Investigation into in vitro anti-leishmanial combinations of calcium channel blockers and current anti-leishmanial drugs

The need for drug combinations to treat visceral leishmaniasis (VL) arose because of resistance to antimonials, the toxicity of current treatments and the length of the course of therapy. Calcium channel blockers (CCBs) have shown anti-leishmanial activity; therefore their use in combination with standard drugs could provide new alternatives for the treatment of VL. In this work, in vitro isobolograms of Leishmania (Leishmania) chagasi using promastigotes or intracellular amastigotes were utilised to identify the interactions between five CCBs and the standard drugs pentamidine, amphotericin B and glucantime. The drug interactions were assessed with a fixed ratio isobologram method and the fractional inhibitory concentrations (FICs), sum of FICs (ΣFICs) and the overall mean ΣFIC were calculated for each combination. Graphical isobologram analysis showed that the combination of nimodipine and glucantime was the most promising in amastigotes with an overall mean ΣFIC value of 0.79. Interactions between CCBs and the anti-leishmanial drugs were classified as indifferent according to the overall mean ΣFIC and the isobologram graphic analysis.

Genetic diversity of NS3 protease from Brazilian HCV isolates and possible implications for therapy with direct-acting antiviral drugs

The hepatitis C virus (HCV) NS3 protease has been one of the molecular targets of new therapeutic approaches. Its genomic sequence variability in Brazilian HCV isolates is poorly documented. To obtain more information on the magnitude of its genetic diversity, 114 Brazilian HCV samples were sequenced and analysed together with global reference sequences. Genetic distance (d) analyses revealed that subtype 1b had a higher degree of heterogeneity (d = 0.098) than subtypes 1a (d = 0.060) and 3a (d = 0.062). Brazilian isolates of subtype 1b were distributed in the phylogenetic tree among sequences from other countries, whereas most subtype 1a and 3a sequences clustered into a single branch. Additional characterisation of subtype 1a in clades 1 and 2 revealed that all but two Brazilian subtype 1a sequences formed a distinct and strongly supported (approximate likelihood-ratio test = 93) group of sequences inside clade 1. Moreover, this subcluster inside clade 1 presented an unusual phenotypic characteristic in relation to the presence of resistance mutations for macrocyclic inhibitors. In particular, the mutation Q80K was found in the majority of clade 1 sequences, but not in the Brazilian isolates. These data demonstrate that Brazilian HCV subtypes display a distinct pattern of genetic diversity and reinforce the importance of sequence information in future therapeutic approaches.

The first report of the qnrB19, qnrS1 and aac(6´)-Ib-cr genes in urinary isolates of ciprofloxacin-resistant Escherichia coli in Brazil

In this study, we investigated the presence of plasmid-mediated quinolone resistance (PMQR) genes among 101 ciprofloxacin-resistant urinary Escherichia coli isolates and searched for mutations in the quinolone-resistance-determining regions (QRDRs) of the DNA gyrase and topoisomerase IV genes in PMQR-carrying isolates. Eight isolates harboured the qnr and aac(6')-Ib-cr genes (3 qnrS1, 1 qnrB19 and 4 aac(6')-Ib-cr). A mutational analysis of the QRDRs in qnr and aac(6')-Ib-cr-positive isolates revealed mutations in gyrA, parC and parE that might be associated with high levels of resistance to quinolones. No mutation was detected in gyrB. Rare gyrA, parC and parE mutations were detected outside of the QRDRs. This is the first report of qnrB19, qnrS1 and aac(6')-Ib-cr -carrying E. coli isolates in Brazil.

Trends in yearly prevalence of third-generation cephalosporin and fluoroquinolone resistant Enterobacteriaceae infections and antimicrobial use in Spanish hospitals, Spain, 1999 to 2010.

Escherichia coli, Klebsiella pneumoniae, and Enterobacter spp. are a major cause of infections in hospitalised patients. The aim of our study was to evaluate rates and trends of resistance to third-generation cephalosporins and fluoroquinolones in infected patients, the trends in use for these antimicrobials, and to assess the potential correlation between both trends. The database of national point prevalence study series of infections and antimicrobial use among patients hospitalised in Spain over the period from 1999 to 2010 was analysed. On average 265 hospitals and 60,000 patients were surveyed per year yielding a total of 19,801 E. coli, 3,004 K. pneumoniae and 3,205 Enterobacter isolates. During the twelve years period, we observed significant increases for the use of fluoroquinolones (5.8%-10.2%, p<0.001), but not for third-generation cephalosporins (6.4%-5.9%, p=NS). Resistance to third-generation cephalosporins increased significantly for E. coli (5%-15%, p<0.01) and for K. pneumoniae infections (4%-21%, p<0.01) but not for Enterobacter spp. (24%). Resistance to fluoroquinolones increased significantly for E. coli (16%30%, p<0.01), for K. pneumoniae (5%-22%, p<0.01), and for Enterobacter spp. (6%-15%, p<0.01). We found strong correlations between the rate of fluoroquinolone use and the resistance to fluoroquinolones, third-generation cephalosporins, or co-resistance to both, for E. coli (R=0.97, p<0.01, R=0.94, p<0.01, and R=0.96, p<0.01, respectively), and for K. pneumoniae (R=0.92, p<0.01, R=0.91, p<0.01, and R=0.92, p<0.01, respectively). No correlation could be found between the use of third-generation cephalosporins and resistance to any of the latter antimicrobials. No significant correlations could be found for Enterobacter spp.. Knowledge of the trends in antimicrobial resistance and use of antimicrobials in the hospitalised population at the national level can help to develop prevention strategies.

Variability and resistance mutations in the hepatitis C virus NS3 protease in patients not treated with protease inhibitors

The goal of treatment of chronic hepatitis C is to achieve a sustained virological response, which is defined as exhibiting undetectable hepatitis C virus (HCV) RNA levels in serum following therapy for at least six months. However, the current treatment is only effective in 50% of patients infected with HCV genotype 1, the most prevalent genotype in Brazil. Inhibitors of the serine protease non-structural protein 3 (NS3) have therefore been developed to improve the responses of HCV-infected patients. However, the emergence of drug-resistant variants has been the major obstacle to therapeutic success. The goal of this study was to evaluate the presence of resistance mutations and genetic polymorphisms in the NS3 genomic region of HCV from 37 patients infected with HCV genotype 1 had not been treated with protease inhibitors. Plasma viral RNA was used to amplify and sequence the HCV NS3 gene. The results indicate that the catalytic triad is conserved. A large number of substitutions were observed in codons 153, 40 and 91; the resistant variants T54A, T54S, V55A, R155K and A156T were also detected. This study shows that resistance mutations and genetic polymorphisms are present in the NS3 region of HCV in patients who have not been treated with protease inhibitors, data that are important in determining the efficiency of this new class of drugs in Brazil.

Monitoring resistance to Bacillus thuringiensis subsp. israelensis in the field by performing bioassays with each Cry toxin separately

Bacillus thuringiensis subsp. israelensis (Bti) is increasingly used worldwide for mosquito control and is the only larvicide used in the French Rhône-Alpes region since decades. The artificial selection of mosquitoes with field-persistent Bti collected in breeding sites from this region led to a moderate level of resistance to Bti, but to relatively high levels of resistance to individual Bti Cry toxins. Based on this observation, we developed a bioassay procedure using each Bti Cry toxin separately to detect cryptic Bti-resistance evolving in field mosquito populations. Although no resistance to Bti was detected in none of the three mosquito species tested (Aedes rusticus, Aedes sticticus and Aedes vexans), an increased tolerance to Cry4Aa (3.5-fold) and Cry11Aa toxins (8-fold) was found in one Ae. sticticus population compared to other populations of the same species, suggesting that resistance to Bti may be arising in this population. This study confirms previous works showing a lack of Bti resistance in field mosquito populations treated for decades with this bioinsecticide. It also provides a first panorama of their susceptibility status to individual Bti Cry toxins. In combination with bioassays with Bti, bioassays with separate Cry toxins allow a more sensitive monitoring of Bti-resistance in the field.

Postischemic treatment of neonatal cerebral ischemia should target autophagy.

OBJECTIVE: To evaluate the contributions of autophagic, necrotic, and apoptotic cell death mechanisms after neonatal cerebral ischemia and hence define the most appropriate neuroprotective approach for postischemic therapy. METHODS: Rats were exposed to transient focal cerebral ischemia on postnatal day 12. Some rats were treated by postischemic administration of pan-caspase or autophagy inhibitors. The ischemic brain tissue was studied histologically, biochemically, and ultrastructurally for autophagic, apoptotic, and necrotic markers. RESULTS: Lysosomal and autophagic activities were increased in neurons in the ischemic area from 6 to 24 hours postinjury, as shown by immunohistochemistry against lysosomal-associated membrane protein 1 and cathepsin D, by acid phosphatase histochemistry, by increased expression of autophagosome-specific LC3-II and by punctate LC3 staining. Electron microscopy confirmed the presence of large autolysosomes and putative autophagosomes in neurons. The increases in lysosomal activity and autophagosome formation together demonstrate increased autophagy, which occurred mainly in the border of the lesion, suggesting its involvement in delayed cell death. We also provide evidence for necrosis near the center of the lesion and apoptotic-like cell death in its border, but in nonautophagic cells. Postischemic intracerebroventricular injections of autophagy inhibitor 3-methyladenine strongly reduced the lesion volume (by 46%) even when given >4 hours after the beginning of the ischemia, whereas pan-caspase inhibitors, carbobenzoxy-valyl-alanyl-aspartyl(OMe)-fluoromethylketone and quinoline-val-asp(OMe)-Ch2-O-phenoxy, provided no protection. INTERPRETATION: The prominence of autophagic neuronal death in the ischemic penumbra and the neuroprotective efficacy of postischemic autophagy inhibition indicate that autophagy should be a primary target in the treatment of neonatal cerebral ischemia.

The Tertiary structural and thermal evolution of the central Alps - Compressional and extensional structures in an orogenic belt

The Western Alpine Are has been created during the Cretaceous and the Tertiary orogenies. The interference patterns of the Tertiary structures suggest their formation during continental collision of the European and the Adriatic Plates, with an accompanying anticlockwise rotation of the Adriatic indenter. Extensional structures are mainly related to ductile deformation by simple shear. These structures developed at a deep tectonic level, in granitic crustal rocks, at depths in excess of 10 km. In the early Palaeogene period of the Tertiary Orogeny, the main Tertiary nappe emplacement resulted from a NW-thrusting of the Austroalpine, Penninic and Helvetic nappes. Heating of the deep zone of the Upper Cretaceous and Tertiary nappe stack by geothermal heat flow is responsible for the Tertiary regional metamorphism, reaching amphibolite-facies conditions in the Lepontine Gneiss Dome (geothermal gradient 25 degrees C/ km). The Tertiary thrusting occurred mainly during prograde metamorphic conditions with creation of a penetrative NW-SE-oriented stretching lineation, X(1) (finite extension), parallel to the direction of simple shear. Earliest cooling after the culmination of the Tertiary metamorphism, some 38 Ma ago, is recorded by the cooling curves of the Monte Rosa and Mischabel nappes to the west and the Suretta Nappe to the east of the Lepontine Gneiss Dome. The onset of dextral transpression, with a strong extension parallel to the mountain belt, and the oldest S-vergent `'backfolding'' took place some 35 to 30 Ma ago during retrograde amphibolite-facies conditions and before the intrusion of the Oligocene dikes north of the Periadriatic Line. The main updoming of the Lepontine Gneiss Dome started some 32-30 Ma ago with the intrusion of the Bergell tonalites and granodiorites, concomitant with S-vergent backfolding and backthrusting and dextral strike-slip movements along the Tonale and Canavese Lines (Argand's Insubric phase). Subsequently, the center of main updoming migrated slowly to the west, reaching the Simplon region some 20 Ma ago. This was contemporaneous with the westward migration of the Adriatic indenter. Between 20 Ma and the present, the Western Aar Massif-Toce culmination was the center of strong uplift. The youngest S-vergent backfolds, the Glishorn anticline and the Berisal syncline fold the 12 Ma Rb/Sr biotite isochron and are cut by the 11 Ma old Rhone-Simplon Line. The discrete Rhone-Simplon Line represents a late retrograde manifestation in the preexisting ductile Simplon Shear Zone. This fault zone is still active today. The Oligocene-Neogene dextral transpression and extension in the Simplon area were concurrent with thrusting to the northwest of the Helvetic nappes, the Prealpes (35-15 Ma) and with the Jura thin-skinned thrust (11-3 Ma). It was also contemporaneous with thrusting to the south of the Bergamasc (> 35-5 Ma) and Milan thrusts (16-5 Ma).

Genetic diversity of chloroquine-resistant Plasmodium vivax parasites from the western Brazilian Amazon

The molecular basis of Plasmodium vivax chloroquine (CQ) resistance is still unknown. Elucidating the molecular background of parasites that are sensitive or resistant to CQ will help to identify and monitor the spread of resistance. By genotyping a panel of molecular markers, we demonstrate a similar genetic variability between in vitro CQ-resistant and sensitive phenotypes of P. vivax parasites. However, our studies identified two loci (MS8 and MSP1-B10) that could be used to discriminate between both CQ-susceptible phenotypes among P. vivax isolates in vitro. These preliminary data suggest that microsatellites may be used to identify and to monitor the spread of P. vivax-resistance around the world.

Temperamental Dimension and Anxiety Problems in a Clinical Sample of Three- to Six-year old Children: A Study of Variables

In the last few years, many researchers have studied the presence of common dimensions of temperament in subjects with symptoms of anxiety. The aim of this study is to examine the association between temperamental dimensions (high negative affect and activity level) and anxiety problems in clinicalpreschool children. A total of 38 children, ages 3 to 6 years, from the Infant and Adolescent Mental Health Center of Girona and the Center of Diagnosis and Early Attention of Sabadell and Olot were evaluated by parents and psychologists. Their parents completed several screening scales and, subsequently, clinical child psychopathology professionals carried out diagnostic interviews with children from the sample who presented signs of anxiety. Findings showed that children with high levels of negative affect and low activity level have pronounced symptoms of anxiety. However, children with anxiety disorders do not present different temperament styles from their peers without these pathologies

Metabolomic profiles of hepatocellular carcinoma in a European prospective cohort.

BACKGROUND Hepatocellular carcinoma (HCC), the most prevalent form of liver cancer, is difficult to diagnose and has limited treatment options with a low survival rate. Aside from a few key risk factors, such as hepatitis, high alcohol consumption, smoking, obesity, and diabetes, there is incomplete etiologic understanding of the disease and little progress in identification of early risk biomarkers. METHODS To address these aspects, an untargeted nuclear magnetic resonance metabolomic approach was applied to pre-diagnostic serum samples obtained from first incident, primary HCC cases (n = 114) and matched controls (n = 222) identified from amongst the participants of a large European prospective cohort. RESULTS A metabolic pattern associated with HCC risk comprised of perturbations in fatty acid oxidation and amino acid, lipid, and carbohydrate metabolism was observed. Sixteen metabolites of either endogenous or exogenous origin were found to be significantly associated with HCC risk. The influence of hepatitis infection and potential liver damage was assessed, and further analyses were made to distinguish patterns of early or later diagnosis. CONCLUSION Our results show clear metabolic alterations from early stages of HCC development with application for better etiologic understanding, prevention, and early detection of this increasingly common cancer.

Prediction of fat-free mass using bioelectrical impedance analysis in young adults from five populations of African origin.

Background/objectives:Bioelectrical impedance analysis (BIA) is used in population and clinical studies as a technique for estimating body composition. Because of significant under-representation in existing literature, we sought to develop and validate predictive equation(s) for BIA for studies in populations of African origin.Subjects/methods:Among five cohorts of the Modeling the Epidemiologic Transition Study, height, weight, waist circumference and body composition, using isotope dilution, were measured in 362 adults, ages 25-45 with mean body mass indexes ranging from 24 to 32. BIA measures of resistance and reactance were measured using tetrapolar placement of electrodes and the same model of analyzer across sites (BIA 101Q, RJL Systems). Multiple linear regression analysis was used to develop equations for predicting fat-free mass (FFM), as measured by isotope dilution; covariates included sex, age, waist, reactance and height(2)/resistance, along with dummy variables for each site. Developed equations were then tested in a validation sample; FFM predicted by previously published equations were tested in the total sample.Results:A site-combined equation and site-specific equations were developed. The mean differences between FFM (reference) and FFM predicted by the study-derived equations were between 0.4 and 0.6âeuro0/00kg (that is, 1% difference between the actual and predicted FFM), and the measured and predicted values were highly correlated. The site-combined equation performed slightly better than the site-specific equations and the previously published equations.Conclusions:Relatively small differences exist between BIA equations to estimate FFM, whether study-derived or published equations, although the site-combined equation performed slightly better than others. The study-derived equations provide an important tool for research in these understudied populations.

Successes and limitations of targeted therapies in renal cell carcinoma.

Until recently, the standard treatment for metastatic renal cell carcinoma (RCC) was nonspecific immunotherapy based on interleukin-2 or interferon-α. This was associated with a modest survival benefit and with significant clinical toxicities. The understanding of numerous molecular pathways in RCC, including HIF, VEGF, mTOR, and the consecutive use of targeted therapies since the beginning of 2005 have significantly improved outcomes for patients with metastatic RCC with an overall survival greater than 2 years. At present, at least 7 targeted agents are approved for first and consecutive lines of treatment of clear cell metastatic RCC. Long-term benefit and extended survival may be achieved through the optimal use of targeted therapies: optimal dosing, adverse event management and treatment duration and compliance. Advances in the finding of prognostic factors highlight the potential for personalizing treatment for patients with metastatic RCC. Data regarding the best sequencing of targeted therapies, predictive biomarkers, best timing of surgery, patient risk profiles, understanding of resistance mechanisms and safety of targeted therapies are growing and will provide a further step ahead in the management of advanced RCC. In parallel, a new class of therapeutics is emerging in RCC: immunotherapy; in particular check-point blockade antibodies are showing very promising results.

Activities of daily living with reverse prostheses: importance of scapular compensation for functional mobility of the shoulder.

HYPOTHESIS: The nonanatomical design of reverse shoulder prostheses induce medial displacement of the center of rotation, impingements and may reduce the mobility of the shoulder. The aim of this study is to test the hypothesis that during activities of daily living functional mobility of the shoulder can be restored by scapular compensation. MATERIAL AND METHODS: A numerical 3-dimensional model was developed to reproduce the movement of the scapula and humerus, during 4 activities of daily living measured experimentally. This hypothesis was tested in 4 configurations of the aequalis reverse prosthesis (standard 36-mm glenosphere, 42-mm glenosphere, lateralized 36-mm glenosphere, lateralized Bony Increased-Offset Reverse Shoulder Arthroplasty [BIO-RSA]), which were implanted in the virtual model. All impingement positions were evaluated, as the required scapular compensation to avoid impingements. RESULTS: With the 36-mm glenosphere, impingements occurred only for rest of hand to back-pocket positions. The 42-mm partly improved the mobility. The 2 lateralized glenospheres were free of impingement. When impingements occurred, the scapular compensation was less than 10°. CONCLUSION: Most reverse prostheses impingements reported in clinical and biomechanical studies can be avoided, either by scapular compensation or by a glenosphere lateralization. After reverse shoulder arthroplasty, a fraction of the mobility of the gleno-humeral is transferred to the scapulo-thoracic joint.

Cell disposition of raltegravir and newer antiretrovirals in HIV-infected patients: high inter-individual variability in raltegravir cellular penetration.

Objectives The site of pharmacological activity of raltegravir is intracellular. Our aim was to determine the extent of raltegravir cellular penetration and whether raltegravir total plasma concentration (C(tot)) predicts cellular concentration (C(cell)). Methods Open-label, prospective, pharmacokinetic study on HIV-infected patients on a stable raltegravir-containing regimen. Plasma and peripheral blood mononuclear cells were simultaneously collected during a 12 h dosing interval after drug intake. C(tot) and C(cell) of raltegravir, darunavir, etravirine, maraviroc and ritonavir were measured by liquid chromatography coupled to tandem mass spectrometry after protein precipitation. Longitudinal mixed effects analysis was applied to the C(cell)/C(tot) ratio. Results Ten HIV-infected patients were included. The geometric mean (GM) raltegravir total plasma maximum concentration (C(max)), minimum concentration (C(min)) and area under the time-concentration curve from 0-12 h (AUC(0-12)) were 1068 ng/mL, 51.1 ng/mL and 4171 ng·h/mL, respectively. GM raltegravir cellular C(max), C(min) and AUC(0-12) were 27.5 ng/mL, 2.9 ng/mL and 165 ng·h/mL, respectively. Raltegravir C(cell) corresponded to 5.3% of C(tot) measured simultaneously. Both concentrations fluctuate in parallel, with C(cell)/C(tot) ratios remaining fairly constant for each patient without a significant time-related trend over the dosing interval. The AUC(cell)/AUC(tot) GM ratios for raltegravir, darunavir and etravirine were 0.039, 0.14 and 1.55, respectively. Conclusions Raltegravir C(cell) correlated with C(tot) (r = 0.86). Raltegravir penetration into cells is low overall (∼5% of plasma levels), with distinct raltegravir cellular penetration varying by as much as 15-fold between patients. The importance of this finding in the context of development of resistance to integrase inhibitors needs to be further investigated.