Search

Autoimmune Regulator (AIRE) is a transcriptional regulator that is crucial for establishing central tolerance as illustrated by the Mendelian Autoimmune Polyendocrinopathy-Candidiasis-Ectodermal Dystrophy (APECED) syndrome associated with AIRE-inactivating recessive or dominant mutations. Polymorphisms in AIRE have been proposed to be implicated in genetic susceptibility to non-Mendelian organ specific autoimmune diseases. Because there is evidence that in predisposition to Graves' disease (GD) central tolerance is crucial, we investigated whether AIRE polymorphisms could modulate risk of GD. A case-control association study using 29 variants and conducted in 150 GD patients and 200 controls did not detect any significant association. This result is not exceptional: a systematic review of the literature, including GWAS, on the association of AIRE variants with organ specific autoimmune diseases did not show clear associations; similarly heterozygous recessive mutations are not associated to non-Mendelian autoimmunity. Dominant negative mutations of AIRE are associated to autoimmunity but as mild forms of APECED rather than to non-Mendelian organ specific autoimmunity. The lack of association of common AIRE polymorphisms with polygenic autoimmune diseases is counterintuitive as many other genes less relevant for immunological tolerance have been found to be associated. These findings give rise to the intriguing possibility that evolution has excluded functionally modifying polymorphisms in AIRE., (Copyright © 2016 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.)

Graves' disease (GD) is an autoimmune thyroid disease defined by the production of stimulating autoantibodies to the thyroid-stimulating hormone receptor (TSHR) (TSAbs) that induce a sustained state of hyperthyroidism in patients. We previously demonstrated that TSHR, the target of this autoimmune response, is also a key susceptibility gene for GD, probably acting through thymic-dependent central tolerance. We also showed that TSHR is, unexpectedly, expressed in thymocytes. In this report, we confirm the expression of TSHR in thymocytes by protein immunoblotting and quantitative PCR, and show that expression is confined to maturing thymocytes. Using functional assays, we show that thymic TSHR is functional and that TSAbs can stimulate thymocytes through this receptor. This new activity of TSAbs on thymocytes may: 1) explain GD-associated thymic enlargement (hyperplasia), and 2) suggest the provocative hypothesis that the continuous stimulation of thymocytes by TSAbs could lead to a vicious cycle of iterative improvement of the affinity and stimulating capability of initially low-affinity antibacterial (e.g., Yersinia) Abs cross-reactive with TSHR, eventually leading to TSAbs. This may help to fill one of the gaps in our present understanding of unusual characteristics of TSAbs., (Copyright © 2015 by The American Association of Immunologists, Inc.)

Down syndrome (DS), or trisomy of chromosome 21, is the most common genetic disorder associated with autoimmune diseases. Autoimmune regulator protein (AIRE), a transcription factor located on chromosome 21, plays a crucial role in autoimmunity by regulating promiscuous gene expression (pGE). To investigate if autoimmunity in DS is promoted by the reduction of pGE owing to dysregulation of AIRE, we assessed the expression of AIRE and of several peripheral tissue-restricted Ag genes by quantitative PCR in thymus samples from 19 DS subjects and 21 euploid controls. Strikingly, despite the 21 trisomy, AIRE expression was significantly reduced by 2-fold in DS thymuses compared with controls, which was also confirmed by fluorescent microscopy. Allele-specific quantification of intrathymic AIRE showed that despite its lower expression, the three copies are expressed. More importantly, decreased expression of AIRE was accompanied by a reduction of pGE because expression of tissue-restricted Ags, CHRNA1, GAD1, PLP1, KLK3, SAG, TG, and TSHR, was reduced. Of interest, thyroid dysfunction (10 cases of hypothyroidism and 1 of Graves disease) developed in 11 of 19 (57.9%) of the DS individuals and in none of the 21 controls. The thymuses of these DS individuals contained significantly lower levels of AIRE and thyroglobulin, to which tolerance is typically lost in autoimmune thyroiditis leading to hypothyroidism. Our findings provide strong evidence for the fundamental role of AIRE and pGE, namely, central tolerance, in the predisposition to autoimmunity of DS individuals., (Copyright © 2014 by The American Association of Immunologists, Inc.)

Aim: This study sought to develop models to predict survival at 7 and 30 days based on symptoms detected by palliative home care teams (PHCTs)., Materials and Methods: This prospective analytic study included a 6-month recruitment period with patient monitoring until death or 180 days after recruitment. The inclusion criteria consisted of age greater than 18 years, advanced cancer, and treatment provided by participating PHCTs between April and July 2009. The study variables included death at 7 or 30 days, survival time, age, gender, place of residence, type of tumor and extension, presence of 11 signs and symptoms measured with a 0-3 Likert scale, functional and cognitive status, and use of a subcutaneous butterfly needle. The statistics applied included a descriptive analysis according to the percentage or mean±standard deviation. For symptom comparison between surviving and nonsurviving patients, the χ(2) test was used. Classification and regression tree (CART) methodology was used for model development. An internal validation system (cross-validation with 10 partitions) was used to ensure generalization of the models. The area under the receiver operating characteristics (ROC) curve was calculated (with a 95% confidence interval) to assess the validation of the models., Results: A total of 698 patients were included. The mean age of the patients was 73.7±12 years, and 60.3% were male. The most frequent type of neoplasm was digestive (37.6%). The mean Karnofsky score was 51.8±14, the patients' cognitive status according to the Pfeiffer test was 2.6±4 errors, and 8.3% of patients required a subcutaneous butterfly needle. Each model provided 8 decision rules with a probability assignment range between 2.2% and 99.1%. The model used to predict the probability of death at 7 days included the presence of anorexia and dysphagia and the level of consciousness, and this model produced areas under the curve (AUCs) of 0.88 (0.86-0.90) and 0.81 (0.79-0.83). The model used to predict the probability of death at 30 days included the presence of asthenia and anorexia and the level of consciousness, and this model produced AUCs of 0.78 (0.77-0.80) and 0.77 (0.75-0.79)., Conclusion: For patients with advanced cancer treated by PHCTs, the use of classification schemes and decision trees based on specific symptoms can help clinicians predict survival at 7 and 30 days.

Patients attended by palliative care teams: are they always comparable populations? To answer this question we have compared the basic epidemiological characteristics of patients attended by home palliative care teams (HPCT) in two autonomous regions of Spain. We carried out a coordinated analytical, observational and prospective study in two Spanish autonomous regions: Aragon and Catalonia. Data were kept during each home care visit according to patients' needs. Inclusion criteria were: advanced cancer, over 18 years old and first contact with a HPCT. The recruitment period was 6 months. Variables included were: Survival time (days), age, sex, primary disease and extension, place of residence. Functional and cognitive state, and co-morbidity. 10 signs/symptoms: asthenia, anorexia, cachexia, dysphagia, xerostomy, dyspnoea, oedemas, level of consciousness, presence of delirium, presence of pressure ulcers and some treatment data. Others variables considered were: responsible team, origin, destination when discharge, date and place of death, number of visits made and duration of monitoring. We developed a comparison between groups by Chi-squared test or the non-parametric Mann-Whitney U test and a survival analysis by Kaplan-Meier curves and the logrank test to determine differences between factors. The SPSS version 15.0 software package was used. 698 patients were included, 56.2% from Aragon and 43.8% from Catalonia. 60.3% were males, without differences between the regions. Characteristics relative to age, sex, place of residence and extension of oncological diseases were similar for both groups. We found significant differences between the two populations relative to survival time, co-morbidity, functional state, presence and intensity of a number of symptoms and the treatments, patient monitoring and the their destination after discharge. We can conclude that palliative care teams cover different profiles of patients with regard to their co-morbidity, functional, cognitive and symptomatic states. It must be pointed that the organization of palliative care services and their experience appears to condition the profile of patients they attend. There is a need of consensus on the basic descriptors for palliative care patients in order to ensure that results will be comparable.

Processing bodies (PBs) and stress granules (SGs) are two highly conserved cytoplasmic ribonucleoprotein foci that contain translationally repressed mRNAs together with proteins from the mRNA metabolism. Interestingly, they also share some common features with other granules, including the prokaryotic inclusion bodies. Although the function of PBs and SGs remains elusive, major advances have been done in unraveling their composition and assembly by using the yeast Saccharomyces cerevisae.

Inevitably, viruses depend on host factors for their multiplication. Here, we show that hepatitis C virus (HCV) RNA translation and replication depends on Rck/p54, LSm1, and PatL1, which regulate the fate of cellular mRNAs from translation to degradation in the 5'-3'-deadenylation-dependent mRNA decay pathway. The requirement of these proteins for efficient HCV RNA translation was linked to the 5' and 3' untranslated regions (UTRs) of the viral genome. Furthermore, LSm1-7 complexes specifically interacted with essential cis-acting HCV RNA elements located in the UTRs. These results bridge HCV life cycle requirements and highly conserved host proteins of cellular mRNA decay. The previously described role of these proteins in the replication of 2 other positive-strand RNA viruses, the plant brome mosaic virus and the bacteriophage Qss, pinpoint a weak spot that may be exploited to generate broad-spectrum antiviral drugs.

Short interfering RNAs (siRNAs) targeting viral or cellular genes can efficiently inhibit human immunodeficiency virus type 1 (HIV-1) replication. Nevertheless, optimal HIV-1 gene silencing by siRNA requires precise complementarity with most of the target sequence. The emergence of mutations in the targeted gene could lead to rapid viral escape from the siRNA. In the present study, Escherichia coli endoribonuclease III (RNase III) or mammalian Dicer was used to cleave double-stranded RNA into endoribonuclease-prepared siRNA (esiRNA). esiRNAs generate a variety of siRNAs which can efficiently and specifically target multiple sites in the cognate RNA. esiRNAs targeting the region encoding the HIV-1 reverse transcriptase (RT) reduced viral replication by 90%. The inhibition was dose dependent and sequence specific because several irrelevant esiRNAs did not inhibit HIV-1 replication. Importantly, esiRNAs obtained from the prototypic RT sequence of the HXB2 strain and from highly mutated RT sequences showed similar degrees of viral inhibition, suggesting that the heterogeneous population of esiRNAs could overcome individual mismatches in the RT sequence. Finally, esiRNAs generated by Dicer cleavage were five times more potent than those generated by bacterial RNase III digestion. These results show that esiRNAs are potent HIV-1 inhibitors. Moreover, sequence targets do not need to be highly conserved to reach a high level of viral replication inhibition.

Short interfering RNAs (siRNAs) targeting viral or cellular genes can efficiently inhibit human immunodeficiency virus type 1 (HIV-1) replication. Nevertheless, the emergence of mutations in the gene being targeted could lead to the rapid escape from the siRNA. Here, we simulate viral escape by systematically introducing single-nucleotide substitutions in all 19 HIV-1 residues targeted by an effective siRNA. We found that all mutant viruses that were tested replicated better in the presence of the siRNA than in the presence of the wild-type virus. The antiviral activity of the siRNA was completely abolished by single substitutions in 10 (positions 4 to 11, 14, and 15) out of 16 positions tested (substitution at 3 of the 19 positions explored rendered nonviable viruses). With the exception of the substitution observed at position 12, substitutions at either the 5' end or the 3' end (positions 1 to 3, 16, and 18) were better tolerated by the RNA interference machinery and only in part affected siRNA inhibition. Our results show that optimal HIV-1 gene silencing by siRNA requires a complete homology within most of the target sequence and that substitutions at only a few positions at the 5' and 3' ends are partially tolerated.

The quasispecies nature of hepatitis C virus (HCV) may have important implications concerning resistance to antiviral agents. To determine whether HCV NS5A quasispecies composition and dynamics are related to responsiveness to combined interferon (IFN) and ribavirin therapy, extensive sequence analyses of cloned RT-PCR amplification products of HCV-1b NS5A quasispecies of sequential isolates from 15 treated (nine sustained responders and six non-responders) and three untreated patients were performed. Accumulation of mutations in NS5A during therapy was relatively frequent in the V3 domain, but unusual elsewhere. Amino acid changes were the result of the imposition of minor variants that were already present before treatment and always occurred within the first week of therapy. Before treatment, the complexity and diversity of quasispecies were lower in isolates from responders than in those from non-responders, particularly in the V3 domain, where differences in nucleotide entropy (0.35 vs 0.64, P=0.003), genetic distance (0.0145 vs 0.0302, P=0.05) and non-synonymous substitutions (0.0102 vs 0.0203, P=0.036) were statistically significant. These differences became more apparent during treatment, because complexity and diversity remained stable or tended to increase in non-responders, whereas they tended to decrease in responders. These observations suggest that the composition and dynamics of HCV NS5A quasispecies, particularly in the V3 domain, may play a role in the response to combined IFN/ribavirin therapy.

The NS5A protein of hepatitis C virus (HCV) confers cell growth regulation and has been implicated in viral oncogenesis. Here, we investigated whether highly divergent NS5A proteins obtained from HCV-infected patients presented an oncogenic potential when expressed in mammalian cells. In general, NS5A expression was associated with increased rates of cell growth and culture proliferation. Immortalized primary hepatocyte and immortalized fibroblast cell lines expressing a subset of these sequences exhibited a significant increase in protein synthetic rate, culture saturation density, and a transformed cellular phenotype, as shown by anchorage-independent cell growth and colony formation in soft agar assays. Oncogenic transformation correlated with inhibition of protein kinase R (PKR) activity and concomitant reduction of eukaryotic initiation factor 2alpha (elF2alpha) phosphorylation levels that caused stimulation of mRNA translation. The extent of sequence variation throughout NS5A or within the previously characterized PKR-binding domain was not a predictive indicator of this cellular phenotype, suggesting that sequences outside this region contribute to PKR regulation. Our data indicate that NS5A oncogenic potential is conditional through viral sequence variation. These results provide further evidence to define the PKR pathway as a mediator of cell growth control and suggest that viral regulation of PKR may contribute to hepatocyte growth deregulation during chronic HCV infection.

Unlabelled: The Internal Medicine service of the Hospital General de Vic (Barcelona) takes part in the mortality committee by revising and discussing in-hospital mortality., Background: to establish the characteristics of the deceased, death causes and to revise possible changes in the last six-years time or problems related to the exitus, to evaluate and improve hospitalized patientś assistance., Methodology: Every case was revised following a specific register: demographical data, diagnosis and death cause, hospital death, documentation data, terminal or agonic situation when hospitalized, autopsies and death quality data. Exitus due to hospital problems were analyzed and classified in different groups. The statistical analysis was performed with measures of central tendency and of standard deviation., Results: During the revised six years, there were 819 exitus (5.1%). Global average death age was 79 +/- 1.8 years: 52.5% were men and 47.4% were women; 22.8% died in less than forty-eight hours after hospitalization. The most frequent death causes were cerebrovascular accident (24%), chronic obstructive pulmonary disease (14.4%) and pneumonia (9.6%). There were a small number of autopsies (4.8%). Ratio of exitus due to hospital problems was stable during the six years (0.5%), in which nosocomial infection was the severest problem., Conclusions: The total percentage of exitus was 5.1%, higher than the common standards. Mortality causes coincide with other series. Ratio of exitus due to hospital problems was according to recommended objectives. The number of autopsies was very small. A correct completing and revision of the clinical recording is indispensable to spot a shortage in the hospitalized patientś assistance.

The quasispecies nature of hepatitis C virus (HCV) is thought to play a central role in modulating viral functions. Recent work has linked NS5A protein with viral replication, resistance to interferon (IFN), and control of cellular growth, probably through the interaction of its protein kinase R (double stranded RNA-activated protein kinase, PKR) binding domain (PKR-bd) with cellular PKR, but knowledge of how PKR-bd viral population evolves during disease progression is limited. Since we have previously described an association between amino acid composition of the PKR-bd and the presence of HCC, in this report we further investigated the dynamic behavior of viral population parameters by sequencing an average of 20 clones per sample in 27 samples from 19 untreated patients with different degrees of liver disease, 8 of whom were followed over time. Viral population parameters varied widely from patient to patient, but no differences were observed in the complexity, diversity, types of nucleotide changes, or evolutionary pattern of the quasispecies according to the stage of liver disease. In five samples, we detected "quasispecies-tails"; that is, clones whose minimum genetic distance to the remaining clones of their own quasispecies were higher than the maximum genetic distance found between any other two clones of the same sample. In summary, independent of the degree of liver disease, or the mutations detected within the consensus sequence of the PKR-bd, the NS5A of HCV presents a flexible and variable quasispecies structure that remains largely stable during the natural course of an HCV infection, highlighting the central role of NS5A protein in viral life cycle., (Copyright 2003 Wiley-Liss, Inc.)

In July 2000, symptomatic acute hepatitis C was diagnosed in five patients who had attended the emergency room of a municipal hospital on the same day, about 6 weeks before. Investigation of the remaining 65 patients visited at the emergency room on that day disclosed that 8 patients had a positive anti-hepatitis C virus (anti-HCV) test and 4 of them had biochemical evidence of acute anicteric hepatitis. HCV RNA was detected in 12 of the 13 anti-HCV-positive patients. Phylogenetic analysis of the nonstructural 5A (NS5A) and E2 regions showed that 10 patients, including all 9 with acute hepatitis, were infected with a closely related HCV strain, while the remaining 2 patients harbored unrelated strains. Flushing of intravenous catheters with heparin retrieved from a multidose heparin solution in saline was carried out for all the patients involved in the hepatitis outbreak but in only 1 of 23 (4%) matched controls recruited among HCV-noninfected patients attending the emergency room on the same day, and this was the only significant difference concerning risk factors for HCV infection between patients and controls. Thus, accidental contamination of a multidose heparin solution with blood from an unrecognized HCV carrier was identified as the source of this nosocomial outbreak of hepatitis C.

Recurrence of hepatitis C virus (HCV) infection after liver transplantation is almost universal and usually leads to chronic hepatitis with different degrees of severity. The pathogenic mechanisms underlying the variable outcome of HCV infection recurrence are not well defined, but recent data suggest that the dynamics of HCV quasispecies may be involved. HCV quasispecies evolution was traced by longitudinal single strand conformation polymorphism, direct sequencing, and cloning analyses of pre- and post-transplant HCV-1b isolates from patients with histologically severe (seven cases) or mild or moderate (nine cases) HCV infection recurrence. Differences between the two groups of patients that concerned the level of viremia or the degree of HCV quasispecies complexity and diversity were not observed at any of the three time points analyzed. However, emergence of nucleotide and amino acid changes during the 12 months follow-up was significantly more frequent in patients with mild or moderate than in those with severe HCV infection recurrence. The ratio of non-synonymous to synonymous nucleotide substitutions 12 months after transplantation was also greater in the former, suggesting that the HVR1 of HCV is under stronger selective pressure in these subjects. These findings suggest that the degree of amino acid diversification in the HVR1 of HCV, which probably reflects the strength of immune pressure on HCV, is inversely related to the histological severity of HCV infection recurrence., (Copyright 2001 Wiley-Liss, Inc.)

Two genomic regions of hepatitis C virus (HCV), the interferon sensitivity-determining region (ISDR) of the non-structural 5A gene (NS5A) and the protein kinase-RNA activated (PKR)-eukariotic transcription factor (eIF2-alpha) phosphorylation homology domain (PePHD) of the structural E2 gene, interact in vitro with the interferon-inducible cellular PKR protein kinase. Mutations within these regions might, therefore, influence the response to interferon therapy. Viral load at baseline and sequence heterogeneity of HCV in NS5A and E2 regions was studied in 74 HCV-1b and in 12 HCV-3a infected patients with chronic hepatitis C who were treated with interferon. As previously reported by us, in a smaller series of patients in which the ISDR region was analyzed [Saiz et al. (1998) Journal Infectious Diseases 177:839-847], in the present study a low viral load and a high number of amino acid mutations within the ISDR, but not within the PePHD region, were significantly associated with long-term response to interferon among HCV-1b infected patients. No relationship between these viral features and response to therapy was disclosed in patients infected with HCV-3a., (Copyright 2001 Wiley-Liss, Inc.)

Objectives: To evaluate the prevalence, route of transmission and clinical significance that current co-infection with TT virus (TTV), hepatitis C virus (HCV), and hepatitis G virus (HGV) have in HIV-1-infected patients., Design: Presence of TTV, HCV, and HGV was analyzed in plasma samples from 160 HIV-1-infected patients with parenteral (38 intravenous drug users [IVDUs] and 41 patients with hemophilia) or sexual (39 homosexuals and 42 heterosexuals) risk of exposure, and in 168 volunteer blood donors. Alanine aminotransferase (ALT) levels and CD4+ counts were also analyzed., Methods: HCV and HGV RNA were detected by specific reverse transcriptase (RT) nested polymerase chain reaction (PCR) and TTV DNA by specific heminested PCR., Results: TTV DNA was detected in 39% of the patients and in 14% of the volunteer blood donors. HCV and HGV infections were detected in 42% and in 14% of the patients, and in 0% and 3% of the blood donors, respectively. Prevalences of TTV and HCV infection were higher among patients with parenteral (62% and 68%) than in those with sexual (17% and 16%) risk of exposure. A higher prevalence of TTV infection (but not of HCV or HGV infection) was observed among patients with hemophilia (76%) than IVDUs (47%), and among homosexuals (26%) than among heterosexuals (10%). Abnormal ALT levels were related with the presence of HCV infection, independently of the detection of TTV DNA. TTV infection did not seem to alter the levels of CD4+ T cells., Conclusions: Prevalence of current TTV infection is high among HIV-infected patients with parenteral risk of exposure, but TTV is also transmitted through sexual routes; detection of TTV does not seem to influence the clinical or immune status of HIV-infected patients.

Hepatitis G virus (HGV) RNA was detected in 18 of 133 pregnant women from Tanzania without known risk factors for HGV infection and in 7 of 18 children born to HGV RNA-positive mothers. Molecular evidence of mother-to-infant transmission was obtained only for three of seven children. HGV RNA was also detected in 4 of 42 children born to non-HGV-infected women. Thus, mechanisms other than materno-filial may play an important role in HGV transmission during early childhood.

In patients with chronic hepatitis C, the influence of the genetic heterogeneity of the hepatitis C virus (HCV) on the progression of liver disease and on the responsiveness to interferon therapy is a matter of controversy. In this study we evaluated the genetic complexity of HCV by single-strand conformation polymorphism (SSCP) analysis of amplicons from the hypervariable region 1 (HVR1) in 168 patients with chronic genotype 1b HCV infection, of whom 122 received a single course of interferon therapy (3 MU, three times weekly for 6 months). No correlation was observed between the degree of genetic complexity of HCV (indicated by the number of bands in the SSCP assay) and patient age, serum alanine aminotransferase activity, or serum HCV-RNA concentration, measured by competitive polymerase chain reaction. HCV genomic complexity was not related to gender nor to the presumed source of infection. The number of SSCP bands detected in serum samples from patients with chronic hepatitis, either mild (8.1 +/- 3.9), moderate (8.0 +/- 3.3), or severe (9.2 +/- 3.3), and in patients with liver cirrhosis, either compensated (8.0 +/- 2.9), decompensated (6.3 +/- 2.9), or with superimposed hepatocellular carcinoma (9.5 +/- 2.9), was similar. The number of SSCP bands detected in patients with sustained response (7.5 +/- 3. 9), transient response (8.3 +/- 2.9), or no response (8.2 +/- 3.6) to interferon administration was similar as well. These observations suggest that the genetic complexity of hypervariable region (HVR1) of HCV, as estimated by SSCP analysis, is not related to the severity of liver injury nor to the type of response to interferon therapy. Thus, information offered by SSCP analysis of HVR1 of HCV in chronic HCV genotype 1b infection does not appear to be useful in the clinical management of these patients. (HEPATOLOGY 1999;29:897-903.)

Comparative sequence analysis of different isolates of hepatitis G virus (HGV) has demonstrated significant intersubject genetic heterogeneity, but few data on intrasubject genetic evolution have been reported. To further investigate the genetic diversification of the HGV genome, 36 plasma samples from eleven patients chronically infected with HGV serially obtained 2-4 years apart were analysed. We determined the viral nucleotide sequence of the 5' non-coding (NC) and the NS3 regions by directly sequencing the RT-PCR amplified products obtained from the viral RNAs. Intrasubject sequence variation was found to be 1.3-2.4 x 10(-3) base substitutions per genome site per year within the 5' NC region and 1.3-9.4 x 10(-3) base substitutions per genome site per year within the NS3 region. Depending on the genomic region analysed (i.e. 5' NC or NS3 region), pairwise comparisons and phylogenetic reconstructions showed that intersubject genetic distances were 17.5- to 20.8-fold greater than intrasubject ones. Overall, the evolution rate of HGV in the regions analysed is not significantly different from that found in hepatitis C virus.