Ochratoxin A at nanomolar concentration perturbs the homeostasis of neural stem cells in highly differentiated but not in immature three-dimensional brain cell cultures.

Ochratoxin A (OTA), a fungal contaminant of basic food commodities, is known to be highly cytotoxic, but the pathways underlying adverse effects at subcytotoxic concentrations remain to be elucidated. Recent reports indicate that OTA affects cell cycle regulation. Therefore, 3D brain cell cultures were used to study OTA effects on mitotically active neural stem/progenitor cells, comparing highly differentiated cultures with their immature counterparts. Changes in the rate of DNA synthesis were related to early changes in the mRNA expression of neural stem/progenitor cell markers. OTA at 10nM, a concentration below the cytotoxic level, was ineffective in immature cultures, whereas in mature cultures it significantly decreased the rate of DNA synthesis together with the mRNA expression of key transcriptional regulators such as Sox2, Mash1, Hes5, and Gli1; the cell cycle activator cyclin D2; the phenotypic markers nestin, doublecortin, and PDGFRα. These effects were largely prevented by Sonic hedgehog (Shh) peptide (500ngml(-1)) administration, indicating that OTA impaired the Shh pathway and the Sox2 regulatory transcription factor critical for stem cell self-renewal. Similar adverse effects of OTA in vivo might perturb the regulation of stem cell proliferation in the adult brain and in other organs exhibiting homeostatic and/or regenerative cell proliferation.

Connexin 43 expression in human hypertrophied heart due to pressure and volume overload.

Left ventricular hypertrophy (LVH) is due to pressure overload or mechanical stretch and is thought to be associated with remodeling of gap-junctions. We investigated whether the expression of connexin 43 (Cx43) is altered in humans in response to different degrees of LVH. The expression of Cx43 was analyzed by quantitative polymerase chain reaction, Western blot analysis and immunohistochemistry on left ventricular biopsies from patients undergoing aortic or mitral valve replacement. Three groups were analyzed: patients with aortic stenosis with severe LVH (n=9) versus only mild LVH (n=7), and patients with LVH caused by mitral regurgitation (n=5). Cx43 mRNA expression and protein expression were similar in the three groups studied. Furthermore, immunohistochemistry revealed no change in Cx43 distribution. We can conclude that when compared with mild LVH or with LVH due to volume overload, severe LVH due to chronic pressure overload is not accompanied by detectable changes of Cx43 expression or spatial distribution.

The protective role of transferrin in Müller glial cells after iron-induced toxicity.

PURPOSE: Transferrin (Tf) expression is enhanced by aging and inflammation in humans. We investigated the role of transferrin in glial protection. METHODS: We generated transgenic mice (Tg) carrying the complete human transferrin gene on a C57Bl/6J genetic background. We studied human (hTf) and mouse (mTf) transferrin localization in Tg and wild-type (WT) C57Bl/6J mice using immunochemistry with specific antibodies. Müller glial (MG) cells were cultured from explants and characterized using cellular retinaldehyde binding protein (CRALBP) and vimentin antibodies. They were further subcultured for study. We incubated cells with FeCl(3)-nitrilotriacetate to test for the iron-induced stress response; viability was determined by direct counting and measurement of lactate dehydrogenase (LDH) activity. Tf expression was determined by reverse transcriptase-quantitative PCR with human- or mouse-specific probes. hTf and mTf in the medium were assayed by ELISA or radioimmunoassay (RIA), respectively. RESULTS: mTf was mainly localized in retinal pigment epithelium and ganglion cell layers in retina sections of both mouse lines. hTf was abundant in MG cells. The distribution of mTf and hTf mRNA was consistent with these findings. mTf and hTf were secreted into the medium of MG cell primary cultures. Cells from Tg mice secreted hTf at a particularly high level. However, both WT and Tg cell cultures lose their ability to secrete Tf after a few passages. Tg MG cells secreting hTf were more resistant to iron-induced stress toxicity than those no longer secreted hTf. Similarly, exogenous human apo-Tf, but not human holo-Tf, conferred resistance to iron-induced stress on MG cells from WT mice. CONCLUSIONS: hTf localization in MG cells from Tg mice was reminiscent of that reported for aged human retina and age-related macular degeneration, both conditions associated with iron deposition. The role of hTf in protection against toxicity in Tg MG cells probably involves an adaptive mechanism developed in neural retina to control iron-induced stress.

Enhanced vascular contractility in alpha1-adrenergic receptor-deficient mice.

AIM: Alpha1-adrenergic receptors (alpha1-ARs) are classified into three subtypes: alpha1A-AR, alpha1B-AR, and alpha1D-AR. Triple disruption of alpha1A-AR, alpha1B-AR, and alpha1D-AR genes results in hypotension and produces no contractile response of the thoracic aorta to noradrenalin. Presently, we characterized vascular contractility against other vasoconstrictors, such as potassium, prostaglandin F2alpha (PGF(2alpha)) and 5-hydroxytryptamine (5-HT), in alpha1A-AR, alpha1B-AR, and alpha1D-AR triple knockout (alpha1-AR triple KO) mice. MAIN METHODS: The contractile responses to the stimulation with vasoconstrictors were studied using isolated thoracic aorta. KEY FINDINGS: As a result, the phasic and tonic contraction induced by a high concentration of potassium (20 mM) was enhanced in the isolated thoracic aorta of alpha1-AR triple KO mice compared with that of wild-type (WT) mice. In addition, vascular responses to PGF(2alpha) and 5-HT were also enhanced in the isolated thoracic aorta of alpha1-AR triple KO mice compared with WT mice. Similar to in vitro findings with isolated thoracic aorta, in vivo pressor responses to PGF(2alpha) were enhanced in alpha1-AR triple KO mice. Real-time reverse transcription-polymerase chain reaction analysis and western blot analysis indicate that gene expression of the 5-hydroxytryptamine 2A (5-HT(2A)) receptor was up-regulated in the thoracic aorta of alpha1-AR triple KO mice while the prostaglandin F2alpha receptor (FP) was unchanged. SIGNIFICANCE: These results suggest that loss of alpha1-ARs can lead to enhancement of vascular responsiveness to the vasoconstrictors and may imply that alpha1-ARs and the subsequent signaling regulate the vascular responsiveness to other stimulations such as depolarization, 5-HT and PGF(2alpha).

Ultra-Deep Pyrosequencing Detects Conserved Genomic Sites and Quantifies Linkage of Drug-Resistant Amino Acid Changes in the Hepatitis B Virus Genome

Selection of amino acid substitutions associated with resistance to nucleos(t)ide-analog (NA) therapy in the hepatitis B virus (HBV) reverse transcriptase (RT) and their combination in a single viral genome complicates treatment of chronic HBV infection and may affect the overlapping surface coding region. In this study, the variability of an overlapping polymerase-surface region, critical for NA resistance, is investigated before treatment and under antiviral therapy, with assessment of NA-resistant amino acid changes simultaneously occurring in the same genome (linkage analysis) and their influence on the surface coding region.

The structure of human tRNALys3 anticodon bound to HIV genome is stabilized by modified nucleosides and adjacent mismatch base pairs

Replication of human immunodeficiency virus (HIV) requires base pairing of the reverse transcriptase primer, human tRNA(Lys3), to the viral RNA. Although the major complementary base pairing occurs between the HIV primer binding sequence (PBS) and the tRNA's 3'-terminus, an important discriminatory, secondary contact occurs between the viral A-rich Loop I, 5'-adjacent to the PBS, and the modified, U-rich anticodon domain of tRNA(Lys3). The importance of individual and combined anticodon modifications to the tRNA/HIV-1 Loop I RNA's interaction was determined. The thermal stabilities of variously modified tRNA anticodon region sequences bound to the Loop I of viral sub(sero)types G and B were analyzed and the structure of one duplex containing two modified nucleosides was determined using NMR spectroscopy and restrained molecular dynamics. The modifications 2-thiouridine, s(2)U(34), and pseudouridine, Psi(39), appreciably stabilized the interaction of the anticodon region with the viral subtype G and B RNAs. The structure of the duplex results in two coaxially stacked A-form RNA stems separated by two mismatched base pairs, U(162)*Psi(39) and G(163)*A(38), that maintained a reasonable A-form helix diameter. The tRNA's s(2)U(34) stabilized the interaction between the A-rich HIV Loop I sequence and the U-rich anticodon, whereas the tRNA's Psi(39) stabilized the adjacent mismatched pairs.

Postnatal β-cell maturation is associated with islet-specific microRNA changes induced by nutrient shifts at weaning.

Glucose-induced insulin secretion is an essential function of pancreatic β-cells that is partially lost in individuals affected by Type 2 diabetes. This unique property of β-cells is acquired through a poorly understood postnatal maturation process involving major modifications in gene expression programs. Here we show that β-cell maturation is associated with changes in microRNA expression induced by the nutritional transition that occurs at weaning. When mimicked in newborn islet cells, modifications in the level of specific microRNAs result in a switch in the expression of metabolic enzymes and cause the acquisition of glucose-induced insulin release. Our data suggest microRNAs have a central role in postnatal β-cell maturation and in the determination of adult functional β-cell mass. A better understanding of the events governing β-cell maturation may help understand why some individuals are predisposed to developing diabetes and could lead to new strategies for the treatment of this common metabolic disease.

Abacavir/Lamivudine Versus Tenofovir/Emtricitabine in Virologically Suppressed Patients Switching from Ritonavir-Boosted Protease Inhibitors to Raltegravir

There are few clinical data on the combination abacavir/lamivudine plus raltegravir. We compared the outcomes of patients from the SPIRAL trial receiving either abacavir/lamivudine or tenofovir/emtricitabine at baseline who had taken at least one dose of either raltegravir or ritonavir-boosted protease inhibitors. For the purpose of this analysis, treatment failure was defined as virological failure (confirmed HIV-1 RNA ≥50 copies/ml) or discontinuation of abacavir/lamivudine or tenofovir/emtricitabine because of adverse events, consent withdrawal, or lost to follow-up. There were 143 (72.59%) patients with tenofovir/emtricitabine and 54 (27.41%) with abacavir/lamivudine. In the raltegravir group, there were three (11.11%) treatment failures with abacavir/lamivudine and eight (10.96%) with tenofovir/emtricitabine (estimated difference 0.15%; 95% CI -17.90 to 11.6). In the ritonavir-boosted protease inhibitor group, there were four (14.81%) treatment failures with abacavir/lamivudine and 12 (17.14%) with tenofovir/emtricitabine (estimated difference -2.33%; 95% CI -16.10 to 16.70). Triglycerides decreased and HDL cholesterol increased through the study more pronouncedly with abacavir/lamivudine than with tenofovir/emtricitabine and differences in the total-to-HDL cholesterol ratio between both combinations of nucleoside reverse transcriptase inhibitors (NRTIs) tended to be higher in the raltegravir group, although differences at 48 weeks were not significant. While no patient discontinued abacavir/lamivudine due to adverse events, four (2.80%) patients (all in the ritonavir-boosted protease inhibitor group) discontinued tenofovir/emtricitabine because of adverse events (p=0.2744). The results of this analysis do not suggest that outcomes of abacavir/lamivudine are worse than those of tenofovir/emtricitabine when combined with raltegravir in virologically suppressed HIV-infected adults.

Imaging techniques applied for detection of secretion, transgene delivery and G proteincoupled receptors in reproductive and endocrine cells in vivo and in vitro

Post-testicular sperm maturation occurs in the epididymis. The ion concentration and proteins secreted into the epididymal lumen, together with testicular factors, are believed to be responsible for the maturation of spermatozoa. Disruption of the maturation of spermatozoa in the epididymis provides a promising strategy for generating a male contraceptive. However, little is known about the proteins involved. For drug development, it is also essential to have tools to study the function of these proteins in vitro. One approach for screening novel targets is to study the secretory products of the epididymis or the G protein-coupled receptors (GPCRs) that are involved in the maturation process of the spermatozoa. The modified Ca2+ imaging technique to monitor release from PC12 pheochromocytoma cells can also be applied to monitor secretory products involved in the maturational processes of spermatozoa. PC12 pheochromocytoma cells were chosen for evaluation of this technique as they release catecholamines from their cell body, thus behaving like endocrine secretory cells. The results of the study demonstrate that depolarisation of nerve growth factor -differentiated PC12 cells releases factors which activate nearby randomly distributed HEL erythroleukemia cells. Thus, during the release process, the ligands reach concentrations high enough to activate receptors even in cells some distance from the release site. This suggests that communication between randomly dispersed cells is possible even if the actual quantities of transmitter released are extremely small. The development of a novel method to analyse GPCR-dependent Ca2+ signalling in living slices of mouse caput epididymis is an additional tool for screening for drug targets. By this technique it was possible to analyse functional GPCRs in the epithelial cells of the ductus epididymis. The results revealed that, both P2X- and P2Y-type purinergic receptors are responsible for the rapid and transient Ca2+ signal detected in the epithelial cells of caput epididymides. Immunohistochemical and reverse transcriptase-polymerase chain reaction (RTPCR) analyses showed the expression of at least P2X1, P2X2, P2X4 and P2X7, and P2Y1 and P2Y2 receptors in the epididymis. Searching for epididymis-specific promoters for transgene delivery into the epididymis is of key importance for the development of specific models for drug development. We used EGFP as the reporter gene to identify proper promoters to deliver transgenes into the epithelial cells of the mouse epididymis in vivo. Our results revealed that the 5.0 kb murine Glutathione peroxidase 5 (GPX5) promoter can be used to target transgene expression into the epididymis while the 3.8 kb Cysteine-rich secretory protein-1 (CRISP-1) promoter can be used to target transgene expression into the testis. Although the visualisation of EGFP in living cells in culture usually poses few problems, the detection of EGFP in tissue sections can be more difficult because soluble EGFP molecules can be lost if the cell membrane is damaged by freezing, sectioning, or permeabilisation. Furthermore, the fluorescence of EGFP is dependent on its conformation. Therefore, fixation protocols that immobilise EGFP may also destroy its usefulness as a fluorescent reporter. We therefore developed a novel tissue preparation and preservation techniques for EGFP. In addition, fluorescence spectrophotometry with epididymal epithelial cells in suspension revealed the expression of functional purinergic, adrenergic, cholinergic and bradykinin receptors in these cell lines (mE-Cap27 and mE-Cap28). In conclusion, we developed new tools for studying the role of the epididymis in sperm maturation. We developed a new technique to analyse GPCR dependent Ca2+ signalling in living slices of mouse caput epididymis. In addition, we improved the method of detecting reporter gene expression. Furthermore, we characterised two epididymis-specific gene promoters, analysed the expression of GPCRs in epididymal epithelial cells and developed a novel technique for measurement of secretion from cells.

Clinical outcomes of lung transplant recipients with telomerase mutations.

BACKGROUND: Successful lung transplantation for patients with pulmonary fibrosis from telomerase mutations may be limited by systemic complications of telomerase dysfunction, including myelosuppression, cirrhosis, and malignancy. We describe clinical outcomes in 14 lung transplant recipients with telomerase mutations. METHODS: Subjects underwent lung transplantation between February 2005 and April 2014 at 5 transplant centers. Data were abstracted from medical records, focusing on outcomes reflecting post-transplant treatment effects likely to be complicated by telomerase mutations. RESULTS: The median age of subjects was 60.5 years (interquartile range = 52.0-62.0), 64.3% were male, and the mean post-transplant observation time was 3.2 years (SD ± 2.9). A mutation in telomerase reverse transcriptase was present in 11 subjects, a telomerase RNA component mutation was present in 2 subjects, and an uncharacterized mutation was present in 1 subject. After lung transplantation, 10 subjects were leukopenic and 5 did not tolerate lymphocyte anti-proliferative agents. Six subjects developed recurrent lower respiratory tract infections, 7 developed acute cellular rejection (A1), and 4 developed chronic lung allograft dysfunction. Eight subjects developed at least 1 episode of acute renal failure and 10 developed chronic renal insufficiency. In addition, 3 subjects developed cancer. No subjects had cirrhosis. At data censorship, 13 subjects were alive. CONCLUSIONS: The clinical course for lung transplant recipients with telomerase mutations is complicated by renal disease, leukopenia with intolerance of lymphocyte anti-proliferative agents, and recurrent lower respiratory tract infections. In contrast, cirrhosis was absent, acute cellular rejection was mild, and development of chronic lung allograft dysfunction was comparable to other lung transplant recipients. Although it poses challenges, lung transplantation may be feasible for patients with pulmonary fibrosis from telomerase mutations.

Once-daily dolutegravir versus darunavir plus ritonavir for treatment-naive adults with HIV-1 infection (FLAMINGO): 96 week results from a randomised, open-label, phase 3b study.

BACKGROUND: The primary analysis of the FLAMINGO study at 48 weeks showed that patients taking dolutegravir once daily had a significantly higher virological response rate than did those taking ritonavir-boosted darunavir once daily, with similar tolerability. We present secondary efficacy and safety results analysed at 96 weeks. METHODS: FLAMINGO was a multicentre, open-label, phase 3b, non-inferiority study of HIV-1-infected treatment-naive adults. Patients were randomly assigned (1:1) to dolutegravir 50 mg or darunavir 800 mg plus ritonavir 100 mg, with investigator-selected combination tenofovir and emtricitabine or combination abacavir and lamivudine background treatment. The main endpoints were plasma HIV-1 RNA less than 50 copies per mL and safety. The non-inferiority margin was -12%. If the lower end of the 95% CI was greater than 0%, then we concluded that dolutegravir was superior to ritonavir-boosted darunavir. This trial is registered with ClinicalTrials.gov, number NCT01449929. FINDINGS: Of 595 patients screened, 488 were randomly assigned and 484 included in the analysis (242 assigned to receive dolutegravir and 242 assigned to receive ritonavir-boosted darunavir). At 96 weeks, 194 (80%) of 242 patients in the dolutegravir group and 164 (68%) of 242 in the ritonavir-boosted darunavir group had HIV-1 RNA less than 50 copies per mL (adjusted difference 12·4, 95% CI 4·7-20·2; p=0·002), with the greatest difference in patients with high viral load at baseline (50/61 [82%] vs 32/61 [52%], homogeneity test p=0·014). Six participants (three since 48 weeks) in the dolutegravir group and 13 (four) in the darunavir plus ritonavir group discontinued because of adverse events. The most common drug-related adverse events were diarrhoea (23/242 [10%] in the dolutegravir group vs 57/242 [24%] in the darunavir plus ritonavir group), nausea (31/242 [13%] vs 34/242 [14%]), and headache (17/242 [7%] vs 12/242 [5%]). INTERPRETATION: Once-daily dolutegravir is associated with a higher virological response rate than is once-daily ritonavir-boosted darunavir. Dolutegravir compares favourably in efficacy and safety to a boosted darunavir regimen with nucleoside reverse transcriptase inhibitor background treatment for HIV-1-infected treatment-naive patients. FUNDING: ViiV Healthcare and Shionogi & Co.

Oxidative stress in gastric mucosa of asymptomatic humans infected with Helicobacter pylori: effect of bacterial eradication.

BACKGROUND: Inducible nitric oxide synthase (iNOS) and interleukin 8 (IL-8) are positive in approximately 50% of Helicobacter pylori-related diseases but it is not clear whether oxidative stress is also present in H. pylori asymptomatic humans. Our aim was to study the expression of iNOS, superoxide dismutase, catalase and IL-8 production in H. pylori-infected asymptomatic humans, and to investigate the effect of eradication of H. pylori. MATERIALS AND METHODS: Biopsies of corpus and antrum of asymptomatic H. pylori positive and negative humans served for determination of the gastritis score and H. pylori status; iNOS was measured by reverse transcriptase polymerase chain reaction and immunohistochemistry and superoxide dismutase and catalase by immunohistochemistry. IL-8 in biopsies was assessed by enzyme-linked immunosorbent assay. RESULTS: Immunostaining of iNOS, catalase and superoxide dismutase was significantly associated with H. pylori infection and was localized to inflammatory cells. IL-8 concentrations were greater in the H. pylori positive than H. pylori negative group and decreased after bacterial eradication. A decrease in staining for iNOS and catalase was observed after H. pylori eradication. CONCLUSIONS: INOS and antioxidant enzymes are present in gastric biopsies of asymptomatic H. pylori positive humans. Eradication caused a significant decrease in staining for iNOS and catalase. These results indicate that oxidative stress occurs in asymptomatic patients and can be modulated by H. pylori eradication.

The fusion protein SS18-SSX1 employs core Wnt pathway transcription factors to induce a partial Wnt signature in synovial sarcoma.

Expression of the SS18/SYT-SSX fusion protein is believed to underlie the pathogenesis of synovial sarcoma (SS). Recent evidence suggests that deregulation of the Wnt pathway may play an important role in SS but the mechanisms whereby SS18-SSX might affect Wnt signaling remain to be elucidated. Here, we show that SS18/SSX tightly regulates the elevated expression of the key Wnt target AXIN2 in primary SS. SS18-SSX is shown to interact with TCF/LEF, TLE and HDAC but not β-catenin in vivo and to induce Wnt target gene expression by forming a complex containing promoter-bound TCF/LEF and HDAC but lacking β-catenin. Our observations provide a tumor-specific mechanistic basis for Wnt target gene induction in SS that can occur in the absence of Wnt ligand stimulation.

Cost-effectiveness of Tenofovir / Emtricitabine / Efavirenz versus Zidovudine / Lamivudine / Efavirenz as fist-line regimens for the treatment of HIV-infected patients in low-income settings

The Highly Active Antiretroviral Therapy (HAART) is the combination of at least three antiretroviral compounds. The combination purpose is to reduce the likelihood of drug resistance. However in the long-term the resistance to the first-line combination occurs and leads to treatment failure. Thus, a second-line and even a third-line regimen are recommended in the long run. [...] [P. 5] The two treatment alternatives under comparison: Tenofovir (300 mg) CO-formulated with Emtricitabine (200 mg) and Efavirenz (600 mg) currently known under the brand name Atripla (R) was introduced in July 2006 in the United States market. The excellent safety profile and ease of use make this combination a perfect first-line regimen in low-income settings. Therefore, this treatment option was recommended in WHO 2006 reviewed guidelines. Unfortunately, Tenofovir and Emtricitabine compounds are still costly and not yet widely available. For a matter of simplification this regimen is referred in this report as "the recent" therapy. Initially, we had in mind to consider the most frequently used first-line regimen in low-income countries (Stavudine / Larnivudme / Nevirapine) as a comparator for this economic evaluation. Unfortunately, according to the literature review results (see Annex 3); there was no data available comparing head to head the effectiveness of this regimen with the recent one. Instead, we selected a less frequently but commonly used first-line regimen in low-income countries as a comparator: Zidovudine, Lamivudine, Efavirenz. This combination has extensive experience in durability, safety and toxicity and seems to be an optimal choice for a first-line regimen according to the clinical trial group 384 team. Furthermore, Zidovudine, one of the compounds of this combination is now recommended as one of the preferred NNRTI [Non Nucleoside Reverse Transcriptase Inhibitors] options to be considered by countries instead of Stavudine (the most used NNRTI in limited-income countries). As this combination has been included in the WHO guidelines as a first-line therapy since 2003 when WHO launched the "3 by 5" scaling-up initiative, this combination of drugs is referred in this report as the "old" therapy. Objectives: The primary objective of this economic evaluation is to compare the two first-line HAARTs introduced above, in a low-income setting context. Both of these combinations are recommended by the 2006 WHO guidelines as potential first-line regimens. The secondary objective is to provide a simplified and comprehensible cost-effectiveness modeling tool in order to help policy makers, in resource-limited settings, make decisions about which first-line HAART to fund using the scarce resources available. [P. 6-7]

Estratégias farmacológicas para a terapia anti-AIDS

The replicative cycle of HIV presents several events. The proteins involved in these events can be anticipated as pharmacological targets, aiming to the development of anti viral agents. Presently, there are fifteen commercially available anti-HIV drugs, which act at substrate binding site of reverse transcriptase (zidovudine, didanosine, zalcitabine, stavudine, lamivudine and abacavir), at a non-substrate binding site of reverse transcriptase (nevirapine, delavirdine and efavirenz), or by inhibiting HIV protease activity (saquinavir, ritonavir, indinavir, nelfinavir, amprenavir and lopinavir). The present review focus both on these established classes of drugs and on new classes of compounds acting on other virus specific steps.