ICO Amplicon NGS Data Analysis: A Web Tool for Variant Detection in Common High‐Risk Hereditary Cancer Genes Analyzed by Amplicon GS Junior Next‐Generation Sequencing

Next‐generation sequencing (NGS) has revolutionized genomic research and is set to have a major impact on genetic diagnostics thanks to the advent of benchtop sequencers and flexible kits for targeted libraries. Among the main hurdles in NGS are the difficulty of performing bioinformatic analysis of the huge volume of data generated and the high number of false positive calls that could be obtained, depending on the NGS technology and the analysis pipeline. Here, we present the development of a free and user‐friendly Web data analysis tool that detects and filters sequence variants, provides coverage information, and allows the user to customize some basic parameters. The tool has been developed to provide accurate genetic analysis of targeted sequencing of common high‐risk hereditary cancer genes using amplicon libraries run in a GS Junior System. The Web resource is linked to our own mutation database, to assist in the clinical classification of identified variants. We believe that this tool will greatly facilitate the use of the NGS approach in routine laboratories.     

Migration of human blood dendritic cells across endothelial cell monolayers: adhesion molecules and chemokines involved in subset-specific transmigration

Distinct subsets of dendritic cells (DCs) are present in blood, probably "en route" to different tissues. We have investigated the chemokines and adhesion molecules involved in the migration of myeloid (CD11c(+)) and plasmacytoid (CD123(+)) human peripheral blood DCs across vascular endothelium. Among blood DCs, the CD11c(+) subset vigorously migrated across endothelium in the absence of any chemotactic stimuli, whereas spontaneous migration of CD123(+) DCs was limited. In bare cell migration assays, myeloid DCs responded with great potency to several inflammatory and homeostatic chemokines, whereas plasmacytoid DCs responded poorly to all chemokines tested. In contrast, the presence of endothelium greatly favored transmigration of plasmacytoid DCs in response to CXCL12 (stromal cell-derived factor-1) and CCL5 (regulated on activation, normal T expressed and secreted). Myeloid DCs exhibited a very potent transendothelial migration in response to CXCL12, CCL5, and CCL2 (monocyte chemoattractant protein-1). Furthermore, we explored whether blood DCs acutely switch their pattern of migration to the lymph node-derived chemokine CCL21 (secondary lymphoid-tissue chemokine) in response to microbial stimuli [viral double-stranded (ds)RNA or bacterial CpG-DNA]. A synthetic dsRNA rapidly enhanced the response of CD11c(+) DCs to CCL21, whereas a longer stimulation with CpG-DNA was needed to trigger CD123(+) DCs responsive to CCL21. Use of blocking monoclonal antibodies to adhesion molecules revealed that both DC subsets used platelet endothelial cell adhesion molecule-1 to move across activated endothelium. CD123(+) DCs required beta(2) and beta(1) integrins to transmigrate, whereas CD11c(+) DCs may use integrin-independent mechanisms to migrate across activated endothelium.     

Frequency of delta F508 mutation in Venezuelan patients with cystic fibrosis

Cystic Fibrosis (CF) is the most common and severe autosomal recessive disease in Caucasian populations, with an incidence of 1 in 2500 live births. It is characterized by a generalized disturbance in exocrine glands and it is caused by over one thousand mutations at the cystic fibrosis conductance regulator gene (CFTR) mapped at 7q31. AF508 is the most frequent mutation worldwide and it consists in a deletion of the codon that encodes fenilalanine at the 508 protein's position. The aim of this study was to determine the frequency of the delta F508 mutation in Venezuelan patients with CF using the Polymerase Chain Reaction (PCR). We studied thirty patients of twenty eight families who were diagnosed with CF based on their clinical features and sweat chloride level > 60 mEq/l in two determinations. Detection of the mutation was performed from the amplification of a 98 pair of bases (pb) CF gene segment which contains the codon that encodes fenilalanine in the 508 position by PCR. This PCR product is absent in those who have the mutation. The delta F508 allelic frequency was 26.79%, distributed in six homozygous and seven compound heterozygote delta F508/X. The reminder mutations (no delta F508) represent 73.21%. The delta F508 frequency in our sample is less than the reported in European countries. On the other hand, a delta F508 frequency highly heterogeneous has been observed in Latin-American countries. This variation results from mixed populations with a different genetic background influenced by external migration and CF molecular alterations, which exists in the analyzed populations. In this study, the delta F508 mutation comes mainly from grandparents (79.41%) who were born in Mediterranean countries and Colombia, while the no delta F508 mutations come from grandparents who were born in Venezuela (79.27%) and Colombia (17.07%).     

Sublingual immunotherapy for hazelnut food allergy: a randomized, double-blind, placebo-controlled study with a standardized hazelnut extract

BACKGROUND: Food allergy may be life-threatening, and patients affected need to receive accurate diagnoses and treatment. Hazelnut has often been implicated as responsible for allergic reactions, and trace quantities can induce systemic reactions. OBJECTIVE: The aim of this study was to evaluate the efficacy and tolerance of sublingual immunotherapy with a standardized hazelnut extract in patients allergic to hazelnut. METHODS: This was a randomized, double-blind, placebo-controlled study. Inclusion criteria were a history of hazelnut allergy and positive skin prick test and double-blind placebo-controlled food challenge results. Patients were then randomly assigned into 2 treatment groups (hazelnut immunotherapy or placebo). Efficacy was assessed by double-blind, placebo-controlled food challenge after 8 to 12 weeks of treatment. Blood samples were drawn for measurement of specific IgE, IgG(4), and serum cytokines before and after treatment. RESULTS: Twenty-three patients were enrolled and divided into 2 treatment groups. Twenty-two patients reached the planned maximum dose at 4 days. Systemic reactions were observed in only 0.2% of the total doses administered. Mean hazelnut quantity provoking objective symptoms increased from 2.29 g to 11.56 g (P = .02; active group) versus 3.49 g to 4.14 g (placebo; NS). Moreover, almost 50% of patients who underwent active treatment reached the highest dose (20 g), but only 9% in the placebo. Laboratory data showed an increase in IgG(4) and IL-10 levels after immunotherapy in only the active group. CONCLUSION: Our data confirm significant increases in tolerance to hazelnut after sublingual immunotherapy as assessed by double-blind, placebo-controlled food challenge, and good tolerance to this treatment.     

Trypanosoma cruzi cleaves galectin-3 N-terminal domain to suppress its innate microbicidal activity

Galectin-3 is the best-characterized member of galectins, an evolutionary conserved family of galactoside-binding proteins that play central roles in infection and immunity, regulating inflammation, cell migration and cell apoptosis. Differentially expressed by cells and tissues with immune privilege, they bind not only to host ligands, but also to glycans expressed by pathogens. In this regard, we have previously shown that human galectin-3 recognizes several genetic lineages of the protozoan parasite Trypanosoma cruzi, the causal agent of Chagas’ disease or American trypanosomiasis. Herein we describe a molecular mechanism developed by T. cruzi to proteolytically process galectin-3 that generates a truncated form of the protein lacking its N-terminal domain – required for protein oligomerization – but still conserves a functional carbohydrate recognition domain (CRD). Such processing relies on specific T. cruzi proteases, including Zn-metalloproteases and collagenases, and ultimately conveys profound changes in galectin-3-dependent effects, as chemical inhibition of parasite proteases allows galectin-3 to induce parasite death in vitro. Thus, T. cruzi might have established distinct mechanisms to counteract galectin-3-mediated immunity and microbicide properties. Interestingly, non-pathogenic T. rangeli lacked the ability to cleave galectin-3, suggesting that during evolution two genetically similar organisms have developed different molecular mechanisms that, in the case of T. cruzi, favoured its pathogenicity, highlighting the importance of T. cruzi proteases to avoid immune mechanisms triggered by galectin-3 upon infection. This study provides the first evidence of a novel strategy developed by T. cruzi to abrogate signalling mechanisms associated with galectin-3-dependent innate immunity.     

Long-lasting resistance to haloperidol-induced catalepsy in male rats chronically treated with caffeine

Chronic caffeine consumption has been inversely associated with the risk of developing Parkinson's disease. Here we assessed whether chronic caffeine treatment increases the resistance of male Wistar rats to haloperidol (1 mg/kg, s.c.)-induced catalepsy, measured in the bar test at 15 min intervals during 3 h. Caffeine (5 mg/kg/day) was delivered for 6 months via drinking water. Control rats received only tap water. Treatments began when animals were 3–4 months old. In order to unveil long-lasting catalepsy refractoriness not attributable to the presence of caffeine in the brains of rats, they were evaluated from day 18 to day 27 after caffeine withdrawal, a time that is far in excess for the full excretion of a caffeine dose in this species. The average cataleptic immobility measured in caffeine-treated rats (n = 23) was 1148 ± 140 s, a value 34 ± 8% lower than that recorded in control animals (n = 20), whose mean immobility was 1736 ± 137 s (P = 0.0026, t-test). The percentage of catalepsy reduction measured in caffeine-treated rats evaluated on days 18–20 after caffeine discontinuation (−32 ± 13%, n = 12, P < 0.05) was comparable to the catalepsy decrease recorded in those animals tested on days 21–27 (−36 ± 10%, n = 11, P < 0.02), a finding compatible with the notion that the effect was indeed mediated by enduring changes of brain functioning and not by the physical presence of caffeine or its metabolites. Caffeine-treated rats also had higher catalepsy latency scores compared with control rats (P < 0.01, U-test). The present findings show that chronic consumption of caffeine produces perdurable resistance to catalepsy induced by dopamine receptor blockade, possibly through enhancement of dopamine transmission, giving further support to the epidemiological results indicating that prolonged caffeine consumption affords neuroprotection against Parkinson's disease.     

Most HIV type 1 non-B infections in the Spanish cohort of antiretroviral treatment-naïve HIV-infected patients (CoRIS) are due to recombinant viruses

HIV-1 group M is classified into 9 subtypes, as well as recombinants favored by coinfection and superinfection events with different variants. Although HIV-1 subtype B is predominant in Europe, intersubtype recombinants are increasing in prevalence and complexity. In this study, phylogenetic analyses of pol sequences were performed to detect the HIV-1 circulating and unique recombinant forms (CRFs and URFs, respectively) in a Spanish cohort of antiretroviral treatment-naïve HIV-infected patients included in the Research Network on HIV/AIDS (CoRIS). Bootscanning and other methods were used to define complex recombinants not assigned to any subtype or CRF. A total of 670 available HIV-1 pol sequences from different patients were collected, of which 588 (87.8%) were assigned to HIV-1 subtype B and 82 (12.2%) to HIV-1 non-B variants. Recombinants caused the majority (71.9%) of HIV-1 non-B infections and were found in 8.8% of CoRIS patients. Eleven URFs (accounting for 13.4% of HIV-1 non-B infections), presenting complex mosaic patterns, were detected. Among them, 10 harbored subtype B fragments. Four of the 11 URFs were found in Spanish natives. A cluster of three B/CRF02_AG recombinants was detected. We conclude that complex variants, including unique recombinant forms, are being introduced into Spain through both immigrants and natives. An increase in the frequency of mosaic viruses, reflecting the increasing heterogeneity of the HIV epidemic in our country, is expected.     

The immunodominant T helper 2 (Th2) response elicited in BALB/c mice by the Leishmania LiP2a and LiP2b acidic ribosomal proteins cannot be reverted by strong Th1 inducers

The search for disease-associated T helper 2 (Th2) Leishmania antigens and the induction of a Th1 immune response to them using defined vaccination protocols is a potential strategy to induce protection against Leishmania infection. Leishmania infantum LiP2a and LiP2b acidic ribosomal protein (P proteins) have been described as prominent antigens during human and canine visceral leishmaniasis. In this study we demonstrate that BALB/c mice infected with Leishmania major develop a Th2-like humoral response against Leishmania LiP2a and LiP2b proteins and that the same response is induced in BALB/c mice when the parasite P proteins are immunized as recombinant molecules without adjuvant. The genetic immunization of BALB/c mice with eukaryotic expression plasmids coding for these proteins was unable to redirect the Th2-like response induced by these antigens, and only the co-administration of the recombinant P proteins with CpG oligodeoxynucleotides (CpG ODN) promoted a mixed Th1/Th2 immune response. According to the preponderance of a Th2 or mixed Th1/Th2 responses elicited by the different regimens of immunization tested, no evidence of protection was observed in mice after challenge with L. major. Although alterations of the clinical outcome were not detected in mice presensitized with the P proteins, the enhanced IgG1 and interleukin (IL)-4 response against total Leishmania antigens in these mice may indicate an exacerbation of the disease.     

Correlation between intelligence test scores and executive function measures.

In this study, some executive function measures (Wisconsin Card Sorting Test [WCST], verbal fluency, and Trial Making Test [TMT], Form A and Form B) were correlated with Wechsler Intelligence Scale for Children-Revised (WISC-R) scores. Fifty 13- to 16-year-old normal children were selected. It was found that verbal fluency tests correlated about 0.30 with Verbal Intelligence Quotient (IQ) and Full Scale IQ. In the WCST only Perseverative Errors negatively correlated with Verbal IQ and Full Scale IQ. Two correlations were found to be significant with regard to the TMT: TMT Form B Errors negatively correlated with WISC-R Vocabulary subtest; and TMT Form A Time negatively correlated with Performance IQ. These results support the assumption that traditional intelligence tests are not appropriately evaluating executive functions.     

Multicoil Dixon chemical species separation with an iterative least-squares estimation method.

This work describes a new approach to multipoint Dixon fat-water separation that is amenable to pulse sequences that require short echo time (TE) increments, such as steady-state free precession (SSFP) and fast spin-echo (FSE) imaging. Using an iterative linear least-squares method that decomposes water and fat images from source images acquired at short TE increments, images with a high signal-to-noise ratio (SNR) and uniform separation of water and fat are obtained. This algorithm extends to multicoil reconstruction with minimal additional complexity. Examples of single- and multicoil fat-water decompositions are shown from source images acquired at both 1.5T and 3.0T. Examples in the knee, ankle, pelvis, abdomen, and heart are shown, using FSE, SSFP, and spoiled gradient-echo (SPGR) pulse sequences. The algorithm was applied to systems with multiple chemical species, and an example of water-fat-silicone separation is shown. An analysis of the noise performance of this method is described, and methods to improve noise performance through multicoil acquisition and field map smoothing are discussed.