Generation, establishment and characterization of a pluripotent stem cell line (CVTTHi001-A) from primary fibroblasts isolated from a patient with activated PI3 kinase delta syndrome (APDS2).

APDS2 is caused by mutations in PIK3R1 gene resulting in constitutive PI3Kδ activation. PI3Kδ is predominantly expressed in leukocytes and plays critical roles in regulating immune responses. Here we first derived fibroblast primary cells from a skin biopsy of a patient carrying a heterozygous single T deletion in intron 11 of the PIK3R1 gene. We next present the derivation of an induced pluripotent stem cell (iPS) line using a non-integrative reprogramming technology. Pluripotent-related hallmarks are further shown, including: iPSCs self-renewal and expression of pluripotent and differentiation markers after in vitro differentiation towards embryonic germ layers, assessed by RT-PCR and immunofluorescence.

ANP32E, a Protein Involved in Steroid-Refractoriness in Ulcerative Colitis, Identified by a Systems Biology Approach.

BACKGROUND AND AIMS: Steroid-refractoriness is a common and unpredictable phenomenon in ulcerative colitis [UC], but there are no conclusive studies on the molecular functions involved. We aimed to assess the mechanism of action related to steroid failure by integrating transcriptomic data from UC patients, and updated molecular data on UC and glucocorticoids. METHODS: MicroRNA [miRNA] and mRNA expression were evaluated by sequencing and microarrays, respectively, from rectal biopsies of patients with moderately-to-severe active UC, obtained before and on the third day of steroid treatment. The differential results were integrated into the mathematical models generated by a systems biology approach. RESULTS: This computational approach identified 18 proteins that stand out either by being associated with the mechanism of action or by providing a means to classify the patients according to steroid response. Their biological functions have been linked to inflammation, glucocorticoid-induced transcription and angiogenesis. All the selected proteins except ANP32E [a chaperone which has been linked to the exchange of H2A.z histone and promotes glucocorticoid receptor-induced transcription] had previously been related to UC and/or glucocorticoid-induced biological actions. Western blot and immunofluorescence assays confirmed the implication of this chaperone in steroid failure in patients with active UC. CONCLUSIONS: A systems biology approach allowed us to identify a comprehensive mechanism of action of steroid-refractoriness, highlighting the key role of steroid-induced transcription and the potential implication of ANP32E in this phenomenon.

Loss of Tribbles pseudokinase-3 promotes Akt-driven tumorigenesis via FOXO inactivation.

Tribbles pseudokinase-3 (TRIB3) has been proposed to act as an inhibitor of AKT although the precise molecular basis of this activity and whether the loss of TRIB3 contributes to cancer initiation and progression remain to be clarified. In this study, by using a wide array of in vitro and in vivo approaches, including a Trib3 knockout mouse, we demonstrate that TRIB3 has a tumor-suppressing role. We also find that the mechanism by which TRIB3 loss enhances tumorigenesis relies on the dysregulation of the phosphorylation of AKT by the mTORC2 complex, which leads to an enhanced phosphorylation of AKT on Ser473 and the subsequent hyperphosphorylation and inactivation of the transcription factor FOXO3. These observations support the notion that loss of TRIB3 is associated with a more aggressive phenotype in various types of tumors by enhancing the activity of the mTORC2/AKT/FOXO axis.

Complete Genome Sequence of the Multiresistant Acinetobacter baumannii Strain AbH12O-A2, Isolated during a Large Outbreak in Spain.

We report the complete genome sequence of Acinetobacter baumannii strain AbH12O-A2, isolated during a large outbreak in Spain. The genome has 3,875,775 bp and 3,526 coding sequences, with 39.4% G+C content. The availability of this genome will facilitate the study of the pathogenicity of the Acinetobacter species.

microRNA profiling in duodenal ulcer disease caused by Helicobacter pylori infection in a Western population.

Although the connection of microRNAs (miRNAs) to some diseases is well established, their involvement in chronic infections such as Helicobacter pylori has received less attention. The aim was to compare miRNA expression profiling in patients with duodenal ulcer (DU) due to H. pylori infection with that in infected patients without DU and in uninfected patients. The miRNA expression profile was determined by microarrays in antral mucosal samples from well-characterized dyspeptic patients (n = 46). The most significant set of miRNAs was subsequently analysed in an independent validation group of patients (n = 42). Transcripts for IL8, IL12p40, IL12p35 and IL23p19, the signalling molecules MYD88, GATA6, SOCS2 and STAT6 and H. pylori virulence factors cagA and VacA were analysed. Microarray experiments showed that 17 miRNAs were deregulated in the mucosa of H. pylori-infected patients. No significant differences were observed between normal and DU patients. PCR confirmed the up-regulation of miR-9, miR-146a, miR-155 and miR-650 and the down-regulation of miR-96 and miR-204 in the independent validation set of patients. Importantly, miR-9, miR-96, miR-146a and miR-650 expression was specific to chronic-active gastritis. H. pylori-infected patients showed higher levels of IL8 and IL12p40 mRNAs and lower levels of GATA6 and SOCS2 mRNAs. The antral mucosa of patients with non-active or chronic-active gastritis showed significantly lower levels of GATA6, MYD88, SOCS2 and STAT6 mRNAs compared with patients without gastritis. The down-regulation of these factors was not correlated with the expression of any of the validated miRNAs. The exact role of the miRNA changes observed will require further study.

Exploring the diabetogenicity of the HLA-B18-DR3 CEH: independent association with T1D genetic risk close to HLA-DOA.

The objective of this study was to identify additional diabetes susceptibility markers in the MHC that could be responsible for the differential diabetogenicity of different HLA-DR3 CEHs. High-resolution SNP genotyping of the MHC was carried out in 15 type 1 diabetes (T1D) patients and 39 non-diabetic controls, homozygous for DR3-DQ2 and with one copy of the A(*)30-B(*)18-MICA(*)4-F1C30-DRB1(*)0301-DQB1(*)0201-DPB1(*)0202 HLA haplotype. Significantly associated SNPs were replicated in an independent sample of 554 T1D patients and 841 controls without HLA matching. Electrophoretic mobility shift assay was used to show a functional effect of an associated SNP. Seven SNPs showed evidence of association in the initial discovery experiment. Upon replication, only rs419434 (upstream HLA-DOA gene) remained significant. A functional variant (rs432375) in complete LD with rs419434 was shown to affect USF-1 binding and could be responsible for the association signal in the region. We have identified a new susceptibility locus within the MHC with a modest contribution to T1D (OR=1.93; CI: 1.52-2.44; P=10(-8)) that is independent of HLA-DRB1 locus.

The functional R620W variant of the PTPN22 gene is associated with celiac disease.

The functional (R620W) variant of human PTPN22 (protein tyrosine phosphatase non-receptor 22) gene has been implicated in the risk to several autoimmune disorders, including type 1 diabetes, Graves' disease, rheumatoid arthritis and systemic lupus erythematosus. In an association study of this single nucleotide polymorphism with celiac disease (CD), comparison of 262 young diagnosis patients and 214 adult controls from Spain showed a higher frequency of the minor allele in the CD group (9.7% vs 5.6% in controls; P = 0.018), suggestive of an increased genetic risk to the disease (odds ratio = 1.82; 95% confidence interval 1.1-3.0). These results support the role of PTPN22 as a general autoimmunity locus involved in tolerance induction in the thymus.

Conserved extended haplotypes discriminate HLA-DR3-homozygous Basque patients with type 1 diabetes mellitus and celiac disease.

The major susceptibility locus for type 1 diabetes mellitus (T1D) maps to the human lymphocyte antigen (HLA) class II region in the major histocompatibility complex on chromosome 6p21. In southern European populations, like the Basques, the greatest risk to T1D is associated with DR3 homo- and heterozygosity and is comparable to that of DR3/DR4, the highest risk genotype in northern European populations. Celiac disease (CD) is another DR3-associated autoimmune disorder showing certain overlap with T1D that has been explained by the involvement of common genetic determinants, a situation more frequent in DR3-rich populations, like the Basques. As both T1D- and CD-associated HLA alleles are part of conserved extended haplotypes (CEH), we compared DR3-homozygous T1D and CD patients to determine whether CEHs were equally distributed between both disorders or there was a differential contribution of different haplotypes. We observed a very pronounced distribution bias (P<10(-5)) of the two major DR3 CEHs, with DR3-B18 predominating in T1D and DR3-B8 in CD. Additionally, high-density single nucleotide polymorphism (SNP) analysis of the complete CEH [A*30-B*18-MICA*4-F1C30-DRB1*0301-DQB1*0201-DPB1*0202] revealed extraordinary conservation throughout the 4.9 Mbp analyzed supporting the existence of additional diabetogenic variants (other than HLA-DRB1*0301-DQB1*0201), conserved within the DR3-B18 CEH (but not in other DR3 haplotypes) that could explain its enhanced diabetogenicity.

First detection of Leishmania major in peridomestic Phlebotomus papatasi from Isfahan province, Iran: comparison of nested PCR of nuclear ITS ribosomal DNA and semi-nested PCR of minicircle kinetoplast DNA.

Two PCR methods were compared for their sensitivity in detecting cultured Leishmania major, before being used to estimate infection rates in female sandflies (Phlebotomus papatasi) collected from peridomestic animal shelters and the nearby burrows of the gerbil reservoir hosts, Rhombomys opimus, in Isfahan province, central Iran. A semi-nested PCR was used to amplify a fragment of minicircle kinetoplast (k) DNA with a length and sequence diagnostic for L. major, and a nested PCR was developed to amplify a fragment containing the internal transcribed spacers of the ribosomal RNA genes (ITS-rDNA) with a sequence diagnostic for L. major. The semi-nested PCR was less sensitive than the nested PCR when using DNA extracted from cultured promastigotes of L. major, but it was more sensitive for detecting L. major in wild-caught sandflies. At the edges of two Isfahan villages, infection rates were significantly higher in P.papatasi collected outside gerbil burrows (14/28) compared with those from peridomestic animal shelters (2/21). This is the first record of L. major detected in P.papatasi from peridomestic sites in Isfahan province.

Population differentiation of Phlebotomus perniciosus in Spain following postglacial dispersal.

Comparative sequencing of mitochondrial cytochrome b (Cyt b) and isoenzyme analyses have not resolved the population structure of the Iberian lineage of the sandfly Phlebotomus perniciosus, the most widespread vector of Leishmania infantum (Protozoa, Trypanosomatidae) to humans and dogs in the western Mediterranean subregion. Allelic variation at trinucleotide microsatellite loci was investigated in 13 Spanish populations of P. perniciosus. Four out of five loci showed significant differentiation between (pairwise F(ST)>0.23), but not within (pairwise F(ST)&<0.05), two regional groups of populations (southern and northeastern). All Cyt b sequences belonged to the Iberian lineage, which differs by six fixed nucleotide differences from the typical lineage found in northwest Africa, Malta and Italy. The northeastern group of Spanish populations had a reduced number of microsatellite alleles (16 out of the 29 present in the southern populations), indicating its derivation as a peripheral isolate following the species' dispersal from a southern Ice Age refuge 8000-12 000 years ago. Pairwise F(ST) values did not increase with geographical distance between populations, over distances of 246-850 km (between regions) and 16-491 km (within regions). This suggests that the two regional groups of populations remain isolated, but that within each region there are no significant permanent barriers to gene flow between contiguous populations. These findings will help to predict the capacity of this sandfly to disperse, and originate new foci of leishmaniasis, in response to climate warming.

Detection and identification of Leishmania DNA within naturally infected sand flies by seminested PCR on minicircle kinetoplastic DNA.

A seminested PCR assay was developed in order to amplify the kinetoplast minicircle of Leishmania species from individual sand flies. The kinetoplast minicircle is an ideal target because it is present in 10,000 copies per cell and its sequence is known for most Leishmania species. The two-step PCR is carried out in a single tube using three primers, which were designed within the conserved area of the minicircle and contain conserved sequence blocks. The assay was able to detect as few as 3 parasites per individual sand fly and to amplify minicircle DNA from at least eight Leishmania species. This technique permits the processing of a large number of samples synchronously, as required for epidemiological studies, in order to study infection rates in sand fly populations and to identify potential insect vectors. Comparison of the sequences obtained from sand flies and mammal hosts will be crucial for developing hypotheses about the transmission cycles of Leishmania spp. in areas of endemicity.

Phylogenetic relationships of phlebotomine sandflies inferred from small subunit nuclear ribosomal DNA.

Relationships among seventy specimens, fifteen species and three genera of phlebotomines were inferred from the phylogenetic analysis of small subunit nuclear rDNA, obtained by the PCR amplification and cloning of almost full-length genes. Outgroups included fifteen dipterans, and single representatives of four other insect orders. The more distant the taxa compared, the larger were the regions of ambiguous sequence alignment that needed to be deleted in order to avoid circularity in performing parsimony analyses. Phlebotomine sequences formed a monophyletic clade within the suborder Nematocera, with the progressively more basal sister groups of Diptera being Culicomorpha, Tipulomorpha and the suborder Brachycera. Within Phlebotominae, subgeneric relationships were resolved and the genus Phlebotomus was shown to be monophyletic, but markers for intraspecific geographical populations were not found and intergeneric relationships were not resolved.

Typing of sandflies from Greece and Cyprus by DNA polymorphism of 18S rRNA gene.

A simple and reliable technique was developed to distinguish Phlebotomine sandflies by restriction fragment length polymorphism of PCR-amplified (PCR-RFLP) 18S rDNAs. Seven morphologically identified sandflies species from several localities of Greece and Cyprus were studied, and specific patterns were developed by double digesting amplified 18S rDNAs with HpaII and RsaI. Three additional species of the subgenus Larroussius were distinguished by a second double digestion with AccI and BanI. We have successfully applied the method on samples in which morphological characters were badly distinguished due to poor storage conditions and in larval stages.