in maintaining the SMC nucleus in its correct position and the SMC spindle in the correct orientation relative to the division site previously delineated by the preprophase band. Because these MFs thus appear to assure that the SMC phragmoplast begins to form in the correct orientation near the division site to which it needs to grow, we suggest that MFs are involved in control of correct placement and orientation of the new cell wall of the subsidiary cell.Abbreviations CB cytochalasin B - DIC differential interference contrast - DMSO dimethylsulfoxide - MBS m-maleimidobenzoyl-N-hydroxylsuccinimide ester - MF microfilament - MT microtubule - PBS phosphate buffered saline - SMC subsidiary cell mother cell Dedicated to the memory of Professor Oswald Kiermayer     

Human dermal microvascular endothelial but not human umbilical vein endothelial cells express CD36 in vivo and in vitro.

CD36 is an 88-kDa glycoprotein that has been identified on platelets, monocytes, and some endothelial cells. Experimental evidence suggests that CD36 mediates the binding of Plasmodium falciparum-infected RBC to a variety of cells, and therefore may play a role in the vascular complications associated with malaria. Additionally, CD36 may also bind the extracellular matrix proteins thrombospondin and collagen. Human umbilical vein endothelial cells have been used in in vitro models examining the binding of P. falciparum RBC to endothelial cells, but they do not consistently express cell surface CD36. Inasmuch as human dermal microvascular endothelial cells (HDMEC) differ in a variety of ways from large vessel endothelial cells, we have examined HDMEC for cell surface expression of CD36 in vivo and in vitro. Direct immunofluorescence of skin showed bright staining of HDMEC with antibody recognizing CD36 and flow cytometric analysis of cultured HDMEC revealed cell surface expression. In contrast, large vessel endothelial cells were not stained with antibody recognizing CD36 in vivo and cultured cells derived from umbilical vein failed to express cell surface CD36 in vitro. Western immunoblots of lysates of HDMEC but not human umbilical vein endothelial cells demonstrated an 88-kDa protein that comigrated with CD36 from platelets. Functional studies demonstrated that adherence of PRBC to HDMEC was inhibited up to 66% by mAb recognizing CD36. Furthermore, the expression of CD36 on HDMEC was increased in a dose- and time-dependent manner by IFN-gamma, and was decreased by protein kinase C agonists. These data demonstrate that HDMEC express functionally active CD36 and this expression can be positively and negatively regulated by soluble factors. This study demonstrates that HDMEC are useful in the study of CD36-mediated binding of PRBC to endothelial cells in vitro and provides further evidence of distinct phenotypic differences between HDMEC and large vessel endothelial cells.     

A graphical method for detecting recombination in phylogenetic data sets

Current phylogenetic tree reconstruction methods assume that there is a single underlying tree topology for all sites along the sequence. The presence of mosaic sequences due to recombination violates this assumption and will cause phylogenetic methods to give misleading results due to the imposition of a single tree topology on all sites. The detection of mosaic sequences caused by recombination is therefore an important first step in phylogenetic analysis. A graphical method for the detection of recombination, based on the least squares method of phylogenetic estimation, is presented here. This method locates putative recombination breakpoints by moving a window along the sequence. The performance of the method is assessed by simulation and by its application to a real data set.      

bilbo, a non-LTR retrotransposon of Drosophila subobscura: a clue to the evolution of LINE-like elements in Drosophila

We used the repetitive character of transposable elements to isolate a non-LTR retrotransposon in Drosophila subobscura. bilbo, as we have called it, has homology to TRIM and LOA elements. Sequence analysis showed a 5' untranslated region (UTR), an open reading frame (ORF) with no RNA-binding domains, a downstream ORF that had structural homology to that of the I factor, and, finally, a 3' UTR which ended in several 5-nt repeats. The results of our phylogenetic and structural analyses shed light on the evolution of Drosophila non-LTR retrotransposons and support the hypothesis that an ancestor of these elements was structurally complex.