Large-scale amplification, cloning and sequencing of near full-length HIV-1 subtype C genomes

Full-length HIV-1 genome sequencing provides important data needed to address several vaccine design, molecular epidemiologic and pathogenesis questions. A protocol is presented for obtaining near full-length genomes (NFLGs) from subjects infected with HIV-1 subtype C. This protocol was used to amplify NFLGs from 244 of 366 (67%) samples collected at two clinics in Durban, South Africa (SK and PS). Viral load was directly associated with frequency of successful NFLG amplification for both cohorts (PS; p = 0.005 and SK; p < 0.001). Seventeen of 38 initially NFLG-negative SK samples had variation within the PCR primer binding sites, however only 3 of these were successfully re-amplified using re-designed primers homologous to the target viruses. NFLGs were obtained from 7 of 24 PBMC samples processed from subjects whose plasma did not yield a NFLG. Stable plasmid clones were obtained from all 244 NFLG-positive PCR products, and both strands of each genome were sequenced, using a primary set of 46 primers. These methods thus allow the large-scale collection of HIV-1 NFLGs from populations infected primarily with subtype C. The methods are readily adaptable to other HIV-1 subtypes, and provide materials for viral functional analyses and population-based molecular epidemiology studies that include analysis of viral genome chimerization.     

Bioactive xanthones with effect on P-glycoprotein and prediction of intestinal absorption

Our research group has been focusing in the discovery of potential antitumor small molecules based on the xanthone scaffold. However, a serious obstacle in the field of cancer therapy is the multidrug resistance (MDR) phenotype, most often caused by the overexpression of P-glycoprotein (P-gp). Another limitation to development of such drug candidates is the reduced information available about the bioavailability of these compounds. We have previously identified four interesting compounds as inhibitors of tumor cell growth namely two dihydroxyxanthones, a xanthonolignoid and a pyranoxanthone. Based on these considerations, it was our aim to: (i) investigate their effect on the P-gp activity; and (ii) estimate their intestinal absorption using Caco-2 cell monolayers as an intestinal model. An HPLC analysis from the in vitro permeation assay with Caco-2 cells monolayer was performed to predict the intestinal permeability of xanthonic derivatives. A rhodamine (Rh123) accumulation assay using P-gp overexpressing leukemia cells, K562Dox, incubated with the four xanthonic derivatives, was performed to investigate their P-gp inhibitory activity. A luminescence-based ATPase assay was performed to differentiate between competitive and noncompetitive P-gp inhibitors. The xanthonolignoid and the pyranoxanthone were found to increase the accumulation of Rh123 in K562Dox cell line, and both were acting by a noncompetitive P-gp inhibitory mechanism. Transport of the four xanthones occurred in the absorptive direction (P_app, 0.012–2.8 nm/s). The behavior of the xanthonolignoid and the pyranoxanthone as P-gp inhibitors and their high apparent permeability coefficients make them promising hit compounds to pursue with further studies.     

Modulation of P2X7 receptor expression in macrophages from mineral oil-injected mice

P2X7 receptor activation is involved in a number of pro-inflammatory responses in macrophages and other immune cells. Their expression can be positively modulated with lipopolysaccharide (LPS) and TNFalpha, reinforcing their role during inflammation. We investigated the effect of substances capable of recruiting macrophages into the peritoneal cavity of mice (mineral oil and thioglycolate) on P2X7 receptor expression and function, addressing whether these stimuli can interfere with multinucleated giant cell (MGC) formation, ATP-induced apoptosis, plasma membrane permeabilization and nitric oxide production. It was demonstrated that mineral oil treatment reduces P2X7-dependent MGC formation, whereas thioglycolate treatment does not. Mineral oil treatment reduced P2X7 receptor expression, down-modulating ATP-induced apoptosis, permeabilization and nitric oxide production. In conclusion, mineral oil down modulated P2X7 expression and consequently P2X7-associated phenomena, but thioglycolate did not. These effects might be associated with the unpleasant side effects already described during long-term administration of mineral oil for cosmetic purposes or as a laxative and could be useful in understanding the mechanism of recycling and modulation of P2 receptors present in other situations of immunopathological interest.     

Portable miniaturized extracorporeal membrane oxygenation systems for H1N1-related severe acute respiratory distress syndrome: A case series

Background

Technological advances improved the practice of “modern” extracorporeal membrane oxygenation (ECMO). In the present report, we describe the experience of a referral ECMO center using portable miniaturized ECMO systems for H1N1-related severe acute respiratory distress syndrome (ARDS).

Methods

An observational study of all patients with H1N1-associated ARDS treated with ECMO in Hospital S. João (Porto, Portugal) between November 2009 and April 2011 was performed. Extracorporeal membrane oxygenation support was established using either ELS or Cardiohelp systems (Maquet-Cardiopulmonary-AG, Hirrlingen, Germany).

Results

Ten adult patients with severe ARDS secondary to H1N1 infection (Pao₂/fraction of inspired oxygen, 69 mm Hg [56-84]; Murray score, 3.5 [3.5-3.8]) were included, and 60% survived to hospital discharge. Five patients were uneventfully transferred on ECMO from referring hospitals to our center by ambulance. Six patients were treated during the first postpandemic influenza season. All patients were treated with oseltamivir, and 1 received in addition zanamivir. Four patients received corticosteroids. Nosocomial infection was the most common complication (40%). Of the 4 deaths, 2 were caused by hemorrhagic shock; 1, by irreversible multiple organ failure; and 1, by refractory septic shock.

Conclusion

In our experience, ECMO support was a valuable therapeutic option for H1N1-related severe ARDS. The use of portable miniaturized systems allowed urgent rescue of patients from referring hospitals and safe interhospital and intrahospital transport during ECMO support.     

Assessment of body fat composition disturbances by bioimpedance analysis in HIV-infected adults

HIV-lipodystrophy syndrome is characterized by different patterns of body fat distribution (BFD) which are identified by clinical and body composition (BC) assessment, including bioimpedance analysis (BIA). Our aim was to compare BC in HIV-infected patients on combination antiretroviral therapy (cART) according to 4 distinct phenotypes of BFD (G1-no lipodystrophy, G2-isolated central fat accumulation, G3-lipoatrophy, G4-mixed forms of lipodystrophy) and assessed factors associated with them. Anthropometry and BIA were performed in 344 HIV-1 patients. G2 and G4 phenotype patients had significantly higher fat mass (FM) but no differences were observed in fat-free mass (FFM) and total body water among the 4 phenotypes. Significant negative associations were found between the presence of lipoatrophy and female gender, body mass index (BMI), waist (WC), hip (HC) and thigh circumferences, and total body FM estimated by BIA. After adjustment for gender, cART duration and BMI, G3 had significant lower WC [odds ratio (OR)=0.84; 0.78- 0.90] and HC (OR=0.88; 0.81-0.96) mean. Independently of gender, cART duration and BMI, G2 remained significantly associated with higher WC (OR=1.11; 1.05-1.18) and HC (OR=1.15; 1.07-1.23) mean, and with FM estimated by BIA [FM as %, OR=1.17 (1.09-1.26); and FM as kg, OR=1.15 (1.06- 1.25)]. There was a significant positive association between G4 and female gender (OR=1.66; 1.01-2.75), BMI (OR=1.10; 1.04-1.17) and WC (OR=1.15; 1.09-1.21). The similar FFM along the BFD spectrum describes the actual BC of these patients without sarcopenia. In a clinical setting, BIA is an easy and useful tool to evaluate fat mass and FFM and gives us a picture of BC that was not possible with anthropometry.     

H7 avian influenza virus vaccines protect chickens against challenge with antigenically diverse isolates

Vaccination has been a critical tool in the control of some avian influenza viruses (AIV) and has been used routinely in Pakistan to help control sporadic outbreaks of highly pathogenic (HP) H7 AIV since 1995. During that time, several AIV isolates were utilized as inactivated vaccines with varying degrees of success. In order to evaluate which H7 AIV strains may serve as optimal vaccines for diverse H7 AIVs from Pakistan we conducted vaccination-challenge studies with five H7 vaccines against challenge with two HPAIVs: A/chicken/Murree/NARC-1/1995 H7N3 and A/chicken/Karachi/SPVC-4/2004 H7N3. To further characterize the isolates antigenic cartography was used to visually demonstrate the antigenic relationships among the isolates. All vaccines provided similar protection against mortality, morbidity and shedding of challenge virus from the respiratory tract. However, some minor (not statistically significant) differences were observed and correlated with antibody levels induced by the vaccine prior to challenge.