Search

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. [ABSTRACT FROM AUTHOR]

Unregulated logging in tropical forests promotes invasion of secondary vegetation that compete with species of medium and slow growth, limiting their natural regeneration. Many reforestation efforts fail for this situation endangering the ecosystem balance by differences in density and distribution of species. In order to promote a greater growth in shorter time of the timber species (Tabebuia donnell-smithii Rose) primavera under nursery conditions, the effect of inoculation with Rhizophagus intraradices (Schenck et Sm.) Walker et Schuessler, Glomus spp., and Azospirillum brasilense Tarrand, Krieg et Döbereiner was evaluated. Nursery was established with seeds collected in the Soconusco, Chiapas, Mexico, in plastic bags of 5 kg with a mixture of Andosolmollic soil and river sand washed (1:1) with 2 g of inoculant at the time of transplantation. Azospirillum brasilense had a concentration of 9×106 bacteria g-1 and R. intraradices 40 spores g-1 of soil with 95 % of colonization to the root system. The experimental design was completely randomized treatments were individual, combined microorganisms, and a control with four replications. Morphological and physiological variables of yield, percentage of mycorrhizal root colonization were recorded every 28 d from the 56 d to 168 d after sowing. Plants with R. intraradices, double symbiosis A. brasilense+R. intraradices and Glomus sp. (V) showed higher increase of biomass at 112 d (p⩽0.05), whereas the highest nitrogen content was found in roots of plants inoculated with R. intraradices and in the shoot with A. brasilense. The phosporus content was higher with A. brasilense and R. intraradices on the shoot. The final significant differences in plant height were 6 to 8 cm among inoculated treatments and of 16 cm of these with respect to the control. [ABSTRACT FROM AUTHOR]

La explotación forestal sin regulación en bosques tropicales promueve la invasión de vegetación secundaria que compite con especies de mediano y lento crecimiento, limitando su regeneración natural. Muchos esfuerzos de reforestación fracasan por esta situación poniendo en riesgo el equilibrio del ecosistema por diferencias en densidad y distribución de especies. Con el fin de promover un mayor crecimiento en menor tiempo de la especie maderable primavera (Tabebuia donnell-smithii Rose) en condiciones de vivero, se evaluó el efecto de la inoculación con Rhizophagus intraradices (Schenck et Sm.) Walker et Schuessler, Glomus spp., y Azospirillum brasilense Tarrand, Krieg et Döbereiner. El vivero se estableció con semillas recolectadas en el Soconusco, Chiapas, México, en bolsas de plástico de 5 kg con una mezcla de suelo andosol-mólico y arena de río lavada (1:1) con 2 g de inoculante al momento del trasplante. Azospirillum brasilense tuvo una concentración de 9×106 bacterias g-1 y R. intraradices 40 esporas g-1 de suelo con 95 % de colonización al sistema radical. El diseño experimental fue completamente al azar y los tratamientos fueron los microorganismos individuales, combinados y un testigo, con cuatro repeticiones. Las variables morfológicas y fisiológicas del rendimiento, porcentaje de colonización radical micorrízica se registraron cada 28 d desde los 56 d hasta 168 d después de siembra. Las plantas con R. intraradices, la simbiosis doble A. brasilense+R. intraradices y Glomus sp. (V) presentaron mayor aumento de biomasa a 112 d (p⩽0.05), mientras que el mayor contenido de nitrógeno se encontró en raíces de plantas inoculadas con R. intraradices y en el vástago con A. brasilense. El contenido de fósforo fue mayor con R. intraradices y A. brasilense en el vástago. Las diferencias significativas finales en altura de planta fueron de 6 a 8 cm entre tratamientos inoculados y de 16 cm de éstos respecto al testigo. [ABSTRACT FROM AUTHOR]

The Cl-/HCO-3 anion 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-3 exchange 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. [ABSTRACT FROM AUTHOR]

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 ± 0.8 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 13.0 ± 5.5 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 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. [ABSTRACT FROM AUTHOR]

Objective. 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. Methods. 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). Results. 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' IgE > 700 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. Conclusion. 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 levels >700 IU/ml. [ABSTRACT FROM AUTHOR]

Extracellular acidification by osteoclasts is essential to bone resorption. During proton pumping, intracellular pH (pHi) is thought to be kept at a near-neutral level by chloride/bicarbonate exchange. Here we show that the Na+-independent chloride/bicarbonate anion exchanger 2 (Ae2) is relevant for this process in the osteoclasts from the long bones of Ae2a,b-/- mice (deficient in the main isoforms Ae2a, Ae2b1, and Ae2b2). Although the long bones of these mice had normal numbers of multinucleated osteoclasts, these cells lacked a ruffled border and displayed impaired bone resorption activity, resulting in an osteopetrotic pheno-type of long bones. Moreover, in vitro osteoclastogenesis assays using long-bone marrow cells from Ae2a,b-/- mice suggested a role for Ae2 in osteoclast formation, as fusion of preosteoclasts for the generation of active multinucleated osteoclasts was found to be slightly delayed. In contrast to the abnormalities observed in the long bones, the skull of Ae2a,b-/- mice showed no alterations, indicating that calvaria osteoclasts may display normal resorptive activity. Microfluorimetric analysis of osteoclasts from normal mice showed that, in addition to Ae2 activity, calvaria osteoclasts---but not long-bone osteoclasts--possess a sodium-dependent bicarbonate transporting activity. Possibly, this might compensate for the absence of Ae2 in calvaria osteoclasts of Ae2a,b-/- mice. [ABSTRACT FROM AUTHOR]

Background & Aims: Cl−/HCO3 − anion exchanger 2 (AE2) is involved in intracellular pH (pHi) regulation and transepithelial acid-base transport, including secretin-stimulated biliary bicarbonate excretion. AE2 gene expression was found to be reduced in liver biopsy specimens and blood mononuclear cells from patients with primary biliary cirrhosis (PBC), a disease characterized by chronic nonsuppurative cholangitis associated with antimitochondrial antibodies (AMA) and other autoimmune phenomena. In mice with widespread Ae2 gene disruption, we previously reported altered spermiogenesis and reduced gastric acid secretion. We now describe the hepatobiliary and immunologic changes observed in these Ae2a.b -deficient mice. Methods: In this murine model, splenocyte pHi and T-cell populations were studied by flow cytometry. CD3-stimulated cytokine secretion was estimated using cytokine arrays. AMA were evaluated by immunoblotting and proteomics. Hepatobiliary changes were assessed by immunohistopathology, flow cytometry, and serum biochemistry. Cholangiocyte gene expression was analyzed by real-time polymerase chain reaction. Results: Ae2a,b −/− mice exhibit splenomegaly, elevated pHi in splenocytes, increased production of interleukin-12p70 and interferon gamma, expanded CD8+ T-cell population, and under represented CD4+FoxP3+/regulatory T cells. Most Ae2a,b −/− mice tested positively for AMA, showing increased serum levels of immunoglobulin M and G, and liver-specific alkaline phosphatase. About one third of Ae2a,b −/− mice had extensive portal inflammation with CD8+ and CD4+ T lymphocytes surrounding damaged bile ducts. Cholangiocytes isolated from Ae2a,b −/− mice showed gene expression changes compatible with oxidative stress and increased antigen presentation. Conclusions: Ae2 deficiency alters pHi homeostasis in immunocytes and gene expression profile in cholangiocytes, leading to immunologic and hepatobiliary changes that resemble PBC. [Copyright &y& Elsevier]

Human fibroblasts in cell culture converted the epoxide intermediate leukotriene A4 into the potent chemotaxin leukotriene B4. The identity of leukotriene B4 was ascertained by its mobility in reverse-phase high performance liquid chromatography, ultraviolet spectroscopy and gas chromatography/mass spectrometry. The presence of the enzyme responsible for the conversion (i.e. leukotriene A4 hydrolase), as well as the corresponding mRNA, were demonstrated by Western and Northern blot analyses. Leukotriene-A4-hydrolase enzyme activity, protein and mRNA were all enhanced (approximately threefold) in human fibroblasts that had been transformed by simian virus 40. [ABSTRACT FROM AUTHOR]

Ralstonia solanacearum (Rs) causes the disease quarantine called bacterial wilt in potatoes. Mexico importseed from United States of America (USA) and Canada, which is a significant aspect to eventual introduction of the disease in areas with large tracts of potato. Sonora is an important region in relation to potato production. Therefore, this research was conducted, with the following objectives: a) production of antibody for Rs; b) diagnosis of Rs in foreign tuber, and in tuber for human consumption which is used for seed; c) detection of Rs in vegetative growth of potato lots in Sonora, Mexico. We analyzed seed, seedling, leaf and tuber; the detection methods used were: specific means, ELISA, antiserum produced and pathogenicity tests. The positive results have shown the presence of Rs in tubers for consumption; in foreign tuber and vegetative stages it was negative. The separate testing should not be used as a unique method of detection; the presence of Rs, represents a risk of eventual manifestation of the disease, so it is necessary to undertake activities with preventive phytosanitary control. [ABSTRACT FROM AUTHOR]

Primary biliary cirrhosis (PBC) is a cholestatic disease associated with autoimmune phenomena and alterations in both biliary bicarbonate excretion and expression of the bicarbonate carrier AE2. The bile acid ursodeoxycholic acid (UCDA) is currently used in treatment of cholestatic liver diseases and is the treatment of choice in PBC; however, a subset of PBC patients respond poorly to UDCA monotherapy. In these patients, a combination of UDCA and glucocorticoid therapy appears to be beneficial. To address the mechanism of this benefit, we analyzed the effects of UDCA and dexamethasone on AE2 gene expression in human liver cells from hepatocyte and cholangiocyte lineages. The combination of UDCA and dexamethasone, but not UDCA or dexamethasone alone, increased the expression of liver-enriched alternative mRNA isoforms AE2b1 and AE2b2 and enhanced AE2 activity. Similar effects were obtained after replacing UDCA with UDCA conjugates. In in vitro and in vivo reporter assays, we found that a UDCA/dexamethasone combination upregulated human AE2 alternate overlapping promoter sequences from which AE2b1 and AE2b2 are expressed. In chromatin immunoprecipitation assays, we demonstrated that combination UCDA/dexamethasone treatment induced p300-related interactions between HNF1 and glucocorticoid receptor on the AE2 alternate promoter. Our data provide a potential molecular explanation for the beneficial effects of the combination of UDCA and glucocorticoids in PBC patients with inadequate response to UDCA monotherapy. [ABSTRACT FROM AUTHOR]

AE2 (SLC4A2) is the member of the Na+-independent anion exchanger (AE) family putatively involved in the secretion of bicarbonate to bile. In humans, three variants of AE2 mRNA have been described: the full-length transcript AE2a (expressed from the upstream promoter in most tissues), and alternative transcripts AE2b1 and AE2b2 (driven from alternate promoter sequences in a tissue-restricted manner, mainly in liver and kidney). These transcripts would result in AE protein isoforms with short N-terminal differences. To ascertain their translation, functionality, and membrane sorting, we constructed expression vectors encoding each AE2 isoform fused to GFP at the C-terminus. Transfected HEK293 cells showed expression of functional GFP-tagged AE2 proteins, all three isoforms displaying comparable AE activities. Primary rat hepatocytes transfected with expression vectors and repolarized in a collagen-sandwich configuration showed a microtubule-dependent apical sorting of each AE2 isoform. This shared apical sorting is liver-cell specific, as sorting of AE2 isoforms was basolateral in control experiments on polarized kidney MDCK cells. Hepatocytic apical targeting of AE2 isoforms suggests that they all may participate in the canalicular secretion of bicarbonate to bile. [Copyright &y& Elsevier]

Background: Improving the mating competitiveness and survival of sterile males are direct means to increase the effectiveness of the sterile insect technique (SIT). Some insecticide growth regulators, such as the juvenile hormone analogue (JHA) methoprene, have been used to improve the mating competitiveness of male tephritid flies by reducing their sexual maturation period. However, the application of methoprene reduces fly resistance to stress and decreases survival. Here, we compared the effects of methoprene and pyriproxyfen (PPF), another JHA, in Anastrepha ludens males. PPF is an insect growth regulator that exhibits higher negative effects on the larval molting process than methoprene or natural juvenile hormone. Both compounds were administered at two doses (0.05% and 0.10%) via the male diet immediately after emergence., Results: Our results show that both PPF and methoprene reduced male sexual maturation. However, PPF-treated males exhibited a shorter maturation period and obtained more matings at a given age than methoprene-treated males. No significant differences were observed between the two PPF doses tested (0.05% and 0.10%). Male survival was equally reduced by the two compounds., Conclusion: Our results demonstrate that PPF accelerated sexual development without reducing the mating propensity of sterile male flies and can be used as a suitable alternative for methoprene. © 2023 Society of Chemical Industry., (© 2023 Society of Chemical Industry.)

Nitrogen gas (N 2 ) fixation in the anode-respiring bacterium Geobacter sulfurreducens occurs through complex, multistep processes. Optimizing ammonium (NH 4 + ) production from this bacterium in microbial electrochemical technologies (METs) requires an understanding of how those processes are regulated in response to electrical driving forces. In this study, we quantified gene expression levels (via RNA sequencing) of G. sulfurreducens growing on anodes fixed at two different potentials (-0.15 V and +0.15 V versus standard hydrogen electrode). The anode potential had a significant impact on the expression levels of N 2 fixation genes. At -0.15 V, the expression of nitrogenase genes, such as nifH , nifD , and nifK , significantly increased relative to that at +0.15 V, as well as genes associated with NH 4 + uptake and transformation, such as glutamine and glutamate synthetases. Metabolite analysis confirmed that both of these organic compounds were present in significantly higher intracellular concentrations at -0.15 V. N 2 fixation rates (estimated using the acetylene reduction assay and normalized to total protein) were significantly larger at -0.15 V. Genes expressing flavin-based electron bifurcation complexes, such as electron-transferring flavoproteins (EtfAB) and the NADH-dependent ferredoxin:NADP reductase (NfnAB), were also significantly upregulated at -0.15 V, suggesting that these mechanisms may be involved in N 2 fixation at that potential. Our results show that in energy-constrained situations (i.e., low anode potential), the cells increase per-cell respiration and N 2 fixation rates. We hypothesize that at -0.15 V, they increase N 2 fixation activity to help maintain redox homeostasis, and they leverage electron bifurcation as a strategy to optimize energy generation and use. IMPORTANCE Biological nitrogen fixation coupled with ammonium recovery provides a sustainable alternative to the carbon-, water-, and energy-intensive Haber-Bosch process. Aerobic biological nitrogen fixation technologies are hindered by oxygen gas inhibition of the nitrogenase enzyme. Electrically driving biological nitrogen fixation in anaerobic microbial electrochemical technologies overcomes this challenge. Using Geobacter sulfurreducens as a model exoelectrogenic diazotroph, we show that the anode potential in microbial electrochemical technologies has a significant impact on nitrogen gas fixation rates, ammonium assimilation pathways, and expression of genes associated with nitrogen gas fixation. These findings have important implications for understanding regulatory pathways of nitrogen gas fixation and will help identify target genes and operational strategies to enhance ammonium production in microbial electrochemical technologies.

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., (Copyright © 2021 The Author(s). Published by Wolters Kluwer Health, Inc.)

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 - /HCO 3 - 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., (© 2019 The Author(s).)

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 - /HCO 3 - 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., (Copyright © 2018 Ferrata Storti Foundation.)

Applying microbial electrochemical technologies for the treatment of highly saline or thermophilic solutions is challenging due to the lack of proper inocula to enrich for efficient exoelectrogens. Brine pools from three different locations (Valdivia, Atlantis II and Kebrit) in the Red Sea were investigated as potential inocula sources for enriching exoelectrogens in microbial electrolysis cells (MECs) under thermophilic (70°C) and hypersaline (25% salinity) conditions. Of these, only the Valdivia brine pool produced high and consistent current 6.8±2.1A/m 2 -anode in MECs operated at a set anode potential of +0.2V vs. Ag/AgCl (+0.405V vs. standard hydrogen electrode). These results show that exoelectrogens are present in these extreme environments and can be used to startup MEC under thermophilic and hypersaline conditions. Bacteroides was enriched on the anode of the Valdivia MEC, but it was not detected in the open circuit voltage reactor seeded with the Valdivia brine pool., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

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., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/)

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., (© 2014 Eur J Oral Sci.)

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.

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., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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.

Unlabelled: Cl(-) /HCO3- 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., (Copyright © 2012 American Association for the Study of Liver Diseases.)