Novel (60%) and recurrent (40%) androgen receptor gene mutations in a series of 59 patients with a 46,XY disorder of sex development.

BACKGROUND Androgen receptor (AR) gene mutations are the most frequent cause of 46,XY disorders of sex development (DSD) and are associated with a variety of phenotypes, ranging from phenotypic women [complete androgen insensitivity syndrome (CAIS)] to milder degrees of undervirilization (partial form or PAIS) or men with only infertility (mild form or MAIS). OBJECTIVE The aim of the study was to characterize the contribution of the AR gene to the molecular cause of 46,XY DSD in a series of Spanish patients. SETTING We studied a series of 133 index patients with 46,XY DSD in whom gonads were differentiated as testes, with phenotypes including varying degrees of undervirilization, and in whom the AR gene was the first candidate for a molecular analysis. METHODS The AR gene was sequenced (exons 1 to 8 with intronic flanking regions) in all patients and in family members of 61% of AR-mutated gene patients. RESULTS AR gene mutations were found in 59 individuals (44.4% of index patients), of whom 46 (78%) were CAIS and 13 (22%) PAIS. Fifty-seven different mutations were found: 21.0% located in exon 1, 15.8% in exons 2 and 3, 57.9% in exons 4-8, and 5.3% intronic. Twenty-three mutations (40.4%) had been previously described and 34 (59.6%) were novel. CONCLUSIONS AR gene mutation is the most frequent cause of 46,XY DSD, with a clearly higher frequency in the complete phenotype. Mutations spread along the whole coding sequence, including exon 1. This series shows that 60% of mutations detected during the period 2002-2009 were novel.

Differences in osteogenic and apoptotic genes between osteoporotic and osteoarthritic patients.

BACKGROUND Osteoporosis is a metabolic disorder characterized by a reduction in bone mass and deterioration in the microarchitectural structure of the bone, leading to a higher risk for spontaneous and fragility fractures.The main aim was to study the differences between human bone from osteoporotic and osteoarthritic patients about gene expression (osteogenesis and apoptosis), bone mineral density, microstructural and biomechanic parameters. METHODS We analyzed data from 12 subjects: 6 with osteoporotic hip fracture (OP) and 6 with hip osteoarthritis (OA), as the control group. All subjects underwent medical history, analytical determinations, densitometry, histomorphometric and biochemical study. The expression of 86 genes of osteogenesis and 86 genes of apoptosis was studied in pool of bone samples from patients with OP and OA by PCR array. RESULTS We observed that most of the genes of apoptosis and osteogenesis show a decrease in gene expression in the osteoporotic group in comparison with the osteoarthritic group. The histomorphometric study shows a lower bone quality in the group of patients with hip fractures compared to the osteoarthritic group. CONCLUSIONS The bone tissue of osteoporotic fracture patients is more fragile than the bone of OA patients. Our results showed an osteoporotic bone with a lower capacities for differentiation and osteoblastic activity as well as a lower rate of apoptosis than osteoarthritic bone. These results are related with structural and biochemical parameters.

Amplicon DNA melting analysis for the simultaneous detection of Brucella spp and Mycobacterium tuberculosis complex. Potential use in rapid differential diagnosis between extrapulmonary tuberculosis and focal complications of brucellosis.

Some sites of extrapulmonary tuberculosis and focal complications of brucellosis are very difficult to differentiate clinically, radiologically, and even histopathologically. Conventional microbiological methods for the diagnosis of extrapulmonary tuberculosis and complicated brucellosis not only lack adequate sensitivity, they are also time consuming, which could lead to an unfavourable prognosis. The aim of this work was to develop a multiplex real-time PCR assay based on SYBR Green I to simultaneously detect Brucella spp and Mycobacterium tuberculosis complex and evaluate the efficacy of the technique with different candidate genes. The IS711, bcsp31 and omp2a genes were used for the identification of Brucella spp and the IS6110, senX3-regX3 and cfp31 genes were targeted for the detection of the M. tuberculosis complex. As a result of the different combinations of primers, nine different reactions were evaluated. A test was defined as positive only when the gene combinations were capable of co-amplifying both pathogens in a single reaction tube and showed distinguishable melting temperatures for each microorganism. According to the melting analysis, only three combinations of amplicons (senX3-regX3+bcsp31, senX3-regX3+IS711 and IS6110+IS711) were visible. Detection limits of senX3-regX3+bcsp31 and senX3-regX3+IS711 were of 2 and 3 genome equivalents for M. tuberculosis complex and Brucella while for IS6110+IS711 they were of 200 and 300 genome equivalents, respectively. The three assays correctly identified all the samples, showing negative results for the control patients. The presence of multicopy elements and GC content were the components most influencing the efficiency of the test; this should be taken into account when designing a multiplex-based SYBR Green I assay. In conclusion, multiplex real time PCR assays based on the targets senX3-regX3+bcsp31 and senX3-regX3+IS711 using SYBR Green I are highly sensitive and reproducible. This may therefore be a practical approach for the rapid differential diagnosis between extrapulmonary tuberculosis and complicated brucellosis.

Novel Chlamydiales strains isolated from a water treatment plant.

Chlamydiae are obligate intracellular bacteria infecting free-living amoebae, vertebrates and some invertebrates. Novel members are regularly discovered, and there is accumulating evidence supporting a very important diversity of chlamydiae in the environment. In this study, we investigated the presence of chlamydiae in a drinking water treatment plant. Samples were used to inoculate Acanthamoeba monolayers (Acanthamoeba co-culture), and to recover autochthonous amoebae onto non-nutritive agar. Chlamydiae were searched for by a pan-chlamydia 16S rRNA gene PCR from both Acanthamoeba co-cultures and autochthonous amoebae, and phylotypes determined by 16S rRNA gene sequencing. Autochthonous amoebae also were identified by 18S rRNA gene amplification and sequencing. From a total of 79 samples, we recovered eight chlamydial strains by Acanthamoeba co-culture, but only one of 28 amoebae harboured a chlamydia. Sequencing results and phylogenetic analysis showed our strains belonging to four distinct chlamydial lineages. Four strains, including the strain recovered within its natural host, belonged to the Parachlamydiaceae; two closely related strains belonged to the Criblamydiaceae; two distinct strains clustered with Rhabdochlamydia spp.; one strain clustered only with uncultured environmental clones. Our results confirmed the usefulness of amoeba co-culture to recover novel chlamydial strains from complex samples and demonstrated the huge diversity of chlamydiae in the environment, by identifying several new species including one representing the first strain of a new family.

Evolutionary simulations to detect functional lineage-specific genes.

MOTIVATION: Supporting the functionality of recent duplicate gene copies is usually difficult, owing to high sequence similarity between duplicate counterparts and shallow phylogenies, which hamper both the statistical and experimental inference. RESULTS: We developed an integrated evolutionary approach to identify functional duplicate gene copies and other lineage-specific genes. By repeatedly simulating neutral evolution, our method estimates the probability that an ORF was selectively conserved and is therefore likely to represent a bona fide coding region. In parallel, our method tests whether the accumulation of non-synonymous substitutions reveals signatures of selective constraint. We show that our approach has high power to identify functional lineage-specific genes using simulated and real data. For example, a coding region of average length (approximately 1400 bp), restricted to hominoids, can be predicted to be functional in approximately 94-100% of cases. Notably, the method may support functionality for instances where classical selection tests based on the ratio of non-synonymous to synonymous substitutions fail to reveal signatures of selection. Our method is available as an automated tool, ReEVOLVER, which will also be useful to systematically detect functional lineage-specific genes of closely related species on a large scale. AVAILABILITY: ReEVOLVER is available at http://www.unil.ch/cig/page7858.html.

In vitro methods for diagnosing nonimmediate hypersensitivity reactions to drugs.

Nonimmediate drug hypersensitivity reactions (DHRs) are difficult to manage in daily clinical practice, mainly owing to their heterogeneous clinical manifestations and the lack of selective biological markers. In vitro methods are necessaryto establish a diagnosis, especially given the low sensitivity of skin tests and the inherent risks of drug provocation testing. In vitro evaluation of nonimmediate DHRs must include approaches that can be applied during the different phases of the reaction. During the acute phase, monitoring markers in both skin and peripheral blood helps to discriminate between immediate and nonimmediate DHRs with cutaneous responses and to distinguish between reactions that, although they present similar clinical symptoms, are produced by different immunological mechanisms and therefore have a different treatment and prognosis. During the resolution phase, in vitro testing is used to detect the response of T cells to drug stimulation; however, this approach has certain limitations, such as the lack of validated studies assessing sensitivity. Moreover, in vitro tests indicate an immune response that is not always related to a DHR. In this review, members of the Immunology and Drug Allergy Committee of the Spanish Society of Allergy and Clinical Immunology (SEAIC) provide an overview of the most widely used in vitro tests for evaluating nonimmediate DHRs.

Nevirapine vs efavirenz in virologically supressed patients: Differences in lipoprotein subclasses and inflammatory biomarkers.

Background: The interaction between lipid disturbances and inflammatory markers is not well known in patients on antiretroviral therapy (ART). As nevirapine (NVP) is associated with a better lipid profile than efavirenz (EFV), we investigated the relationships between lipid profiles, lipoprotein subclasses and inflammatory biomarkers in patients with prolonged viral suppression with either NVP or EFV and no obvious clinical inflammation. Methods: 122 clinically stable HIV-infected patients with HIV-1 RNA <20 copies longer than 6 months on NNRTI therapy were studied. 72 (59%) were on EFV and 50 (41%) on NVP. Any potentially inflammatory co-morbid diseases (concurrent viral hepatitis, diabetes, hypertension, chronic liver or renal diseases), or statin treatment, were exclusion criteria. Inflammatory biomarkers included hsCRP, LpPLA2, sCD40L, IL-6, IL-8, t-PA, MCP-1, p-selectin and VCAM-1. Lipoprotein subclass measures (VLDL, LDL, IDL and HDL particle number and size) were obtained by the use of proton nuclear magnetic resonance spectroscopy. Results: 82% were male; median age 45 years. Median CD4 count 550/μL (IQR 324). Median time since HIV diagnosis 96 months (IQR 102) and accumulated time on ART 50 months (IQR 101). Patients on NVP had higher time since HIV diagnosis (126.9 [66.7] vs 91.3 [6.6] months, p=0.008) a prolonged time on ART (89.6 [54.6] vs 62.3 [52.2] months, p=0.01) and were older (47.7 vs 40.7 years, p=0.001) than those on EFV. NVP-treated patients presented increased HDL-c (55.8 [16] vs 48.8 [10.7] mg/dL, p=0.007) and apoA1 levels (153.4 [31.9] vs 141.5 [20.5] mg/dL, p=0.02), and reduced apoB/apoA1 ratio (0.68 [0.1] vs 0.61 [0.1], p=0.003) than EFV-treated patients. No differences in inflammatory markers or lipoprotein subclasses were found between NVP and EFV. In patients with extreme lipid values (less favorable: 75th percentiles of LDL, small/dense LDLp and small HDLp, or more favorable: HDL p75 and apoB/apoA1 ratio p25), no consistent differences in inflammatory biomarkers were found. Conclusions: Patients with prolonged viral suppression on NVP present significantly higher HDL and apoA1 levels and reduced apoB/apoA1 ratios than those on EFV, but no differences were found in lipoprotein particles nor inflammatory biomarkers. Relationships between lipid parameters and inflammatory biomarkers in NNRTItreated patients are complex and do not show a linear relationship in this study.

Safety and immunomodulatory effects of three probiotic strains isolated from the feces of breast-fed infants in healthy adults: SETOPROB study.

We previously described the isolation and characterization of three probiotic strains from the feces of exclusively breast-fed newborn infants: Lactobacillus paracasei CNCM I-4034, Bifidobacterium breve CNCM I-4035 and Lactobacillus rhamnosus CNCM I-4036. These strains were shown to adhere to intestinal mucus in vitro, to be sensitive to antibiotics and to resist biliary salts and low pH. In the present study, a multicenter, randomized, double-blind, placebo-controlled trial with 100 healthy volunteers in three Spanish cities was carried out to evaluate the tolerance, safety, gut colonization and immunomodulatory effects of these three probiotics. Volunteers underwent a 15-day washout period, after which they were randomly divided into 5 groups that received daily a placebo, a capsule containing one of the 3 strains or a capsule containing a mixture of two strains for 30 days. The intervention was followed by another 15-day washout period. Patients did not consume fermented milk for the entire duration of the study. Gastrointestinal symptoms, defecation frequency and stool consistency were not altered by probiotic intake. No relevant changes in blood and serum, as well as no adverse events occurred during or after treatment. Probiotic administration slightly modified bacterial populations in the volunteers' feces. Intestinal persistence occurred in volunteers who received L. rhamnosus CNCM I-4036. Administration of B. breve CNCM I-4035 resulted in a significant increase in fecal secretory IgA content. IL-4 and IL-10 increased, whereas IL-12 decreased in the serum of volunteers treated with any of the three strains. These results demonstrate that the consumption of these three bacterial strains was safe and exerted varying degrees of immunomodulatory effects.

Preparation of bacterial DNA template by boiling and effect of immunoglobulin G as an inhibitor in real-time PCR for serum samples from patients with brucellosis.

Real-time PCR is a widely used tool for the diagnosis of many infectious diseases. However, little information exists about the influences of the different factors involved in PCR on the amplification efficiency. The aim of this study was to analyze the effect of boiling as the DNA preparation method on the efficiency of the amplification process of real-time PCR for the diagnosis of human brucellosis with serum samples. Serum samples from 10 brucellosis patients were analyzed by a SYBR green I LightCycler-based real-time PCR and by using boiling to obtain the DNA. DNA prepared by boiling lysis of the bacteria isolated from serum did not prevent the presence of inhibitors, such as immunoglobulin G (IgG), which were extracted with the template DNA. To identify and confirm the presence of IgG, serum was precipitated to separate and concentrate the IgG and was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. The use of serum volumes above 0.6 ml completely inhibited the amplification process. The inhibitory effect of IgG in serum samples was not concentration dependent, and it could be eliminated by diluting the samples 1/10 and 1/20 in water. Despite the lack of the complete elimination of the IgG from the template DNA, boiling does not require any special equipment and it provides a rapid, reproducible, and cost-effective method for the preparation of DNA from serum samples for the diagnosis of brucellosis.

Clinical and Virological Efficacy of Etravirine Plus Two Active Nucleos(t)ide Analogs in an Heterogeneous HIV-Infected Population.

Etravirine (ETV) is recommended in combination with a boosted protease inhibitor plus an optimized background regimen for salvage therapy, but there is limited experience with its use in combination with two nucleos(t)ide reverse-transcriptase inhibitors (NRTIs). This multicenter study aimed to assess the efficacy of this combination in two scenarios: group A) subjects without virologic failure on or no experience with non-nucleoside reverse-transcriptase inhibitors (NNRTIs) switched due to adverse events and group B) subjects switched after a virologic failure on an efavirenz- or nevirapine-based regimen. The primary endpoint was efficacy at 52 weeks analysed by intention-to-treat. Virologic failure was defined as the inability to suppress plasma HIV-RNA to <50 copies/mL after 24 weeks on treatment, or a confirmed viral load >200 copies/mL in patients who had previously achieved a viral suppression or had an undetectable viral load at inclusion. Two hundred eighty seven patients were included. Treatment efficacy rates in group A and B were 88.0% (CI95, 83.9-92.1%) and 77.4% (CI95, 65.0-89.7%), respectively; the rates reached 97.2% (CI95, 95.1-99.3%) and 90.5% (CI95, 81.7-99.3), by on-treatment analysis. The once-a-day ETV treatment was as effective as the twice daily dosing regimen. Grade 1-2 adverse events were observed motivating a treatment switch in 4.2% of the subjects. In conclusion, ETV (once- or twice daily) plus two analogs is a suitable, well-tolerated combination both as a switching strategy and after failure with first generation NNRTIs, ensuring full drug activity. TRIAL REGISTRATION ClinicalTrials.gov NCT01437241.

Selection of Reference Genes for Quantitative Real Time PCR (qPCR) Assays in Tissue from Human Ascending Aorta.

Dilatation of the ascending aorta (AAD) is a prevalent aortopathy that occurs frequently associated with bicuspid aortic valve (BAV), the most common human congenital cardiac malformation. The molecular mechanisms leading to AAD associated with BAV are still poorly understood. The search for differentially expressed genes in diseased tissue by quantitative real-time PCR (qPCR) is an invaluable tool to fill this gap. However, studies dedicated to identify reference genes necessary for normalization of mRNA expression in aortic tissue are scarce. In this report, we evaluate the qPCR expression of six candidate reference genes in tissue from the ascending aorta of 52 patients with a variety of clinical and demographic characteristics, normal and dilated aortas, and different morphologies of the aortic valve (normal aorta and normal valve n = 30; dilated aorta and normal valve n = 10; normal aorta and BAV n = 4; dilated aorta and BAV n = 8). The expression stability of the candidate reference genes was determined with three statistical algorithms, GeNorm, NormFinder and Bestkeeper. The expression analyses showed that the most stable genes for the three algorithms employed were CDKN1β, POLR2A and CASC3, independently of the structure of the aorta and the valve morphology. In conclusion, we propose the use of these three genes as reference genes for mRNA expression analysis in human ascending aorta. However, we suggest searching for specific reference genes when conducting qPCR experiments with new cohort of samples.

The future is genome-wide.

A report of the annual meeting of the European Society of Human Genetics, Amsterdam, 6-9 May 2006.

Reactivation of transcription from a vaccinia virus early promoter late in infection.

We have studied the kinetics of RNA synthesis from the vaccinia virus 7,500-molecular-weight gene (7.5K gene) which is regulated by early and late promoters arranged in tandem. Unexpectedly, after a first burst of RNA synthesis early in infection, transcription was reactivated late in infection. Reactivation was not dependent on the location of the promoter in the genome or on the presence of the upstream late regulatory sequences. The mRNA synthesized from the reactivated promoter in the late phase had the same 5' and 3' ends as the molecules transcribed in the early phase. Interestingly, these molecules were efficiently translated despite the absence of the poly(A) leader characteristic of late mRNAs. Reactivation appears to be dependent on virus assembly since it is prevented by rifampin, a specific inhibitor of morphogenesis. Finally, analysis of various other early genes showed that reactivation is not unique to the 7.5K early promoter.

High level expression of human neuropeptide Y receptors in mammalian cells infected with a recombinant vaccinia virus.

Neuropeptide Y (NPY) is a 36 amino acid peptide present in the central and peripheral nervous system. Numerous studies point to a role of NPY in cardiovascular regulation. NPY effects are mediated through stimulation of specific cell surface G protein-coupled receptors. To allow biochemical studies of the receptor and of its interaction with the ligand, we have developed a potent expression system for NPY receptors using a recombinant vaccinia virus. A human NPY receptor cDNA was fused to a strong vaccinia virus promoter and inserted into the viral genome by homologous recombination. Recombinant viruses were isolated and tested for their ability to induce NPY binding site expression following infection of mammalian cell lines. Using saturation and competition binding experiments we measured a Bmax of 5-10 x 10(6) NPY binding sites per cell. The Kd for the binding of NPY is about 20 nM. Labelling of infected cells with a fluorochrome-labelled NPY indicated that the recombinant protein integrates into the cell membrane.

Biomarkers of therapeutic responses in chronic Chagas disease: state of the art and future perspectives

The definition of a biomarker provided by the World Health Organization is any substance, structure, or process that can be measured in the body, or its products and influence, or predict the incidence or outcome of disease. Currently, the lack of prognosis and progression markers for chronic Chagas disease has posed limitations for testing new drugs to treat this neglected disease. Several molecules and techniques to detect biomarkers inTrypanosoma cruzi-infected patients have been proposed to assess whether specific treatment with benznidazole or nifurtimox is effective. Isolated proteins or protein groups from different T. cruzistages and parasite-derived glycoproteins and synthetic neoglycoconjugates have been demonstrated to be useful for this purpose, as have nucleic acid amplification techniques. The amplification of T. cruziDNA using the real-time polymerase chain reaction method is the leading test for assessing responses to treatment in a short period of time. Biochemical biomarkers have been tested early after specific treatment. Cytokines and surface markers represent promising molecules for the characterisation of host cellular responses, but need to be further assessed.