Plasma angiotensin-(1-8) octapeptide measurement to assess acute angiotensin-converting enzyme inhibition with captopril administered parenterally to normal subjects.

The blood pressure (BP), heart rate (HR), and humoral effects of single intravenous (i.v.) doses of the angiotensin-converting enzyme (ACE) inhibitor captopril was investigated in five normotensive healthy volunteers. Each subject received at 1-week intervals a bolus dose of either captopril (1, 5, and 25 mg) or its vehicle. The study was conducted in a single-blind fashion, and the order of treatment phases was randomized. The different doses of captopril had no acute effect on BP and HR. They induced a dose-dependent decrease in plasma ACE activity and plasma angiotensin II levels. The angiotensin-(1-8) octapeptide was isolated by solid-phase extraction and high-performance liquid chromatography (HPLC) prior to radioimmunoassay (RIA). All three doses of captopril reduced circulating angiotensin II levels within 15 min of drug administration. Only with the 25-mg dose was the angiotensin II concentration below the detection limit at 15 min and still significantly reduced 90 min after drug administration. Simultaneous and progressive decreases in plasma aldosterone levels were observed both with ACE inhibition and during vehicle injection, but the relative fall was more pronounced after captopril administration. No adverse reaction was noticed. These results demonstrate that captopril given parenterally blocks the renin-angiotensin system in a dose-dependent manner. Only with the dose of 25 mg was the inhibition of plasma-converting enzyme activity and the reduction of plasma angiotensin II sustained for at least 1 1/2 h.

Genomic organization, fine-mapping, and expression of the human islet-brain 1 (IB1)/c-Jun-amino-terminal kinase interacting protein-1 (JIP-1) gene.

Islet-brain 1 (IB1), a regulator of the pancreatic beta-cell function in the rat, is homologous to JIP-1, a murine inhibitor of c-Jun amino-terminal kinase (JNK). Whether IB1 and JIP-1 are present in humans was not known. We report the sequence of the 2133-bp human IB1 cDNA, the expression, structure, and fine-mapping of the human IB1 gene, and the characterization of an IB1 pseudogene. Human IB1 is 94% identical to rat IB1. The tissue-specific expression of IB1 in human is similar to that observed in rodent. The IB1 gene contains 12 exons and maps to chromosome 11 (11p11.2-p12), a region that is deleted in DEFECT-11 syndrome. Apart from an IB1 pseudogene on chromosome 17 (17q21), no additional IB1-related gene was found in the human genome. Our data indicate that the sequence and expression pattern of IB1 are highly conserved between rodent and human and provide the necessary tools to investigate whether IB1 is involved in human diseases.

Prevalence of HIV type 1 drug resistance mutations in treatment-naïve and experienced patients from resource-limited settings with universal access to antiretroviral therapy: a survey in two small Brazilian cities

Concerns have been raised that universal availability of antiretroviral agents in resource-limited settings might lead to the emergence and spread of resistant strains. We present the largest survey on human immunodeficiency virus type 1 (HIV-1) resistance among treatment-naïve and experienced patients followed in small, relatively underprivileged cities in Brazil with universal availability to standard of care antiretroviral combinations. Samples were collected between 2004 and 2006 from 95 patients followed in the cities of Saquarema and Santo Antonio de Pádua, state of Rio de Janeiro. A proviral fragment encompassing protease and reverse transcriptase (RT) regions was generated and drug susceptibility level was inferred. Among 50 strains from drug-naïve subjects, one (2%) had intermediate-level resistance to RT inhibitors. Among 38 patients on therapy as of sampling, 28 (73.7%) had plasma viral load (PVL) below detection limit (26 of whom without evidence of resistance mutations) and 11 (28.9%) harbored strains with reduced susceptibility. Only two strains harbored both protease and RT inhibitor mutations. Among seven patients who were off-treatment as of sampling, two (28.5%) harbored strains with reduced susceptibility to RT inhibitors. The relatively high frequency of undetectable PVL among patients on treatment and the overall low prevalence of resistance-associated mutations are reassuring. Continued surveillance, however, is necessary.

Effect of the chitin synthesis inhibitor triflumuron on the development, viability and reproduction of Aedes aegypti

The control of Aedes aegypti is impaired due to the development of resistance to chemical insecticides. Insect Growth Regulators (IGR) exhibit distinct mechanisms of action and are considered potential vector control alternatives. Studies regarding the effects of sublethal IGR doses on the viability of resulting adults will contribute to eval-uating their impact in the field. We analyzed several aspects of Ae. aegypti adults surviving exposure to a partially lethal dose of triflumuron, a chitin synthesis inhibitor. A highly significant difference in the proportion of males and females was noted in the triflumuron-exposed group (65.0% males) compared to the controls (50.2% males). Triflumuron affected adult longevity, particularly for females; after 16 days, only 29.2% of males and 13.8% of females were alive, in contrast with 94% survival of the control mosquitoes. The locomotor activity was reduced and the blood-feeding ability of the treated females was also affected (90.4% and 48.4% of the control and triflumuron-exposed females, respectively, successfully ingested blood). Triflumuron-surviving females ingested roughly 30% less blood and laid 25% fewer eggs than the control females. The treated males and females exhibited a diminished ability to copulate, resulting in less viable eggs.

Prevalence and resistance mutations of non-B HIV-1 subtypes among immigrants in Southern Spain along the decade 2000-2010.

Most of the non-B HIV-1 subtypes are predominant in Sub-Saharan Africa and India although they have been found worldwide. In the last decade, immigration from these areas has increased considerably in Spain. The objective of this study was to evaluate the prevalence of non-B subtypes circulating in a cohort of HIV-1-infected immigrants in Seville, Southern Spain and to identify drug resistance-associated mutations. METHODS: Complete protease and first 220 codons of the reverse transcriptase coding regions were amplified and sequenced by population sequencing. HIV-1 subtypes were determined using Stanford University Drug Resistance Database, and phylogenetic analysis was performed comparing multiple reported sequences. Drug resistance mutations were defined according to the International AIDS Society-USA. RESULTS: From 2000 to 2010 a total of 1,089 newly diagnosed HIV-1-infected patients were enrolled in our cohort. Of these, 121 were immigrants, of which 98 had ethical approval and informed consent to include in our study. Twenty-nine immigrants (29/98, 29.6%) were infected with non-B subtypes, of which 15/29 (51.7%) were CRF02-AG, mostly from Sub-Saharan Africa, and 2/29 (6.9%) were CRF01-AE from Eastern Europe. A, C, F, J and G subtypes from Eastern Europe, Central-South America and Sub-Saharan Africa were also present. Some others harboured recombinant forms CRF02-AG/CRF01-AE, CRF2-AG/G and F/B, B/C, and K/G, in PR and RT-coding regions. Patients infected with non-B subtypes showed a high frequency of minor protease inhibitor resistance mutations, M36I, L63P, and K20R/I. Only one patient, CRF02_AG, showed major resistance mutation L90M. Major RT inhibitor resistance mutations K70R and A98G were present in one patient with subtype G, L100I in one patient with CRF01_AE, and K103N in another patient with CRF01_AE. Three patients had other mutations such as V118I, E138A and V90I. CONCLUSIONS: The circulation of non-B subtypes has significantly increased in Southern Spain during the last decade, with 29.6% prevalence, in association with demographic changes among immigrants. This could be an issue in the treatment and management of these patients. Resistance mutations have been detected in these patients with a prevalence of 7% among treatment-naïve patients compared with the 21% detected among patients under HAART or during treatment interruption.

Modification of oxidative status in Plasmodium berghei-infected erythrocytes by E-2-chloro-8-methyl-3-[(4'-methoxy-1'-indanoyl)-2'-methyliden]-quinoline compared to chloroquine

E-2-chloro-8-methyl-3-[(4'-methoxy-1'-indanoyl)-2'-methyliden]-quinoline (IQ) is a new quinoline derivative which has been reported as a haemoglobin degradation and ß-haematin formation inhibitor. The haemoglobin proteolysis induced by Plasmodium parasites represents a source of amino acids and haeme, leading to oxidative stress in infected cells. In this paper, we evaluated oxidative status in Plasmodium berghei-infected erythrocytes in the presence of IQ using chloroquine (CQ) as a control. After haemolysis, superoxide dismutase (SOD), catalase, glutathione cycle and NADPH + H+-dependent dehydrogenase enzyme activities were investigated. Lipid peroxidation was also assayed to evaluate lipid damage. The results showed that the overall activities of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase were significantly diminished by IQ (by 53.5% and 100%, respectively). Glutathione peroxidase activity was also lowered (31%) in conjunction with a higher GSSG/GSH ratio. As a compensatory response, overall SOD activity increased and lipid peroxidation decreased, protecting the cells from the haemolysis caused by the infection. CQ shared most of the effects showed by IQ; however it was able to inhibit the activity of isocitrate dehydrogenase and glutathione-S-transferase. In conclusion, IQ could be a candidate for further studies in malaria research interfering with the oxidative status in Plasmodium berghei infection.

Analysis of TNFAIP3, a feedback inhibitor of nuclear factor-κB and the neighbor intergenic 6q23 region in rheumatoid arthritis susceptibility

INTRODUCTION Genome-wide association studies of rheumatoid arthritis (RA) have identified an association of the disease with a 6q23 region devoid of genes. TNFAIP3, an RA candidate gene, flanks this region, and polymorphisms in both the TNFAIP3 gene and the intergenic region are associated with systemic lupus erythematosus. We hypothesized that there is a similar association with RA, including polymorphisms in TNFAIP3 and the intergenic region. METHODS To test this hypothesis, we selected tag-single nucleotide polymorphisms (SNPs) in both loci. They were analyzed in 1,651 patients with RA and 1,619 control individuals of Spanish ancestry. RESULTS Weak evidence of association was found both in the 6q23 intergenic region and in the TNFAIP3 locus. The rs582757 SNP and a common haplotype in the TNFAIP3 locus exhibited association with RA. In the intergenic region, two SNPs were associated, namely rs609438 and rs13207033. The latter was only associated in patients with anti-citrullinated peptide antibodies. Overall, statistical association was best explained by the interdependent contribution of SNPs from the two loci TNFAIP3 and the 6q23 intergenic region. CONCLUSIONS Our data are consistent with the hypothesis that several RA genetic factors exist in the 6q23 region, including polymorphisms in the TNFAIP3 gene, like that previously described for systemic lupus erythematosus.

Effects of highly active antiretroviral therapy with nelfinavir in vertically HIV-1 infected children: 3 years of follow-up. Long-term response to nelfinavir in children.

BACKGROUND Antiretroviral treatment (ART) in children has special features and consequently, results obtained from clinical trials with antiretroviral drugs in adults may not be representative of children. Nelfinavir (NFV) is an HIV-1 Protease Inhibitor (PI) which has become as one of the first choices of PI for ART in children. We studied during a 3-year follow-up period the effects of highly active antiretroviral therapy with nelfinavir in vertically HIV-1 infected children. METHODS Forty-two vertically HIV-infected children on HAART with NFV were involved in a multicentre prospective study. The children were monitored at least every 3 months with physical examinations, and blood sample collection to measure viral load (VL) and CD4+ cell count. We performed a logistic regression analysis to determinate the odds ratio of baseline characteristics on therapeutic failure. RESULTS Very important increase in CD4+ was observed and VL decreased quickly and it remained low during the follow-up study. Children with CD4+ <25% at baseline achieved CD4+ >25% at 9 months of follow-up. HIV-infected children who achieved undetectable viral load (uVL) were less than 40% in each visit during follow-up. Nevertheless, HIV-infected children with VL >5000 copies/ml were less than 50% during the follow-up study. Only baseline VL was an important factor to predict VL control during follow-up. Virological failure at defined end-point was confirmed in 30/42 patients. Along the whole of follow-up, 16/42 children stopped HAART with NFV. Baseline characteristics were not associated with therapeutic change. CONCLUSION NFV is a safe drug with a good profile and able to achieve an adequate response in children.

The impact of the nelfinavir resistance-conferring mutation D30N on the susceptibility of HIV-1 subtype B to other protease inhibitors

The human immunodeficiency virus type 1 (HIV-1) protease mutation D30N is exclusively selected by the protease inhibitor (PI) nelfinavir and confers resistance to this drug. We demonstrate that D30N increases the susceptibility to saquinavir (SQV) and amprenavir in HIV-1 subtype B isolates and that the N88D mutation in a D30N background neutralizes this effect. D30N also suppresses indinavir (IDV) resistance caused by the M46I mutation. Interestingly, in patients with viruses originally containing the D30N mutation who were treated with IDV or SQV, the virus either reversed this mutation or acquired N88D, suggesting an antagonistic effect of D30N upon exposure to these PIs. These findings can improve direct salvage drug treatment in resource limited countries where subtype B is epidemiologically important and extend the value of first and second line PIs in these populations.

Effects of MDL 100,240, a dual inhibitor of angiotensin-converting enzyme and neutral endopeptidase on the vasopressor response to exogenous angiotensin I and angiotensin II challenges in healthy volunteers.

MDL 100,240, a dual inhibitor of angiotensin-converting enzyme (ACE) and neutral endopeptidase (NEP), was administered intravenously to two panels of four healthy males in a four-period, dose-increasing (0, 1.56, 6.25, and 25 mg, and 0, 3.13, 12.5, and 50 mg, respectively) double-blind, placebo-controlled study. Plasma ACE activity and blood-pressure response to exogenous angiotensin I and angiotensin II i.v. challenges and safety and tolerance were assessed over a 24-h period. MDL 100,240 induced a rapid, dose-related, and sustained inhibition of ACE (>70% over 24 h at doses > or =12.5 mg). The time integral of ACE inhibition was related to the dose but with near-maximal values already attained at doses > or =12.5 mg. Systolic and diastolic blood-pressure responses to exogenous angiotensin I challenges were inhibited in a dose-dependent fashion, whereas the effects of angiotensin II remained unaffected. Mean supine blood pressure decreased transiently (3 h) at doses > or =3.125 mg and < or =24 h with the 25- and 50-mg doses, but not significantly. MDL 100,240 was well tolerated. In healthy subjects, MDL 100,240 exerts a dose-dependent and long-lasting ACE-blocking activity, also expressed by the inhibition of the pressor responses to exogenous angiotensin I challenges. The baroreceptor reflex, assessed by the response to exogenous angiotensin II challenge, remains unaltered.

Targeting renal cell carcinoma with NVP-BEZ235, a dual PI3K/mTOR inhibitor, in combination with sorafenib.

Background: Targeted therapies for metastatic renal cell carcinoma (RCC), including mammalian target of rapamycin (mTOR) inhibitors and small-molecule multikinase inhibitors, have produced clinical effects. However, most patients acquire resistance over time. Thus, new therapeutic strategies need to be developed. Here, we evaluated the effect of the dual PI3K/mTOR inhibitor NVP-BEZ235, in combination with the multikinase inhibitor sorafenib on renal cancer cell proliferation and survival in vitro as well as on tumor growth in vivo.Methods: The renal carcinoma cell lines 786-0 and Caki-1 were treated with NVP-BEZ235 or sorafenib, either alone or in combination. Tumor cell proliferation and apoptosis were investigated in vitro. The anticancer efficacy of NVP-BEZ235 alone, or in combination with sorafenib, was also evaluated on RCC xenografts in nude mice.Results: Treatment of 786-0 and Caki-1 cells with NVP-BEZ235 or sorafenib resulted in reduced tumor cell proliferation and increased tumor cell apoptosis in vitro. The combination of NVP-BEZ235 and sorafenib was more effective than each compound alone. Similarly, in vivo, NVP-BEZ235 or sorafenib reduced the growth of xenografts generated from 786-0 or Caki-1 cells. The antitumor efficacy of NVP-BEZ235 in combination with sorafenib was superior to NVP-BEZ235 or sorafenib alone.Conclusions: Our findings indicate that the simultaneous use of NVP-BEZ235 and sorafenib has greater antitumor benefit compared to either drug alone and thus provides a treatment strategy in RCC.

Factors associated with the emergence of K65R in patients with HIV-1 infection treated with combination antiretroviral therapy containing tenofovir.

BACKGROUND: The human immunodeficiency virus type 1 reverse-transcriptase mutation K65R is a single-point mutation that has become more frequent after increased use of tenofovir disoproxil fumarate (TDF). We aimed to identify predictors for the emergence of K65R, using clinical data and genotypic resistance tests from the Swiss HIV Cohort Study. METHODS: A total of 222 patients with genotypic resistance tests performed while receiving treatment with TDF-containing regimens were stratified by detectability of K65R (K65R group, 42 patients; undetected K65R group, 180 patients). Patient characteristics at start of that treatment were analyzed. RESULTS: In an adjusted logistic regression, TDF treatment with nonnucleoside reverse-transcriptase inhibitors and/or didanosine was associated with the emergence of K65R, whereas the presence of any of the thymidine analogue mutations D67N, K70R, T215F, or K219E/Q was protective. The previously undescribed mutational pattern K65R/G190S/Y181C was observed in 6 of 21 patients treated with efavirenz and TDF. Salvage therapy after TDF treatment was started for 36 patients with K65R and for 118 patients from the wild-type group. Proportions of patients attaining human immunodeficiency virus type 1 loads <50 copies/mL after 24 weeks of continuous treatment were similar for the K65R group (44.1%; 95% confidence interval, 27.2%-62.1%) and the wild-type group (51.9%; 95% confidence interval, 42.0%-61.6%). CONCLUSIONS: In settings where thymidine analogue mutations are less likely to be present, such as at start of first-line therapy or after extended treatment interruptions, combinations of TDF with other K65R-inducing components or with efavirenz or nevirapine may carry an enhanced risk of the emergence of K65R. The finding of a distinct mutational pattern selected by treatment with TDF and efavirenz suggests a potential fitness interaction between K65R and nonnucleoside reverse-transcriptase inhibitor-induced mutations.

Dual control of hydrogen cyanide biosynthesis by the global activator GacA in Pseudomonas aeruginosa PAO1.

The global response regulator GacA of Pseudomonas aeruginosa PAO1 positively controls the production of the quorum sensing signal molecule N-butanoyl-homoserine-lactone (C4-HSL) and hence the synthesis of several C4-HSL-dependent virulence factors, including hydrogen cyanide (HCN). This study presents evidence that GacA positively influences the transcription of the rhlI gene, specifying C4-HSL synthase, explaining the quorum sensing-dependent transcriptional control of the HCN biosynthetic genes (hcnABC). In addition, GacA was found to modulate hcn gene expression positively at a post-transcriptional level involving the hcnA ribosome-binding site. Thus, the activating effect of GacA on cyanogenesis results from both transcriptional and post-transcriptional mechanisms.

The generation and analysis of combined legless 1 and 2 knock-out mice

Summary The Wnt signaling pathway plays an important role during development and also for maintaining tissue homeostasis due to its function in proliferation, differentiation and cell fate decisions. Wnt ligands bind to Frizzled receptors and activate a signaling cascade that results in the stabilization of β-Catenin, a key component of the pathway. β-Catenin translocates to the nucleus, where, together with a transcription factor of the Tcf/Lef family, it activates the expression of target genes. Legless and Pygopus are two recently discovered essential components of the Wnt pathway in Drosophila, which may mediate the nuclear import and retention of beta-Catenin and/or contribute directly to the activation of Wnt target genes. To address the function of Legless in the mouse, we have generated compound constitutive and conditional knockout alleles of the two homologues legless 'I (bc1-9) and 2. We have induced the deletion of legless in self-renewing tissues such as the gastrointestinal tract, the mammary gland and the skin during adulthood and constitutively in the embryo. The present thesis focused on the consequences of the inactivation of legless in epithelial homeostasis as well as in a regeneration model and its comparison to pygopus. Deletion of neither legless nor pygopus in the adult small intestine resulted in any apparent anomaly, contrasting expectations from the phenotype caused by over-expression of Dickkopf, a Wnt inhibitor (Pinto et al., 2003). These observations indicate that canonical Wnt signaling might not be indispensable for normal gastrointestinal epithelium homeostasis, or that, in this context, Legless and Pygopus are not essential components of the Wnt pathway. However, the regeneration of the colonic epithelium after DSS induced damage was markedly impaired in legless, but not in pygopus deficient mice. Thus, unlike in Drosophila, deletion of mammalian legless and pygopus resulted in different phenotypes, suggesting that Legless might interact with as yet unidentified partners in addition to Pygopus. Resumé La voie de signalisation Wnt joue un rôle important au cours du développement ainsi que pour le maintien de l' homéostase tissulaire due à sa fonction durant la prolifération, la différentiation et les décisions sur l'avenir des cellules. Les ligands de Wnt se lient aux récepteurs Frizzled et activent une cascade de signalisation résultant en la stabilisation de β-Catenin, un composant central de cette voie. β-Catenin est transloquée dans le noyau ou, avec l'aide des facteurs de transcription de la famille Tcf/lef, elle active la transcription des gènes cibles. Legless et Pygopus sont deux composants récemment découverts et essentiels de la voie de signalisation Wnt chez la Drosophile qui pourraient être des médiateurs de l'import et de la rétention nucléaire de bêta-catenin et/ou contribuer directement a l'activation des gènes cibles. Afin de comprendre la fonction de Legless chez la souris, nous avons généré simultanément les allèles « knock-out » constitutifs et conditionnels des deux homologues legless 1 (bc1-9) et 2. Nous avons induit la délétion de legless dans des tissus capables de s'auto renouveler comme le tract gastro-intestinal, la glande mammaire et la peau chez l'adulte et nous avons supprimé constitutivement legless chez l'embryon. La présente thèse est concentrée sur les conséquences de l'inactivation de legless au cours de l' homéostase épithéliale ainsi que dans un modèle de régénération et sur sa comparaison avec pygopus. Ni la délétion de legless ni celle de pygopus dans l'intestin adulte n'ont résulté en quelque anomalie, contrastant nos attentes provenant des phénotypes causes par la surexpression de Dickkpof, un inhibiteur de Wnt (Pinto et al., 2003). Ces observations indiquent que la voie de signalisation Wnt/β-Catenin pourrait ne pas être indispensable à l' homéostase normale du tract gastro-intestinal, ou que, dans ce contexte, Legless et Pygopus ne sont pas des composants essentiels de la vole Wnt. Cependant, la régénération de l'épithélium du colon après induction de son endommagement au DSS fut dramatiquement diminuée chez legless mais pas chez les souris mutantes pour pygopus. Ainsi, a la différence de chez la Drosophile, la délétion de legless et pygopus chez les mammifères a résulté en des phénotypes différents, suggérant que Legless pourrait interagir avec d'autres partenaires, encore non identifies, que Pygopus.

A Microtus fortisprotein, serum albumin, is a novel inhibitor of Schistosoma japonicumschistosomula

Schistosomiasis is an endemic parasite disease and praziquantel is the only drug currently in use to control this disease. Experimental and epidemiological evidence strongly suggests that Microtus fortis( Mf) is a naturally resistant vertebrate host of Schistosoma japonicum. In the present study, we found that Mfserum albumin ( Mf-albumin) and the conditioned medium of pcDNA3.1- Mf-albumin caused 46.2% and 38.7% schistosomula death rates in 96 h, respectively, which were significantly higher than that of the negative control (p < 0.05). We also found that mice injected with Mf-albumin had a 43.5% reduction in worm burden and a 48.1% reduction in liver eggs per gram (p < 0.05) in comparison to the control animals. To characterise the mechanisms involved in clearance, schistosomula were incubated with fluorescein isothiocyanate-labelled Mf-albumin and fluorescent enrichment effects were found in the gut lumen of schistosomula after 48 h of incubation. Next, digestive tract excretions from schistosomula were collected and the sensitivity of Mf-albumin to digestive tract excretions was evaluated. The results indicated that schistosomula digestive tract excretions showed indigestibility of Mf-albumin. The death of schistosomula could be partially attributed to the lack of digestion of Mf-albumin by digestive tract excretions during the development of the schistosomula stage. Therefore, these data indicate the potential of Mf-albumin as one of the major selective forces for schistosomiasis.