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Resumen Objetivo El objetivo del estudio fue conocer la tendencia de las variables relacionadas con la tuberculosis (TB) en Espana a partir del registro del Programa Integrado de Investigacion en Tuberculosis (PII-TB) de la Sociedad Espanola de Neumologia y Cirugia Toracica (SEPAR) y evaluar el PII-TB mediante indicadores relacionados con sus objetivos cientificos. Metodo Estudio transversal multicentrico de base poblacional de casos nuevos de TB registrados prospectivamente por el PII-TB entre 2006 y 2016. La tendencia temporal de variables cuantitativas se realizo mediante un modelo de regresion lineal y las cualitativas mediante la prueba de χ2 de tendencia lineal. Resultados Se analizaron 6.892 casos de TB con una mediana anual de 531. La tendencia general fue significativamente decreciente en mujeres, inmigrantes, privados de libertad y en tratados inicialmente con 3 farmacos. Se incrementaron significativamente la tendencia de grupos de 40 -50 anos y > 50 anos, primera atencion por especialista de zona, hospitalizacion, retraso diagnostico, localizacion diseminada y extrapulmonar unica, cultivo (+), realizacion de antibiogramas, resistencia a farmacos, tratamiento directamente observado, prolongacion del tratamiento y muerte por otra causa. Los objetivos cientificos del PII-TB que incrementaron significativamente fueron las publicaciones alcanzando un maximo de 8 en 2016 y con un factor de impacto total de 49,664, y tambien mejoraron los proyectos iniciados anualmente, presentaciones en congresos y las tesis o tesinas. Conclusiones El PII-TB proporciona informacion relevante sobre la TB y sus factores asociados en Espana. Se ha formado un amplio equipo de investigadores y se han detectado aspectos cientificos positivos y otros mejorables.

Purpose of review Primary biliary cholangitis (PBC) is characterized by autoimmune damage of intrahepatic bile ducts associated with a loss of tolerance to mitochondrial antigens. PBC etiopathogenesis is intriguing because of different perplexing features, namely: a) although mitochondria are present in all cell types and tissues, the damage is mainly restricted to biliary epithelial cells (BECs); b) despite being an autoimmune disorder, it does not respond to immunosuppressive drugs but rather to ursodeoxycholic acid, a bile salt that induces HCO3- rich choleresis; c) the overwhelming female preponderance of the disease remains unexplained. Here we present an etiopathogenic view of PBC which sheds light on these puzzling facts of the disease. Recent findings PBC develops in patients with genetic predisposition to autoimmunity in whom epigenetic mechanisms silence the Cl-/HCO3- exchanger AE2 in both cholangiocytes and lymphoid cells. Defective AE2 function can produce BECs damage as a result of decreased biliary HCO3- secretion with disruption of the protective alkaline umbrella that normally prevents the penetration of toxic apolar bile salts into cholangiocytes. AE2 dysfunction also causes increased intracellular pH (pHi) in cholangiocytes, leading to the activation of soluble adenylyl cyclase, which sensitizes BECs to bile salt-induced apoptosis. Recently, mitophagy was found to be inhibited by cytosolic alkalization and stimulated by acidification. Accordingly, we propose that AE2 deficiency may disturb mitophagy in BECs, thus, promoting the accumulation of defective mitochondria, oxidative stress and presentation of mitochondrial antigens to the immune cells. As women possess a more acidic endolysosomal milieu than men, mitophagy might be more affected in women in an AE2-defective background. Apart from affecting BECs function, AE2 downregulation in lymphocytes may also contribute to alter immunoregulation facilitating autoreactive T-cell responses. Summary PBC can be considered as a disorder of Cl-/HCO3- exchange in individuals with genetic predisposition to autoimmunity.

ObjectiveTo determine the association between endoplasmic reticulum aminopeptidase (ERAP)1 and ERAP2 single-nucleotide polymorphisms (SNPs) and human leukocyte antigens (HLA)-B27+ or HLA-B15+ patients with spondyloarthritis (SpA).Methods104 patients with SpA according to Assessment of Spondyloarthritis International Society criteria were included in the study. HLA typing was performed by PCR. The polymorphisms were determined by real-time PCR on genomic DNA using customised probes for SNPs rs27044, rs17482078, rs10050860 and rs30187 in ERAP1, and rs2910686, rs2248374 and rs2549782 in ERAP2.Results70 of the104 patients with SpA were HLA-B27+ and 34 were HLA-B15+. The distribution of ERAP1 and ERAP2 SNPs between the HLA-B15+ and HLA-B27+ patients with SpA did not reveal differences. Likewise, no differences in the frequencies of ERAP1 SNP haplotypes and alleles HLA-B15 or HLA-B27 were found. Interestingly, however, the frequencies of three particular haplotypes formed by ERAP2 SNPs rs2549782/rs2248374/rs2910686 varied between HLA-B15+ and HLA-B27+ patients: the ERAP2 SNPs haplotype TGT was more common in HLA-B15+ patients with SpA (OR 2.943, 95% CI 1.264 to 6.585; P=0.009), whereas the ERAP2 SNP haplotypes TGC and CAT were more associated with HLA-B27+ patients with SpA: (OR 4.483, 95% CI 1.524 to 13.187; p=0.003) and (OR 9.014, 95% CI 1.181 to 68.807; p=0.009), respectively.ConclusionAn association was found between HLA-B15+ patients with SpA and haplotype TGT of ERAP2 SNPs. On the other hand, HLA-B27+ patients with SpA were associated with ERAP2 haplotypes TGC and CAT. These associations could be related to the clinical presentation of the disease, specifically with a peripheral or axial predominance, respectively.

The objective of the study was to determine the trend of variables related to tuberculosis (TB) from the Integrated Tuberculosis Research Program (PII-TB) registry of the Spanish Society of Pulmonology and Thoracic Surgery (SEPAR), and to evaluate the PII-TB according to indicators related to its scientific objectives.Cross-sectional, population-based, multicenter study of new TB cases prospectively registered in the PII-TB between 2006 and 2016. The time trend of quantitative variables was calculated using a lineal regression model, and qualitative variables using the χy test for lineal trend.A total of 6,892 cases with an annual median of 531 were analyzed. Overall, a significant downward trend was observed in women, immigrants, prisoners, and patients initially treated with 3 drugs. Significant upward trends were observed in patients aged 40-50 and50 years, first visit conducted by a specialist, hospitalization, diagnostic delay, disseminated disease and single extrapulmonary location, culture(+), sensitivity testing performed, drug resistance, directly observed treatment, prolonged treatment, and death from another cause. The scientific objectives of the PII-TB that showed a significant upward trend were publications, which reached a maximum of 8 in 2016 with a total impact factor of 49,664, numbers of projects initiated annually, presentations at conferences, and theses.PII-TB provides relevant information on TB and its associated factors in Spain. A large team of researchers has been created; some scientific aspects of the registry were positive, while others could have been improved.

Background Anti-dengue virus (DENV) immunoglobulin M (IgM) seroconversion has been the reference standard for dengue diagnosis. However, paired specimens are rarely obtained, and the interval for this testing negates its usefulness in guiding clinical case management. The presence of DENV viremia and appearance of IgM during the febrile phase of dengue provides the framework for dengue laboratory diagnosis by using a single serum specimen. Methods Archived paired serum specimens (n = 1234) from patients with laboratory-confirmed dengue from 2005 through 2011 were used to determine the diagnostic performance of real-time reverse transcription polymerase chain reaction (RT-PCR), for detection of DENV serotypes 1-4, and enzyme-linked immunosorbent assays (ELISAs), for detection of DENV nonstructural protein 1 (NS1) antigen and anti-DENV IgM. Results During 1-3 days after illness onset, real-time RT-PCR and NS1 antigen testing detected 82%-69% and 90%-84% of cases, respectively, as viremia levels declined, while anti-DENV IgM ELISA detected 5%-41% of cases as antibody appeared. Over the 10-day period of the febrile phase of dengue, the cumulative effect of using these 3 types of tests in a diagnostic algorithm confirmed ≥90% of dengue cases. Conclusions The use of molecular or NS1 antigen tests to detect DENV and one to detect anti-DENV IgM in a single serum specimen collected during the first 10 days of illness accurately identified ≥90% of dengue primary and secondary cases.

Background & Aims Patients with primary biliary cholangitis (PBC) exhibit reduced AE2/SLC4A2 gene expression in the liver and peripheral blood mononuclear cells (PBMCs). AE2 encodes a Cl–/HCO3– exchanger involved in biliary bicarbonate secretion and intracellular pH regulation. Reduced AE2 expression in PBC may be pathogenic, as Ae2-knockout mice reproduce characteristic PBC features. Herein, we aimed to identify CpG-methylation abnormalities in AE2 promoter regions that might contribute to the reduced gene transcription in PBC livers and PBMCs. Methods CpG-cytosine methylation rates were interrogated at 1-base pair resolution in upstream and alternate AE2 promoter regions through pyrosequencing of bisulphite-modified genomic DNA from liver specimens and PBMCs. AE2a and alternative AE2b1 and AE2b2 mRNA levels were measured by real-time PCR. Human lymphoblastoid-T2 cells were treated with 5-aza-2´-deoxycytidine for demethylation assays. Results AE2 promoters were found to be hypermethylated in PBC livers compared to normal and diseased liver specimens. Receiver operating characteristic (ROC) curve analysis showed that minimal CpG-hypermethylation clusters of 3 AE2a-CpG sites and 4 alternate-AE2b2-CpG sites specifically differentiated PBC from normal and diseased controls, with mean methylation rates inversely correlating with respective transcript levels. Additionally, in PBMCs a minimal cluster of 3 hypermethylated AE2a-CpG sites distinguished PBC from controls, and mean methylation rates correlated negatively with AE2a mRNA levels in these immune cells. Alternate AE2b2/AE2b1 promoters in PBMCs were constitutively hypermethylated, in line with absent alternative mRNA expression in diseased and healthy PBMCs. Demethylation assays treating lymphoblastoid-T2 cells with 5-aza-2´-deoxycytidine triggered AE2b2/AE2b1 expression and upregulated AE2a-promoter expression. Conclusions Disease-specific hypermethylation of AE2 promoter regions and subsequent downregulation of AE2-gene expression in the liver and PBMCs of patients with PBC might be critically involved in the pathogenesis of this complex disease. Lay summary Primary biliary cholangitis (PBC) is a chronic immune-associated cholestatic liver disease with unclear complex/multifactorial etiopathogenesis affecting mostly middle-aged women. Patients with PBC exhibit reduced expression of the AE2/SLC4A2 gene. Herein, we found that AE2 promoter regions are hypermethylated in the liver and peripheral blood mononuclear cells of patients with PBC. This increased methylation is associated with downregulated AE2-gene expression, which might contribute to the pathogenesis of PBC. Therefore, novel epigenetic targets may improve treatment in patients with PBC who respond poorly to current pharmacological therapies., Graphical abstract Unlabelled Image, Highlights • Patients with PBC have higher AE2 CpG methylation in upstream AE2a and/or AE2b2/AE2b1 promoter regions in liver and PBMCs. • Combined methylation rates of 2 minimal CpG-clusters in the liver and 1 minimal CpG-cluster in PBMCs specifically distinguished PBC from normal and diseased controls. • Methylation rates of AE2 promoter regions inversely correlated with levels of respective AE2 mRNAs in liver and PBMCs. • Alternate AE2b2/AE2b1 promoter regions were found to be densely methylated in both normal and diseased PBMC samples.

// Axel R. Concepcion 1 , January T. Salas 2 , Elena Saez 1 , Sarai Sarvide 1 , Alex Ferrer 3 , Ainhoa Portu 1 , Iker Uriarte 1 , Sandra Hervas-Stubbs 1 , Ronald P.J. Oude Elferink 4 , Jesus Prieto 1 and Juan F. Medina 1 1 Center for Applied Medical Research (CIMA), School of Medicine and Clinic University of Navarra, and Ciberehd, Pamplona, Spain 2 Department of Pathology and Laboratory Medicine, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA 3 Department of Immunology, Mayo Clinic College of Medicine, Rochester, MN, USA 4 Tytgat Institute for Liver and Intestinal Research, Academic Medical Center, Amsterdam, The Netherlands Correspondence to: Juan F. Medina, email: // Jesus Prieto, email: // Keywords : Na + -independent Cl − /HCO3 − anion exchanger AE2, mouse model of autoimmune cholangitis, intracellular pH homeostasis, age-related changes, self-tolerance breakdown, Immunology and Microbiology Section, Immune response, Immunity Received : August 24, 2015 Accepted : August 31, 2015 Published : September 15, 2015 Abstract Primary biliary cirrhosis (PBC) is a chronic cholestatic disease of unknown etiopathogenesis showing progressive autoimmune-mediated cholangitis. In PBC patients, the liver and lymphocytes exhibit diminished expression of AE2/SLC4A2, a Cl − /HCO 3 − anion exchanger involved in biliary bicarbonate secretion and intracellular pH regulation. Decreased AE2 expression may be pathogenic as Ae2 a,b –/– mice reproduce hepatobiliary and immunological features resembling PBC. To understand the role of AE2 deficiency for autoimmunity predisposition we focused on the phenotypic changes of T cells that occur over the life-span of Ae2 a,b –/– mice. At early ages (1-9 months), knockout mice had reduced numbers of intrahepatic T cells, which exhibited increased activation, programmed-cell-death (PD)-1 expression, and apoptosis. Moreover, young knockouts had upregulated PD-1 ligand (PD-L1) on bile-duct cells, and administration of neutralizing anti-PD-L1 antibodies prevented their intrahepatic T-cell deletion. Older (≥10 months) knockouts, however, showed intrahepatic accumulation of cytotoxic CD8 + T cells with downregulated PD-1 and diminished apoptosis. In-vitro DNA demethylation with 5-aza-2’-deoxycytidine partially reverted PD-1 downregulation of intrahepatic CD8 + T cells from aged knockouts. Conclusion: Early in life, AE2 deficiency results in intrahepatic T-cell activation and PD-1/PD-L1 mediated deletion. With aging, intrahepatic CD8 + T cells epigenetically suppress PD-1, and their consequential expansion and further activation favor autoimmune cholangitis.

Primary biliary cirrhosis (PBC) is a chronic and progressive cholestatic liver disease of unknown etiopathogenesis that mainly affects middle-aged women. Patients show non-suppurative cholangitis with damage and destruction of small- and medium-sized intrahepatic bile ducts. Characteristically, the disease is strongly associated with autoimmune phenomena such as the appearance of serum antimitochondrial autoantibodies (AMA) and portal infiltrates with autoreactive T cells which recognize the inner lipoyl domain of the E2 component of the pyruvate dehydrogenase complex (PDC-E2). Here we review the major characteristics of a series of inducible and genetically modified animal models of PBC and analyze their similarities and differences with PBC features in humans.

Regulatory T (Treg) cells can weaken antitumor immune responses, and inhibition of their function appears to be a promising therapeutic approach in cancer patients. Mice with targeted deletion of the gene encoding the Cl-/HCO3- anion exchanger AE2 (also termed SLC4A2), a membrane-bound carrier involved in intracellular pH regulation, showed a progressive decrease in the number of Treg cells. We therefore challenged AE2 as a potential target for tumor therapy, and generated linear peptides designed to bind the third extracellular loop of AE2, which is crucial for its exchange activity. Peptide p17AE2 exhibited optimal interaction ability and indeed promoted apoptosis in mouse and human Treg cells, while activating effector T-cell function. Interestingly, this linear peptide also induced apoptosis in different types of human leukemia, lymphoma and multiple myeloma cell lines and primary malignant samples, while it showed only moderate effects on normal B lymphocytes. Finally, a macrocyclic AE2 targeting peptide exhibiting increased stability in vivo was effective in mice xenografted with B-cell lymphoma. These data suggest that targeting the anion exchanger AE2 with specific peptides may represent an effective therapeutic approach in B-cell malignancies.

Background Since 1973, the association of HLA-B27 and spondyloarthritis (SpA) is well known, however in Colombian population it is present in only 40% of patients and HLA-B15 is present almost in 25%. A mechanism of polygenic mechanism has been proposed as an explanation for the development of SpA. Endoplasmic reticulum aminopeptidase (ERAP) genes 1 and 2 have been implicated. ERAP1 is strongly associated with HLA-B27 positive patients and ankylosing spondylitis, but not with ERAP2 Objectives To determine the association between ERAP polymorphisms and HLA-B27 or HLA-B15 positive SpA patients Methods 178 patients with SpA according to ASAS criteria were included in the study. HLA typing was performed by the PCR technique using the Biorad® HLA-SSP plates. The polymorphisms were determined by the RT-PCR technique using Roche® probes for ERAP1 rs27044, rs17482078, rs10050860, and rs30187. For ERAP2 the probes used were rs2910686, rs2248374 and rs2549782. The allele and genotype frequencies polymorphisms were obtained by direct counting. In each group the Hardy-Weinberg equilibrium was evaluated using the 2 test. Associations were assessed using odds ratio (OR). Stata v.12.0 program was used to analyse data. The construction and analysis of haplotypes was performed using Haploview v.4.2 Results In total 70 patients were HLA-B27 positive and 34 were HLA-B15 positive. 78 were women and 100 were men. Linkage disequilibrium map of the ERAP gene is depicted in figure 1. When analysed by ERAP2 haplotype it is observed that there is a statistically significant association with the combinations described in table 1. No associations were observed between ERAP1 haplotypes and HLA-B15 or B27 Conclusions In the group of patients analysed, a statistically significant association was found between patients with SpA HLA-B15 positive and the haplotype TGT of ERAP2. Also HLA-B27 positive SpA patients were associated with haplotype TGC and CAT of ERAP2 with statistical significance Disclosure of Interest None declared

Background A main challenge in spondyloartritis (SpA) management was the availability of reliable biomarkers related with disease activity, or predicting joint damage and the response to treatment. Although erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) are currently used as biomarkers for disease activity, they lack sensitivity, specificity and reproducibility. With the understanding of SpA pathogenesis, additional biomarkers like metalloproteinase 3 (MMP-3), interleukin (IL)-1a, IL-6, lipopolysaccharide-binding protein (LBP), tumour necrosis factor a (TNFa), macrophage colony stimulating factor (M-CSF), interferon gamma (INF-g), IL-17 and IL-23, had been proposed Objectives We aimed to evaluate the associations of MMP-3, IL-1a, IL-6, M-CSF, LPB, IL-17 and IL-23 levels in SpA patients positive for HLA-B27 or HLA-B15 Methods 178 patients (100 men and 78 women) with SpA according to ASAS criteria were included in the study. HLA typing was performed by PCR using the Biorad® HLA-SSP ABDR plates. The levels of TNFa, IL-1a, IL-6, INF-g and IL-17 were measured by a cytometric bead-array (CBA Flex Set) using a FACS Canto II Flow CytometerO. Enzyme-linked immunosorbent assay (ELISA) was used to determine serum levels of IL-23, M-CSFand MMP-3. CRP and LBP levels were measured by chemiluminescence. Statistical analysis was made with SPSS v19. For comparison of quantitative variables with a normal distribution we used the Student9s t-test. Categorical variables were presented in frequency charts and percentages, and the Chi-squared test and Fisher9s exact test were used when necessary, for comparing groups. Two-tailed P-value Results Of the 178 patients, 70 were positive for HLA-B27, 34 for HLA-B15 and 74 had other HLA-B. According to ASAS classification criteria, 152 patients had axial SpA (axSpA) manifestations, 161 had peripheral SpA (pSpA) manifestations, and 148 patients had mixed axial and peripheral manifestations. Figure 1 shows the mean levels of inflammatory serologic biomarkers in these subgroups of patients Conclusions High levels of IL-17 and IL-23 were associated with the presence of HLA-B27, which mainly correlates with an axial presentation of the disease as compared to HLA-B15 patients. Accordingly, the genotype (presence of HLA-B27 or HLA-B15) and phenotype (axial or peripheral involvement) may help physicians when considering a targeted therapy of SpA patients with IL-17 inhibition in a context of personalized medicine Disclosure of Interest None declared

Schizophrenia is a devastating disease that arises on the background of genetic predisposition and environmental risk factors, such as early life stress (ELS). In this study, we show that ELS-induced schizophrenia-like phenotypes in mice correlate with a widespread increase of histone-deacetylase 1 (Hdac1) expression that is linked to altered DNA methylation. Hdac1 overexpression in neurons of the medial prefrontal cortex, but not in the dorsal or ventral hippocampus, mimics schizophrenia-like phenotypes induced by ELS. Systemic administration of an HDAC inhibitor rescues the detrimental effects of ELS when applied after the manifestation of disease phenotypes. In addition to the hippocampus and prefrontal cortex, mice subjected to ELS exhibit increased Hdac1 expression in blood. Moreover, Hdac1 levels are increased in blood samples from patients with schizophrenia who had encountered ELS, compared with patients without ELS experience. Our data suggest that HDAC1 inhibition should be considered as a therapeutic approach to treat schizophrenia.

Background & Aims Both hepatocytes and cholangiocytes release ATP into the bile, where it acts as a potent autocrine/paracrine stimulus that activates biliary secretory mechanisms. ATP is known to be metabolized into multiple breakdown products, ultimately yielding adenosine. However, the elements implicated in the adenosine-dependent purinergic regulation of cholangiocytes are not known. Methods Normal rat cholangiocytes (NRCs) were used to study the expression of adenosine receptors and transporters and their functional interactions at the apical and basolateral membrane domains of polarized cholangiocytes. Results We found that: (1) cholangiocytes exclusively express two concentrative nucleoside transporters (CNT) known to be efficient adenosine carriers: CNT3, located at the apical membrane, and CNT2, located at apical and basolateral membrane domains; (2) in both domains, NRCs also express the high affinity adenosine receptor A 2A , which modulated the activity of apical CNT3 in a domain-specific manner; (3) the regulation exerted by A 2A on CNT3 was dependent upon the cAMP/PKA/ERK/CREB axis, intracellular trafficking mechanisms and AMPK phosphorylation; (4) secretin increased the activity of the apically-located CNT3, and promoted additional basolateral CNT3-related activity; and (5) extracellular ATP (a precursor of adenosine) was able to exert an inhibitory effect on the apical activity of both CNT3 and CNT2. Conclusions This study uncovered the functional expression of nucleoside transporters in cholangiocytes and provides evidence for direct crosstalks between adenosine transporters and receptors for adenosine and its natural extracellular precursor, ATP. Our data anticipate the possibility of adenosine playing a major role in the physiopathology of the biliary epithelia.

Mitogenic stimulation of lymphocytes involves alkalinization of intracellular pH (pHi ). Subsequent pHi regulation may involve HCO3 (-) extrusion through Cl(-) /HCO3 (-) exchangers and/or Na(+) -HCO3 (-) co-transporters with acid-loading capability. Abnormalities in these mechanisms could result in immune dysfunctions, as suggested by the CD8(+) T-cell expansion encountered in mice lacking Ae2 (a widely expressed acid loader with electroneutral and Na(+) -independent Cl(-) /HCO3 (-) anion-exchange activity). Here we report that CD8(+) T cells but not CD4(+) T cells or other lymphocyte populations, are crucially dependent on Ae2 for pHi regulation. While total lymphocytes (including isolated CD4(+) T cells) exhibit Ae1 expression and Na(+) -HCO3 (-) co-transport with acidifying potential, CD8(+) T cells lack these acid-loading mechanisms. In Ae2-KO mice, CD4(+) but not CD8(+) T cells upregulate these potential Ae2 surrogates. As a consequence, Ae2-KO CD8(+) T cells exhibit alkalinized pHi , and dramatically increase their pHi upon CD3 stimulation. Moreover, stimulated Ae2-deficient CD8(+) T cells show enhanced intracellular production of IL-2 and membrane expression of its receptor IL-2Rα, together with increased cell proliferation and activation. These findings demonstrate that CD8(+) T cells are critically dependent on Ae2 for pHi homeostasis and tuning of cell proliferation and activation. Ae2 thus constitutes a novel target to modulate CD8(+) T-cell responses.

Enamel fluorosis is an irreversible structural enamel defect following exposure to supraoptimal levels of fluoride during amelogenesis. We hypothesized that fluorosis is associated with excess release of protons during formation of hypermineralized lines in the mineralizing enamel matrix. We tested this concept by analyzing fluorotic enamel defects in wild-type mice and mice deficient in anion exchanger-2a,b ( Ae2a,b), a transmembrane protein in maturation ameloblasts that exchanges extracellular Cl− for bicarbonate. Defects were more pronounced in fluorotic Ae2a,b−/− mice than in fluorotic heterozygous or wild-type mice. Phenotypes included a hypermineralized surface, extensive subsurface hypomineralization, and multiple hypermineralized lines in deeper enamel. Mineral content decreased in all fluoride-exposed and Ae2a,b−/− mice and was strongly correlated with Cl−. Exposure of enamel surfaces underlying maturation-stage ameloblasts to pH indicator dyes suggested the presence of diffusion barriers in fluorotic enamel. These results support the concept that fluoride stimulates hypermineralization at the mineralization front. This causes increased release of protons, which ameloblasts respond to by secreting more bicarbonates at the expense of Cl− levels in enamel. The fluoride-induced hypermineralized lines may form barriers that impede diffusion of proteins and mineral ions into the subsurface layers, thereby delaying biomineralization and causing retention of enamel matrix proteins.

Aim. To evaluate the effectiveness of omalizumab in non-atopic asthma. Methods. Using data from a multicenter registry of severe asthma, we evaluated and compared the clinical outcome of 29 omalizumab-treated severe non-atopic asthmatics with 266 omalizumab-treated severe allergic asthmatics. Effectiveness was assessed by considering severe exacerbations, pulmonary function, the Global Evaluation of Treatment Effectiveness (GETE) scale, and Asthma Control Test (ACT). Results. Omalizumab demonstrated significant improvement in the clinical status of non-atopic asthmatics as measured by GETE, which rose from 1.6 ± 1.1 to 2.8 ± 0.9 [corrected] at 4 months (p = .0215) to 2.9 ± 0.9 at 1 year (p = .0093) and to 3.4 ± 0.6 at 2 years (p = .0078), and by the ACT, which increased from 13.3 ± 5.5 [corrected] to 17.5 ± 5.4 at 4 months (p = .0236) to 17.9 ± 4.8 at 1 year (p = .0136) and to 20.6 ± 3.9 at 2 years (p = .0024). Forced expiratory volume in 1 second (FEV1) improved from 61.0 ± 19.4% to 65.1 ± 17.2 at 4 months to 64.1 ± 24.7 at 1 year and to 67.3 ± 23.0% [corrected] at 2 years, but without significant differences between initial and follow-up measurements (p = .52, .91, and .45, respectively) and exacerbations decreased from 3.1 ± 3.5 to 1.9 ± 2.8 at 1 year (p = .1709) to 1.8 ± 4.4 at 2 years (p = .2344). The results were not significantly different from those obtained in atopic asthmatics. Conclusion. Anti-IgE therapy can be effective in non-atopic severe asthma.

Cl−/HCO anion exchanger 2 (AE2) participates in intracellular pH homeostasis and secretin-stimulated biliary bicarbonate secretion. AE2/SLC4A2 gene expression is reduced in liver and blood mononuclear cells from patients with primary biliary cirrhosis (PBC). Our previous findings of hepatic and immunological features mimicking PBC in Ae2-deficient mice strongly suggest that decreased AE2 expression might be involved in the pathogenesis of PBC. Here, we tested the potential role of microRNA 506 (miR-506) — predicted as candidate to target AE2 mRNA — for the decreased expression of AE2 in PBC. Real-time quantitative polymerase chain reaction showed that miR-506 expression is increased in PBC livers versus normal liver specimens. In situ hybridization in liver sections confirmed that miR-506 is up-regulated in the intrahepatic bile ducts of PBC livers, compared with normal and primary sclerosing cholangitis livers. Precursor-mediated overexpression of miR-506 in SV40-immortalized normal human cholangiocytes (H69 cells) led to decreased AE2 protein expression and activity, as indicated by immunoblotting and microfluorimetry, respectively. Moreover, miR-506 overexpression in three-dimensional (3D)-cultured H69 cholangiocytes blocked the secretin-stimulated expansion of cystic structures developed under the 3D conditions. Luciferase assays and site-directed mutagenesis demonstrated that miR-506 specifically may bind the 3′untranslated region (3′UTR) of AE2 messenger RNA (mRNA) and prevent protein translation. Finally, cultured PBC cholangiocytes showed decreased AE2 activity, together with miR-506 overexpression, compared to normal human cholangiocytes, and transfection of PBC cholangiocytes with anti-miR-506 was able to improve their AE2 activity. Conclusion: miR-506 is up-regulated in cholangiocytes from PBC patients, binds the 3′UTR region of AE2 mRNA, and prevents protein translation, leading to diminished AE2 activity and impaired biliary secretory functions. In view of the putative pathogenic role of decreased AE2 in PBC, miR-506 may constitute a potential therapeutic target for this disease. (HEPATOLOGY 2012)

Maturation stage ameloblasts of rodents express vacuolar type-H-ATPase in the ruffled border of their plasma membrane in contact with forming dental enamel, similar to osteoclasts that resorb bone. It has been proposed that in ameloblasts this v-H-ATPase acts as proton pump to acidify the enamel space, required to complete enamel mineralization. To examine whether this v-H-ATPase in mouse ameloblasts is a proton pump, we determined whether these cells express the lysosomal, T-cell, immune regulator I (Tcirg1, v-H-Atp6v(0)a(3)), which is an essential part of the plasma membrane proton pump that is present in osteoclasts. Mutation of this subunit in Tcirg1 null (or oc/oc) mice leads to severe osteopetrosis. No immunohistochemically detectable Tcirg1 was seen in mouse maturation stage ameloblasts. Strong positive staining in secretory and maturation stage ameloblasts however was found for another subunit of v-H-ATPase, subunit b, brain isoform (v-H-Atp6v(1)b(2)). Mouse osteoclasts and renal tubular epithelium stained strongly for both Tcirg1 and v-H-Atp6v(1)b(2). In Tcirg1 null mice osteoclasts and renal epithelium were negative for Tcirg1 but remained positive for v-H-Atp6v(1)b(2). The bone in these mutant mice was osteopetrotic, tooth eruption was inhibited or delayed, and teeth were often morphologically disfigured. However, enamel formation in these mutant mice was normal, ameloblasts structurally unaffected and the mineral content of enamel similar to that of wild type mice. We concluded that Tcirg1, which is essential for osteoclasts to pump protons into the bone, is not appreciably expressed in maturation stage mouse ameloblasts. Our data suggest that the reported v-H-ATPase in maturation stage ameloblasts is not the typical osteoclast-type plasma membrane associated proton pump which acidifies the extracellular space, but rather a v-H-ATPase potentially involved in intracellular acidification. (C) 2012 Elsevier Inc. All rights reserved. (Less)

The essential anion exchanger (AE) involved in biliary bicarbonate secretion is AE2/SLC4A2, a membrane protein which has also been recognized to be relevant for the regulation of the intracellular pH (pHi) in several cell types. Previously, we reported that the expression of AE2 mRNA is diminished in liver biopsies and peripheral blood mononuclear cells from patients with primary biliary cirrhosis (PBC). Immunohistochemical studies indicated that the expression of the AE2 protein is decreased in the bile ducts and hepatocytes in PBC livers. Moreover, we found that bile duct cells isolated from PBC patients and cultured for a few passages exhibit defective Na+-independent Cl–/HCO3– exchange. Interestingly, positron emission tomography studies have shown that PBC patients, even at early stages of the disease, fail to secrete bicarbonate to bile in response to secretin, a defect that can be partially reversed after several months of treatment with ursodeoxycholic acid. Altogether, these findings sustain our hypothesis that dysfunctions related to AE2 might have a role in the pathogenesis of PBC. Inadequate AE2 function in lymphocytes may disturb pHi regulation in these cells and alter immune homeostasis leading to autoimmunity. On the other hand, reduced AE2 in cholangiocytes could cause cholestasis and oxidative stress of bile duct cells. Cholangiocyte changes, together with altered immune homeostasis, could favor the development of antimitochondrial antibodies and the autoimmune attack on biliary ducts. Our recent findings that Ae2a,b-deficient mice indeed display most of these features strongly support the notion that AE2 abnormalities may be involved in the pathogenesis of PBC.

Bicarbonate secretion from cholangiocytes is required for appropriate adjustment of primary canalicular bile along the biliary tract. In human and rat cholangiocytes, bicarbonate secretion is mediated by anion exchanger (AE) 2, an electroneutral Na(+)-independent Cl(-)/HCO(3) (-) AE also involved in intracellular pH (pH(i)) regulation. In Ae2(a,b)-deficient mice, pH(i) is increased in lymphocytes and fibroblasts, whereas it is surprisingly normal in cholangiocytes. Here, we analyze the mechanisms for HCO(3) (-) secretion in cultured Ae2(a,b) (+/+) and Ae2(a,b) (-/-) mouse cholangiocytes by microfluorimetric measurement of pH(i) changes upon established perfusion maneuvers. Cl(-) withdrawal by isethionate-based perfusions showed that Ae2(a,b) (+/+) but not Ae2(a,b) (-/-) mouse cholangiocytes can display Cl(-)/HCO(3) (-) exchange, which is therefore entirely mediated by Ae2. Nevertheless, simultaneous withdrawal of Cl(-) and Na(+) revealed that mouse cholangiocytes possess an additional transport activity for HCO(3) (-) secretion not observed in control rat cholangiocytes. Propionate-based maneuvers indicated that this supplemental Na(+)-driven HCO(3) (-)-secreting activity is Cl(-)-independent, consistent with a Na(+)-HCO(3) (-) cotransport (NBC). NBC activity is greater in Ae2(a,b) (-/-) than Ae2(a,b) (+/+) mouse cholangiocytes, and membrane-depolarization experiments showed that it is electrogenic. Consistent with the potential role of Slc4a4/Nbc1 as the involved transporter, Ae2(a,b) (-/-) mouse cholangiocytes exhibit up-regulated expression of this electrogenic NBC carrier. Whereas Ae2-mediated Cl(-)/HCO(3) (-) exchange in Ae2(a,b) (+/+) mouse cholangiocytes is stimulated by cyclic adenosine monophosphate (cAMP) and acetylcholine, the NBC activity is down-regulated by cAMP and adenosine triphosphate (ATP) in Ae2(a,b) (-/-) mouse cholangiocytes. Polarized Ae2(a,b) (-/-) mouse cholangiocytes placed in Ussing chambers show decreased (but not abolished) cAMP-dependent Cl(-) current and increased ATP-dependent/Ca(2+)-activated Cl(-) secretion, which run in parallel with decreased cystic fibrosis transmembrane conductance regulator messenger RNA expression and increased intracellular Ca(2+) levels. Conclusion: Bicarbonate secretion in mouse cholangiocytes involves two differentially regulated activities: Ae2-mediated Cl(-)/HCO(3) (-) exchange and Na(+)-HCO(3) (-) cotransport.

To explore the functions of the anion exchanger 2 (Ae2) in the development of bones and teeth we examined the distribution of Ae2 in cells involved in the formation of teeth and surrounding bone in young hamsters, mice and rats. In all three species strongest immunostaining for Ae2 was obtained in basolateral membranes of maturation ameloblasts and in osteoclasts resorbing bone. In hamsters a weaker staining was also seen in the Golgi apparatus of secretory ameloblasts, young osteoblasts and osteocytes, odontoblasts and fibroblasts of the forming periodontal ligament. In adult Ae2(a,b)(-/-) mice, in which Ae2-targeted disruption precluded the expression of Ae2a, Ae2b1 and Ae2b2 isoforms, the immunostaining for Ae2 in ameloblasts and osteoclasts was totally abolished. The enamel formation was abnormal but teeth erupted, osteoclasts in jaw bone were functional and structure of dentin and bone was normal. In another mouse model, Ae2(-/-) mice in which the expression of all five Ae2 isoforms was disrupted, teeth failed to erupt and the alveolar bone proved poorly formed with giant but apparently functional osteoclasts. Our data indicate that basolaterally located Ae2a, Ae2b1 or Ae2b2 (or a combination of these) is present in maturation ameloblasts critical for the cells' normal functioning. Although isoforms of Ae2 were also present in basolateral membranes of osteoclasts, they proved to be not critical to osteoclast resorption of orofacial bone. Poorly formed bone and the failure of teeth to erupt seen in the Ae2(-/-) mice with gene disruption affecting all isoforms may result from secondary (systemic) changes that are different from Ae2(a,b)(-/-) mice. J. Exp. Zool. (Mol. Dev. Evol.) 312B:375-387, 2009. (c) 2009 Wiley-Liss, Inc

Background/Objectives In vitro studies have shown that dengue virus (DENV) can thwart the actions of interferon (IFN)-α/β and prevent the development of an antiviral state in infected cells. Clinical studies looking at gene expression in patients with severe dengue show a reduced expression of interferon stimulated genes compared to patients with dengue fever. Interestingly, there are conflicting reports as to the ability of DENV or other flaviviruses to inhibit IFN-α/β signaling. Methodology/Principal Findings In order to determine the relative inhibition of IFN-α/β signaling by DENVs, a method combining flow cytometry and a four-parameter logistic regression model was established. A representative isolate from DENV-1, -3 and -4 and seventeen representative isolates encompassing all DENV-2 genotypes were evaluated. All of the DENVs evaluated in this study were capable of inhibiting IFN-α/β signaling. Most of the strains were able to inhibit IFN-α/β to a degree similar to DENV strain 16681; however, DENV-2 sylvatic strains demonstrated an increased inhibition of phosphorylated signal transducer and activator of transcription (pSTAT1). Surprisingly, we were unable to observe inhibition of pSTAT1 by DENV-2 sylvatic strains or the Asian strain 16681 in non-human primate (NHP) cell lines. Analysis in primary Rhesus macaque dendritic cells suggests that DENVs are capable of inhibiting IFN signaling in these cells. However, contrary to human dendritic cells, production of IFN-α was detected in the supernatant of DENV-infected Rhesus macaque dendritic cells. Conclusions The ability of DENVs to inhibit IFN-α/β signaling is conserved. Although some variation in the inhibition was observed, the moderate differences may be difficult to correlate with clinical outcomes. DENVs were unable to inhibit pSTAT1 in NHP cell lines, but their ability to inhibit pSTAT1 in primary Rhesus macaque dendritic cells suggests that this may be a cell specific phenomena or due to the transformed nature of the cell lines., Author Summary Dengue is a viral illness acquired through the bite of an infected mosquito. This flu-like illness, which in rare instances can be fatal, threatens more than half of the world’s population. Both in vitro and clinical studies looking at how the virus operates have consistently found that the interferon response is modulated by the virus during infection. We looked at the ability of dengue virus (DENV) strains to inhibit phosphorylated signal transducer and activator of transcription (pSTAT1) after IFN-β stimulation and observed that contrary to earlier published reports; all DENVs are capable of inhibiting IFN-α/β signaling. Strains from the DENV-2 sylvatic genotype, which mainly infect non-human primates (NHP), displayed an increased ability to inhibit pSTAT1 compared to the Asian strain 16681. To our surprise, DENVs were only capable of inhibiting pSTAT1 in human cell lines, but not in NHP cell lines. Inhibition of pSTAT1 is observed in both human and NHP primary dendritic cells. These results have important implications in the use of NHP cell lines for studies of IFN-α/β inhibition by DENV in vitro and may be a relevant consideration when using NHPs for DENV pre-clinical studies.

In parietal cells, basolateral Ae2 Cl(-)/HCO(3)(-) exchanger (Slc4a2) appears to compensate for luminal H(+) pumping while providing Cl(-) for apical secretion. In mouse and rat, mRNA variants Ae2a, Ae2b1, Ae2b2, and Ae2c2 are all found in most tissues (although the latter at very low levels), whereas Ae2c1 is restricted to the stomach. We studied the acid secretory function of gastric mucosa in mice with targeted disruption of Ae2a, Ae2b1, and Ae2b2 (but not Ae2c) isoforms. In the oxyntic mucosa of Ae2(a,b)(-/-) mice, total Ae2 protein was nearly undetectable, indicating low gastric expression of the Ae2c isoforms. In Ae2(a,b)(-/-) mice basal acid secretion was normal, whereas carbachol/histamine-stimulated acid secretion was impaired by 70%. These animals showed increased serum gastrin levels and hyperplasia of G cells. Immunohistochemistry and electron microscopy revealed baseline activation of parietal cells with fusion of intracellular H(+)/K(+)-ATPase-containing vesicles with the apical membrane and degenerative changes (but not substantial apoptosis) in a subpopulation of these cells. Increased expression of proliferating cell nuclear antigen in the oxyntic glands suggested enhanced Ae2(a,b)(-/-) parietal cell turnover. These data reveal a critical role of Ae2a-Ae2b1-Ae2b2 isoforms in stimulated gastric acid secretion whereas residual Ae2c isoforms could account to a limited extent for basal acid secretion.

Na + -independent anion exchangers (AE) mediate electroneutral exchange of Cl - for \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{HCO}}_{3}^{-}\end{equation*}\end{document} ions across cell membranes, being involved in intracellular pH and cell volume regulation and in transepithelial hydroionic fluxes. Bicarbonate activation of adenylyl cyclase is known to be necessary for sperm motility and sperm capacitation, and a few studies have suggested a possible role of AE carriers in reproduction. Among the four AE genes identified in mammals thus far, only Ae2 ( Slc4a2 ) has been determined to be expressed in the male reproductive system, especially in developing spermatozoa and in epididymal epithelium. Most AE genes drive alternative transcription, which in mouse Ae2 results in several Ae2 isoforms. Here, we generated mice carrying a targeted disruption of Ae2 that prevents the expression of the three AE2 isoforms (Ae2a, Ae2b 1 , and Ae2b 2 ) normally found in mouse testes. Male Ae2 -/- mice (but not female Ae2 -/- mice) are infertile. Histopathological analysis of Ae2 -/- testes shows an interruption of spermiogenesis, with only a few late spermatids and a complete absence of spermatozoa in the seminiferous tubules. The number of apoptotic bodies is increased in the seminiferous tubules and in the epididymis, which also shows squamous metaplasia of the epididymal epithelium. Our findings reveal an essential role of Ae2 in mouse spermiogenesis and stress the recently postulated involvement of bicarbonate in germ-cell differentiation through the bicarbonate-sensitive soluble-adenylyl-cyclase pathway.

We describe a 47-year-old patient who developed cholelithiasis in adolescence, followed by recurrent intrahepatic cholestasis of pregnancy, and finally biliary cirrhosis in adulthood. In our patient, the consecutive presentation of the 3 mentioned disorders raised the suspicion of a defect of MDR3, the canalicular protein involved in the transport of phospatidylcholine to bile. Mutational analysis in our patient showed a heterozygous missense mutation of the MDR3 gene that has not been described previously, which occurs in exon 14 at codon 535, and results in the substitution of glycine for aspartic acid. Further analysis of 7 members of the family showed the same mutation in her daughter who, on follow-up, developed cholestasis of pregnancy and persisting high serum levels of γ-glutamyl transpeptidase and alkaline phosphatase after delivery. Although biliary cirrhosis associated with MDR3 deficiency typically appears before the age of 25 years, in our case, the relatively mild MDR3 dysfunction allowed for a slower progression of the disease with established, well-advanced cirrhosis in the fifth decade of life. The present case, which accumulates the 3 clinical disorders assocaited with MDR3 deficiency, shows that this condition should be suspected not only in children or young people with high γ-glutamyl transpeptidase cholestasis but also in middle-aged or older patients with chronic idiopathic cholestasis, especially when there is a previous history of cholestasis of pregnancy or juvenile cholelithiasis.

Ameloblasts express transmembrane proteins for transport of mineral ions and regulation of pH in the enamel space. Two major transporters recently identified in ameloblasts are the Na+K+-dependent calcium transporter NCKX4 and the Na+-dependent HPO42– (Pi) cotransporter NaPi-2b. To regulate pH, ameloblasts express anion exchanger 2 (Ae2a,b), chloride channel Cftr, and amelogenins that can bind protons. Exposure to fluoride or null mutation of Cftr, Ae2a,b, or Amelx each results in formation of hypomineralized enamel. We hypothesized that enamel hypomineralization associated with disturbed pH regulation results from reduced ion transport by NCKX4 and NaPi-2b. This was tested by correlation analyses among the levels of Ca, Pi, Cl, Na, and K in forming enamel of mice with null mutation of Cftr, Ae2a,b, and Amelx, according to quantitative x-ray electron probe microanalysis. Immunohistochemistry, polymerase chain reaction analysis, and Western blotting confirmed the presence of apical NaPi-2b and Nckx4 in maturation-stage ameloblasts. In wild-type mice, K levels in enamel were negatively correlated with Ca and Cl but less negatively or even positively in fluorotic enamel. Na did not correlate with P or Ca in enamel of wild-type mice but showed strong positive correlation in fluorotic and nonfluorotic Ae2a,b- and Cftr-null enamel. In hypomineralizing enamel of all models tested, 1) Cl− was strongly reduced; 2) K+ and Na+ accumulated (Na+ not in Amelx-null enamel); and 3) modulation was delayed or blocked. These results suggest that a Na+K+-dependent calcium transporter (likely NCKX4) and a Na+-dependent Pi transporter (potentially NaPi-2b) located in ruffle-ended ameloblasts operate in a coordinated way with the pH-regulating machinery to transport Ca2+, Pi, and bicarbonate into maturation-stage enamel. Acidification and/or associated physicochemical/electrochemical changes in ion levels in enamel fluid near the apical ameloblast membrane may reduce the transport activity of mineral transporters, which results in hypomineralization.

9 páginas Abstract Objective Human leucocyte antigen (HLA) B27 and HLA-B15 are associated with spondyloarthritis (SpA). Recent Assessment of SpondyloArthritis international Society (ASAS) criteria emphasise a distinction between SpA with axial and peripheral patterns. We analysed whether HLA-A, HLA-B and HLA-DRB1 alleles could associate with these patterns. Methods We studied 100 healthy individuals and 178 patients with SpA according to European Spondyloarthropathy Study Group (ESSG) criteria. Patients were then classified according to ASAS criteria, the axial spondyloarthritis pattern (axSpA) being defined by ascertained sacroiliitis and the peripheral pattern (pSpA) by enthesitis and/or arthritis in extremities. A combined ax/p pattern was also considered. Results Only HLA-B27 and HLA-B15 alleles were associated with SpA. ASAS criteria for axSpA were met in 152 patients (12 with isolated axSpA and 140 with a combined ax/p patterns). When the ASAS peripheral criteria were applied, 161 patients met these criteria (13 with isolated pSpA and 148 with a combined ax/p pattern). HLA-B27 was found in 83% of patients with axSpA and 43% of ax/pSpA patients according to axASAS. HLA-B27 occurred in 7% controls but not in any patient with isolated pSpA. HLA-B15 was encountered in 31% of patients with isolated pSpA and 20% of ax/pSpA patients according to pASAS criteria. Moreover, 2 healthy controls, but none of our patients with isolated axSpA were positive for HLA-B15. Conclusions Our data suggest that the presence of HLA-B15 favours the development of isolated/combined peripheral rather than isolated axSpA, while HLA-B27 promotes an isolated/combined axial disease and excludes a peripheral pattern. HLA-B15 should be considered in addition to HLA-B27 when diagnosing patients with SpA according to ASAS criteria.

Formation of crystals in the enamel space releases protons that need to be buffered to sustain mineral accretion. We hypothesized that apical cystic fibrosis transmembrane conductance regulator (CFTR) in maturation ameloblasts transduces chloride into forming enamel as a critical step to secrete bicarbonates. We tested this by determining the calcium, chloride, and fluoride levels in developing enamel of Cftr-null mice by quantitative electron probe microanalysis. Maturation-stage enamel from Cftr-null mice contained less chloride and calcium than did wild-type enamel, was more acidic when stained with pH dyes ex vivo, and formed no fluorescent modulation bands after in vivo injection of the mice with calcein. To acidify the enamel further we exposed Cftr-null mice to fluoride in drinking water to stimulate proton release during formation of hypermineralized lines. In Cftr-deficient mice, fluoride further lowered enamel calcium without further reducing chloride levels. The data support the view that apical CFTR in maturation ameloblasts tranduces chloride into developing enamel as part of the machinery to buffer protons released during mineral accretion.

Primary biliary cirrhosis (PBC) is a disorder of unknown origin with autoimmune features. Recently, impaired biliary secretion of bicarbonate has been shown in patients with PBC. Here we have investigated whether bile duct epithelial cells isolated from PBC patients exhibit defects in transepithelial bicarbonate transport by analyzing the activities of 2 ion exchangers, Cl(-)/HCO3(-) anion exchanger 2 (AE2) and Na(+)/H(+) exchanger (NHE) in isolated cholangiocytes. AE2 and NHE activities were studied in basal conditions and after stimulation with cyclic adenosine monophosphate (cAMP) and extracellular adenosine triphosphate (ATP), respectively. Cholangiocytes were grown from needle liver biopsies from 12 PBC patients, 8 normal controls, and 9 patients with other liver diseases. Also, intrahepatic cholangiocytes were cultured after immunomagnetic isolation from normal liver tissue (n = 6), and from recipients undergoing liver transplantation for end-stage PBC (n = 9) and other forms of liver disease (n = 8). In needle-biopsy cholangiocytes, basal AE2 activity was significantly decreased in PBC as compared with normal livers and disease controls. In addition, we observed that though cAMP increased AE2 activity in cholangiocytes from both normal and non-PBC livers, this effect was absent in PBC cholangiocytes. Similarly, though in cholangiocytes from normal and disease control livers extracellular ATP induced a marked enhancement of NHE activity, cholangiocytes from PBC patients failed to respond to purinergic stimulation. In conclusion, our findings provide functional evidence that PBC cholangiocytes exhibit a widespread failure in the regulation of carriers involved in transepithelial H(+)/HCO3(-) transport, thus, providing a molecular basis for the impaired bicarbonate secretion in this cholestatic syndrome.

Complementary DNA expression of mature human serum albumin was engineered into potato plants under the transcriptional control of patatin B33 promoter and potato proteinase inhibitor II terminator. Protein secretion was achieved by using the signal sequence from potato proteinase inhibitor II. Recombinant albumin accumulated up to 0.2% of total soluble tuber protein in single transformant lines, regardless of the potato cultivar used. Electrophoretic mobility and N-terminal amino acid sequence analysis of partially purified recombinant albumin confirmed proper processing of an immune responsive recombinant albumin, and revealed that the proteinase inhibitor II signal sequence was correctly removed. No further optimisation of these yields was obtained by HSA expression in patatin antisense plants (line Pas58). Subcellular localisation showed that recombinant protein was successfully targeted to the apoplast. Potato tubers may be used, by applying this technology, to produce other heterologous proteins of interest in the biopharmaceutical industry.

During the formation of dental enamel, maturation-stage ameloblasts express ion-transporting transmembrane proteins. The SLC4 family of ion-transporters regulates intra- and extracellular pH in eukaryotic cells by cotransporting HCO3 − with Na+. Mutation in SLC4A4 (coding for the sodium-bicarbonate cotransporter NBCe1) induces developmental defects in human and murine enamel. We have hypothesized that NBCe1 in dental epithelium is engaged in neutralizing protons released during crystal formation in the enamel space. We immunolocalized NBCe1 protein in wild-type dental epithelium and examined the effect of the NBCe1-null mutation on enamel formation in mice. Ameloblasts expressed gene transcripts for NBCe1 isoforms B/D/C/E. In wild-type mice, weak to moderate immunostaining for NBCe1 with antibodies that recognized isoforms A/B/D/E and isoform C was seen in ameloblasts at the secretory stage, with no or low staining in the early maturation stage but moderate to high staining in the late maturation stage. The papillary layer showed the opposite pattern being immunostained prominently at the early maturation stage but with gradually less staining at the mid- and late maturation stages. In NBCe1 −/− mice, the ameloblasts were disorganized, the enamel being thin and severely hypomineralized. Enamel organs of CFTR −/− and AE2a,b −/− mice (CFTR and AE2 are believed to be pH regulators in ameloblasts) contained higher levels of NBCe1 protein than wild-type mice. Thus, the expression of NBCe1 in ameloblasts and the papillary layer cell depends on the developmental stage and possibly responds to pH changes.

Leukotriene A4 hydrolase is a bifunctional Zn2+-containing enzyme catalysing the formation of the potent chemotaxin leukotriene B4. From an analysis of three mutants of Glu-296 we have found that this catalytic residue is critical for the binding of bestatin, a classical aminopeptidase inhibitor. For bestatin, but not for three other tight-binding inhibitors, the IC50 values for inhibition of the epoxide hydrolase activity decreased in the mutants to 0.7-0.003% of the control. Hence Glu-296 is an important structural determinant for binding of bestatin to leukotriene A4 hydrolase; this conclusion might also apply to other members of the M1 family of metallopeptidases.

Previously, we isolated the human AE2 ( SLC4A2 ) gene, a member of the sodium-independent anion exchanger family. Rat ortholog of this gene was reported to drive alternative transcription yielding N-terminal variants of the AE2a message. We thus analyzed the human AE2 gene in this regard. Using HepG2 cells, two alternative first exons, each splicing to exon 3 in alternative transcripts, were found to be transcribed from overlapping sequences of intron 2. Exon 1b 1 corresponds to the rat variant “b” and encodes three initial residues (MTQ) in AE2b 1 isoform that replace the first 17 amino acids of AE2a protein, while the novel exon 1b 2 encodes eight initial residues (MDFLLRPQ) in AE2b 2 isoform. The relative abundance of AE2b 1 and AE2b 2 mRNAs was about 10% of AE2a mRNA each. Alternate promoter sequences have multiple potential binding motifs for liver-enriched factors, and dual-luciferase assays indicated that they possess the ability for driving transcription in transiently transfected HepG2 cells. Tissue survey showed that expression of human AE2b 1 and AE2b 2 transcripts is restricted to liver and kidney, while AE2a mRNA was encountered in all examined tissues. Our findings reveal a characteristic tissue-specific expression of two N-terminal variants of human AE2 from overlapping sequences within intron 2, one of which is a novel isoform.

Background & Aims: Positron emission tomography (PET) allows imaging and quantitative analysis of organ functions in basal and stimulated conditions. We have applied this method to the study of biliary bicarbonate secretion in humans. Methods: PET was performed in 5 healthy subjects and 13 patients with hepatobiliary disorders after intravenous injection of NaH 11 CO 3 . In each case the study was performed in basal conditions and after secretin stimulation. Positron emission from the hepatic area was scanned, and normalized uptake values for parenchymal and hilar regions were estimated. Results: In healthy individuals, the injection of NaH 11 CO 3 resulted in a peak uptake of the label in parenchymal and hilar regions 2–3 minutes after the injection. In both normal and cirrhotic subjects, secretin administration increased bicarbonate uptake in the parenchymal region, followed by accumulation of the label in the perihilar area. Normal basal uptake with absent response to secretin was registered in extrahepatic biliary obstruction and in untreated primary biliary cirrhosis (PBC). The secretin response was present in patients with PBC undergoing treatment with ursodeoxycholic acid. Conclusions: PET allows investigation of biliary bicarbonate secretion in humans. An impaired response to secretin was observed in cholestatic conditions. Preliminary data suggest that ursodeoxycholic acid might improve the response to secretin in PBC. GASTROENTEROLOGY 1999;117:167-172

The Cl(-)/HCO(-) 3anion exchanger 2 (AE2) is known to be involved in intracellular pH (pHi) regulation and transepithelial acid-base transport. Early studies showed that AE2 gene expression is reduced in liver biopsies and blood mononuclear cells from patients with primary biliary cirrhosis (PBC), a disease characterized by chronic non-suppurative cholangitis associated with antimitochondrial antibodies (AMA) and other autoimmune phenomena. Microfluorimetric analysis of the Cl(-)/HCO(-) 3 anion exchange (AE) in isolated cholangiocytes showed that the cAMP-stimulated AE activity is diminished in PBC compared to both healthy and diseased controls. More recently, it was found that miR-506 is upregulated in cholangiocytes of PBC patients and that AE2 may be a target of miR-506. Additional evidence for a pathogenic role of AE2 dysregulation in PBC was obtained with Ae2 (-/-) a,b mice, which develop biochemical, histological, and immunologic alterations that resemble PBC (including development of serum AMA). Analysis of HCO(-) 3 transport systems and pHi regulation in cholangiocytes from normal and Ae2 (-/-) a,b mice confirmed that AE2 is the transporter responsible for the Cl(-)/HCO(-) 3exchange in these cells. On the other hand, both Ae2 (+/+) a,b and Ae2 (-/-) a,b mouse cholangiocytes exhibited a Cl(-)-independent bicarbonate transport system, essentially a Na(+)-bicarbonate cotransport (NBC) system, which could contribute to pHi regulation in the absence of AE2.

Background & Aims Bile salts inhibit their own production by inducing the nuclear receptor small heterodimer partner (SHP) (encoded by NR0B2 ), which contributes to repression of the gene encoding cholesterol 7α-hydroxylase ( CYP7A1 ), a key enzyme for the control of bile salt synthesis. On the other hand, bile salts stimulate hepatic synthesis of nitric oxide. We investigated the role of nitric oxide signaling in the control of CYP7A1 expression and the involvement in this process of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which participates in intracellular propagation of nitric oxide signals. Methods We studied the effects of inhibitors of nitric oxide synthesis (L-NG-nitroarginine methyl ester [L-NAME]) or protein nitrosylation (via dithiothreitol) on bile salt homeostasis in male Wistar rats placed on a cholate-rich diet for 5 days and in cultured primary hepatocytes. S-nitrosylation of GAPDH was assessed using a biotin-switch assay. Interacions of SHP with other proteins and with the Cyp7a1 promoter sequence were studied using immunoprecipitation and chromatin immunoprecipitation (ChIP) assays. We reduced the GAPDH levels in H35 cells with small interfering RNAs. GAPDH nitrosylation was assessed in normal and cholestatic rat and human livers. Results Rats placed on cholate-rich diets and given L-NAME had increased intrahepatic and biliary levels of bile salts, and deficiency in repression of CYP7A1 (at the messenger RNA and protein levels) in liver tissue, despite preserved induction of SHP. In cultured hepatocytes, L-NAME or dithiothreitol blocked cholate-induced down-regulation of CYP7A1 without impairing SHP up-regulation. In hepatocytes, cholate promoted S-nitrosylation of GAPDH and its translocation to the nucleus, accompanied by S-nitrosylation of histone deacetylase 2 (HDAC2) and Sirtuin 1 (SIRT1), deacetylases that participate, respectively, in the formation of Cyp7a1 and Shp repressor complexes. Knockdown of GAPDH prevented repression of CYP7A1 by cholate, and blocking nuclear transport of nitrosylated GAPDH reduced cholate-induced nitrosylation of HDAC2 and SIRT1; this effect was accompanied by abrogation of Cyp7a1 repression. Cholate induced binding of SHP to HDAC2 and its recruitment to the Cyp7a1 promoter; these processes were inhibited by blocking nitric oxide synthesis. Levels of nitrosylated GAPDH and nitrosylated HDAC2 were increased in cholestatic human and rat livers reflecting increased concentrations of bile salts in these conditions. Conclusions In rat liver, excess levels of bile salts activate a GAPDH-mediated transnitrosylation cascade that provides feedback inhibition of bile salt synthesis.

Mitogenic stimulation of lymphocytes involves alkalinization of intracellular pH (pHi ). Subsequent pHi regulation may involve HCO3 (-) extrusion through Cl(-) /HCO3 (-) exchangers and/or Na(+) -HCO3 (-) co-transporters with acid-loading capability. Abnormalities in these mechanisms could result in immune dysfunctions, as suggested by the CD8(+) T-cell expansion encountered in mice lacking Ae2 (a widely expressed acid loader with electroneutral and Na(+) -independent Cl(-) /HCO3 (-) anion-exchange activity). Here we report that CD8(+) T cells but not CD4(+) T cells or other lymphocyte populations, are crucially dependent on Ae2 for pHi regulation. While total lymphocytes (including isolated CD4(+) T cells) exhibit Ae1 expression and Na(+) -HCO3 (-) co-transport with acidifying potential, CD8(+) T cells lack these acid-loading mechanisms. In Ae2-KO mice, CD4(+) but not CD8(+) T cells upregulate these potential Ae2 surrogates. As a consequence, Ae2-KO CD8(+) T cells exhibit alkalinized pHi , and dramatically increase their pHi upon CD3 stimulation. Moreover, stimulated Ae2-deficient CD8(+) T cells show enhanced intracellular production of IL-2 and membrane expression of its receptor IL-2Rα, together with increased cell proliferation and activation. These findings demonstrate that CD8(+) T cells are critically dependent on Ae2 for pHi homeostasis and tuning of cell proliferation and activation. Ae2 thus constitutes a novel target to modulate CD8(+) T-cell responses.

Objective Cholestasis is associated with increased liver injury and morbidity after partial hepatectomy (PH), yet bile acids (BAs) are emerging as important mediators of liver regeneration. Fibroblast growth factor 15 (Fgf15, human FGF19) is a BA-induced ileum-derived enterokine that governs BA metabolism. We evaluated the relevance of Fgf15 in the preservation of BA homeostasis after PH and its potential role in the regenerative process. Design Liver regeneration after PH was studied in Fgf15 −/− and Fgf15 +/+ mice. The effects of the BA sequestrant cholestyramine and adenovirally delivered Fgf15 were examined in this model. The role of Fgf15 in BA-induced liver growth was tested in Fgf15 −/− mice upon cholic acid (CA) feeding. The direct mitogenic effect of Fgf15 was evaluated in cultured mouse hepatocytes and cholangiocytes. Results Fgf15 −/− mice showed marked liver injury and mortality after PH accompanied by persistently elevated intrahepatic BA levels. Cholestyramine feeding and adenovirally delivered Fgf15 reduced BA levels and significantly prevented this lethal outcome. Fgf15 also reduced mortality after extensive hepatectomy in Fgf15 +/+ animals. Liver growth elicited by CA feeding was significantly diminished in Fgf15 −/− mice. Proliferation of hepatocytes and cholangiocytes was also noticeably reduced in CA-fed Fgf15 −/− mice. Fgf15 induced intracellular signalling and proliferation of cultured hepatocytes and cholangiocytes. Conclusions Fgf15 is necessary to maintain BA homeostasis and prevent liver injury during liver regeneration. Moreover, Fgf15 is an essential mediator of the liver growth-promoting effects of BA. Preoperative administration of this enterokine to patients undergoing liver resection might be useful to reduce damage and foster regeneration.

Dengue is a disease caused by infection with one of the four dengue virus serotypes (DENV-1, -2, -3, and -4). The virus is transmitted to humans by Aedes sp. mosquitoes. This enveloped virus contains a positive single-stranded RNA genome. Clinical manifestations of dengue can have a wide range of outcomes varying from a mild febrile illness to a life-threatening condition. New techniques have largely replaced the use of DENV isolation in disease diagnosis. However, virus isolation still serves as the gold standard for detection and serotyping of DENV and is common practice in research and reference laboratories where clinical isolates of the virus are characterized and sequenced, or used for a variety of research experiments. Isolation of DENV from clinical samples can be achieved in mammalian and mosquito cells or by inoculation of mosquitoes. The experimental methods presented here describe the most common procedures used for the isolation, serotyping, propagation, and quantification of DENV.

INTRODUÇÃO: Biomarcadores séricos, tradicionalmente associados à atividade inflamatória e mau prognóstico em doenças reumáticas, não apresentam a mesma relação nas espondiloartrites. OBJETIVO: Estabelecer uma associação entre os níveis séricos de biomarcadores com a presença de fatores associados com a atividade clínica e com o mau prognóstico nas espondiloartropatias. MÉTODOS: Sessenta e dois pacientes (13 com artrite reativa, 19 com espondilite anquilosante e 30 com espondiloartropatia indiferenciada) foram comparados a 46 controles sadios. Foram realizadas avaliações clínicas, radiológicas e laboratoriais. Os resultados foram analisados de acordo com a presença de uveíte, entesite, lombalgia inflamatória, artrite, HLA-B27 e comprometimento das articulações sacroilíacas. Os biomarcadores utilizados foram: VHS, PCRus, SAA, LBP, FSC-M e MMP-3, além da dosagem dos níveis séricos das citocinas: IL-17, IL-6, IL-1α , TNF-α , IFN-γ, e IL-23. RESULTADOS: Quarenta e três (69,4%) pacientes eram homens. A média de idades foi de 31,9 ± 9,9 anos, enquanto a idade média para o aparecimento dos sintomas foi de 26,9 ± 7,3 anos. HLA-B27 foi positivo em 26 (41,9%) dos pacientes, lombalgia inflamatória esteve presente em 42 (67,7%), artrite em 44 (71,0%) e entesite em 34 (54,8%) pacientes. Os níveis séricos de IL-17, IL-23, TNF-α , IL-6, IL-1α e PCRus foram mais elevados em pacientes com espondiloartropatia em comparação com os controles. Os valores de PCRus (P = 0,04), IL-6 (P = 0,003), IL-1α (P = 0,03), e LBP (P = 0,03) se associaram de maneira significativa com presença de HLA-B27, dor lombar inflamatória e artrite. CONCLUSÃO: O aumento dos níveis séricos de PCRus, IL-6, IL-1α e LBP apresentaram associação com fatores relacionados a atividade clínica e mau prognóstico em pacientes com espondiloartrites. BACKGROUND: Serum biomarkers traditionally associated with inflammatory activity and a poor prognosis in rheumatic diseases do not show the same relationship in spondyloarthritis. OBJECTIVE: To establish the association between serum levels of potential biomarkers with the presence of factors related to clinical activity and poor prognosis in spondyloarthritis. METHODS: Sixty-two patients were included: 13 with reactive arthritis, 19 with ankylosing spondylitis, and 30 with undifferentiated spondyloarthritis. The results were compared with those from 46 healthy controls. Clinical, radiological, and laboratory characteristics were assessed. The results were analyzed based on the presence of uveitis, enthesitis, inflammatory back pain, arthritis, HLA-B27 and sacroiliac involvement. The analyzed biomarkers included ESR, US-CRP, SAA, LBP, FSC-M, and MMP-3; and cytokine serum levels measured were: IL-17, IL-6, IL-1α , TNF-α , IFN-γ, and IL-23. RESULTS: Forty-three (69.4%) patients were male. The average age was 31.9 ± 9.9 years and the age at the onset of symptoms was 26.9 ± 7.3 years. HLA-B27 was positive in 26 (41.9%) patients, inflammatory back pain in 42 (67.7%), arthritis in 44 (71.0%), and enthesitis in 34 (54.8%). IL-17, IL-23, TNF-α , IL-6, IL-1α , and US-CRP levels were significantly higher in patients with SpA when compared to controls. US-CRP (P = 0.04), IL-6 (P = 0.003), IL-1α (P = 0.03), and LBP (P = 0.03) levels were associated with presence of HLA-B27, inflammatory back pain, and arthritis. CONCLUSION: An increase in serum levels of US-CRP, IL-6, IL-1α , and LBP was correlated with factors associated with clinical activity and poor prognosis in spondyloarthritis.

The efficacy of omalizumab in severe asthma has been widely demonstrated. The main objective of this study was to evaluate the efficacy and tolerability of omalizumab in a real-life setting in Spain, particularly in those patients with immunoglobulin E (IgE) levels out of range.Totally 266 uncontrolled severe asthma patients receiving high-dose inhaled corticosteroids (ICSs) plus long-acting β2-agonist (LABA) were recruited. Main efficacy outcomes were asthma exacerbation rate (AER), asthma control test (ACT), and global evaluation of treatment effectiveness (GETE).AER was reduced from 3.6 (3.6) in previous year to 0.67 (1.2) at 4 months (p.05) and to 1.04 (1.8) at 2 years (p.05). ACT increased significantly from 14.3 (4.7) at baseline to 18.4 (4.4) at 4 months (p.05) and to 20.3 (4.0) (p.05) at 2 years. After 4 months, 74.6% of patients had reached a good or excellent rate on the GETE scale (p.05). This rate continued increasing up to 81.6% at 2 years. These efficacy results were similar for patients with "off-label" IgE700 IU/ml. At follow-up, maintenance treatment with oral steroids was discontinued in a considerable number of patients: from 89 to 19 (p.05). Omalizumab was discontinued because of lack of efficacy only in 28/266 (10.5%) patients. Overall, 30 patients (11.4%) reported adverse events. Severe adverse events were not observed.This real-life study confirms that omalizumab is very efficacious and very well tolerated in patients with uncontrolled severe asthma. Results did not vary in the subgroup of patients with IgE levels700 IU/ml.