Interleukin-12 is not essential for silicosis in mice

BACKGROUND: Silicosis features foci of inflammation where macrophages and lymphocytes precede and accompany fibroblast proliferation, alveolar epithelial hyperplasia, and increased deposition of connective tissue matrix material. In the mouse following silica inhalation there is recruitment of natural killer-, B-, and CD4(+ )and CD8(+ )lymphocytes to the alveolar spaces, enlargement of bronchial-associated lymphoid tissues (BALT), and aggregation of lymphocytes surrounding small airways and blood vessels. A substantial fraction of the recruited lung lymphocytes produce interferon-γ (IFN-γ), and IFN-γ gene-deleted mice develop less silicosis than wild-type mice. Interleukin-12 (IL-12) is an important pathway for driving the adaptive immune response towards a TH1-like phenotype. We hypothesized that IL-12 might stimulate lymphocyte activation and the up-regulation of IFN-γ, and consequently be an essential mediator for silicosis. RESULTS: C57Bl/6 wild-type (WT) and IL-12 deficient (IL-12 KO) mice were exposed to sham-air or crystobalite silica (61 mg/m(3)) by inhalation for 5 hours/day for 12 days and then studied from 1 to 112 days after exposure. Mice exposed to sham-air had normal lung histology at all time points. WT mice exposed to titanium dioxide (72 mg/m(3)) showed pulmonary macrophage recruitment but no increase in lung collagen. Both WT and IL-12 KO mice exposed to silica showed similar progressive lung pathology, increased wet lung weight and increased total lung collagen (hydroxyproline). IL-12 p35 mRNA was not increased in either strain after silica exposure; IL-12 p40 mRNA was up-regulated after silica in WT mice and constitutively absent in the IL-12 KO mice. IL-18 mRNA was not increased after silica exposure. The expression of IL-15 (an important driver for innate immunity, Natural Killer cell activation, and IFN-γ production) was abundant in air-exposed mice and was increased slightly in the lungs of mice with silicosis. CONCLUSION: The axis of IL-12 driving IFN-γ production is not essential for the full manifestations of silicosis in mice exposed to a crystobalite silica aerosol

Detection of IgG4-Specific Autoantibodies in Rheumatoid Arthritis Serum Samples

Background: Rheumatoid arthritis (RA) is a chronic multi-system autoimmune disease characterized by inflammatory synovitis. Autoantibodies such as rheumatoid factor (RF) and anti-citrullinated protein antibodies (ACPA) have been implicated in the pathogenesis of RA, and are currently important criteria for diagnosis within the 2010 American College of Rheumatology (ACR)/European League Against Rheumatism (EULAR) classification criteria.1 Yet, many patients diagnosed with RA do not have measurable circulating ACPA or RF which may result in delayed diagnosis and treatment. After IgG1, IgG4 is the second most predominant isotype among ACPA and RF; however it is not detected in currently available diagnostic assays. Recent data have demonstrated that patients deemed “sero-negative” based on standard assays may have high titers of IgG4-specific ACPA and/or RF. Objectives: The purpose of this study is to quantitate and compare IgG1- to IgG4-specific anti-CCP antibodies and rheumatoid factor in patients with rheumatoid arthritis. We will determine the frequency of IgG4 autoantibodies, and examine whether they have a differential expression among RA patients. We will also correlate their presence with disease activity, anti-rheumatic drug therapy, and levels of serum cytokines. Ultimately, this work may help to determine if a diagnostic test that detects IgG4 isotype of ACPA and RF will aid in earlier diagnosis and better characterization of rheumatoid arthritis. Methods: To explore our objectives, we have initiated a cross-sectional study with the goal of enrolling 1,000 patients with a confirmed diagnosis of rheumatoid arthritis, based on the 2010 ACR/EULAR classification criteria.1 We are collecting clinical information about each patient including demographics, current treatments, clinical disease activity, laboratory values, and radiographic results. Concurrently, we are collecting serum samples from each patient that will be analyzed for 1) total levels of IgG4 and IgG1; 2) total ACPA and RF; 3) levels of IgG1-specific and IgG4-specific ACPA and RF; and 4) cytokine levels (IL-6, TNF, IL-1, IL-17, IFNy, IL-21, and G-CSF). Results: To date, we have recruited 102 RA patients including 70 females (68.6%) and 32 males (31.4%). Their ages range from 24 to 85 years (mean 58.4 ± 12.4 years). Acute phase reactant levels were available for 98 of the 102 patients, allowing calculation of the disease activity score using 28 joints (DAS28). The mean DAS28 was 3.67 ± 1.0, which is within the moderate disease activity range. The proportion of patients in each disease category was: remission (12.2%), low disease activity (21.4%), moderate disease activity (61.2%), and high disease activity (6.1%). Based on their medical records, at any point in time, 46.1% (n=47) of the recruited subjects had positive RF titers vs. 39.2% (n=40) without RF; 58.8% (n=60) had ACPA vs 26.5% (n=27) without ACPA. For 14.7% (n=15) of the subjects, RF and/or ACPA were either unknown or untested. Of patients with RF, 91.4% (n=43) also had ACPA; of patients with ACPA, 71.7% % (n=43) also had RF. Of the patients tested for both, 27.9% (n=24) were negative for both RF and ACPA. Conclusion: Subject recruitment and data collection are well underway for this large cross-sectional study that will shed light to the role of IgG4- specific autoantibodies in the pathogenesis and diagnosis of rheumatoid arthritis. Reference: 1Aletaha D Neogi T, Silman A, et al. 2010 Rheumatoid arthritis classification criteria: An American College of Rheumatology/European League Against Rheumatism collaborative initiative. Arthritis Rheum 2010; 62(9):2569-81/Ann Rheum Dis. 2010; 69:1580-8

Inactivation of nuclear GSK3 beta by Ser(389) phosphorylation promotes lymphocyte fitness during DNA double-strand break response

Variable, diversity and joining (V(D)J) recombination and immunoglobulin class switch recombination (CSR) are key processes in adaptive immune responses that naturally generate DNA double-strand breaks (DSBs) and trigger a DNA repair response. It is unclear whether this response is associated with distinct survival signals that protect T and B cells. Glycogen synthase kinase 3 beta (GSK3 beta) is a constitutively active kinase known to promote cell death. Here we show that phosphorylation of GSK3 beta on Ser(389) by p38 MAPK (mitogen-activated protein kinase) is induced selectively by DSBs through ATM (ataxia telangiectasia mutated) as a unique mechanism to attenuate the activity of nuclear GSK3 beta and promote survival of cells undergoing DSBs. Inability to inactivate GSK3 beta through Ser(389) phosphorylation in Ser(389)Ala knockin mice causes a decrease in the fitness of cells undergoing V(D)J recombination and CSR. Preselection-Tcrb repertoire is impaired and antigen-specific IgG antibody responses following immunization are blunted in Ser(389)GSK3 beta knockin mice. Thus, GSK3 beta emerges as an important modulator of the adaptive immune response.We thank Dr T. Honjo and Dr K. Otsu for the generation of the original AID deficient mice and the p38 flox/flox mice, respectively. We thank C. Charland for flow cytometry analysis and cell sorting, the Vermont Cancer Center DNA Sequencing Facility and the University of Vermont College of Med. Microscopy Imaging Center for their services. We thank Dr D.R. Green and Dr R.C. Budd for helpful discussion regarding the mechanisms of cell death and reagents. This work was supported by NIH grant R01 AI051454 (M.R. and T.M.T.), P20 GM103496 (T.M.T.) NIH grant R37 GM41052 (M.S.K.) and Lake Champlain Cancer Research Organization (M.R.).S

The induction of antibody production by IL-6 is indirectly mediated by IL-21 produced by CD4+ T cells

Interleukin (IL) 6 is a proinflammtory cytokine produced by antigen-presenting cells and nonhematopoietic cells in response to external stimuli. It was initially identified as a B cell growth factor and inducer of plasma cell differentiation in vitro and plays an important role in antibody production and class switching in vivo. However, it is not clear whether IL-6 directly affects B cells or acts through other mechanisms. We show that IL-6 is sufficient and necessary to induce IL-21 production by naive and memory CD4+ T cells upon T cell receptor stimulation. IL-21 production by CD4+ T cells is required for IL-6 to promote B cell antibody production in vitro. Moreover, administration of IL-6 with inactive influenza virus enhances virus-specific antibody production, and importantly, this effect is dependent on IL-21. Thus, IL-6 promotes antibody production by promoting the B cell helper capabilities of CD4+ T cells through increased IL-21 production. IL-6 could therefore be a potential coadjuvant to enhance humoral immunity

miR-873-5p targets mitochondrialGNMT-Complex II interface contributing tonon-alcoholic fatty liver disease

Objective:Non-alcoholic fatty liver disease (NAFLD) is a complex pathology in which several dysfunctions, including alterations in metabolicpathways, mitochondrial functionality and unbalanced lipid import/export, lead to lipid accumulation and progression to inflammation andfibrosis.The enzyme glycine N-methyltransferase (GNMT), the most important enzyme implicated in S-adenosylmethionine catabolism in the liver, isdownregulated during NAFLD progression. We have studied the mechanism involved in GNMT downregulation by its repressor microRNA miR-873-5p and the metabolic pathways affected in NAFLD as well as the benefit of recovery GNMT expression.Methods:miR-873-5p and GNMT expression were evaluated in liver biopsies of NAFLD/NASH patients. Differentin vitroandin vivoNAFLD murinemodels were used to assess miR-873-5p/GNMT involvement in fatty liver progression through targeting of the miR-873-5p as NAFLD therapy.Results:We describe a new function of GNMT as an essential regulator of Complex II activity in the electron transport chain in the mitochondria.In NAFLD, GNMT expression is controlled by miR-873-5p in the hepatocytes, leading to disruptions in mitochondrial functionality in a preclinicalmurine non-alcoholic steatohepatitis (NASH) model. Upregulation of miR-873-5p is shown in the liver of NAFLD/NASH patients, correlating withhepatic GNMT depletion. Importantly, NASH therapies based on anti-miR-873-5p resolve lipid accumulation, inflammation andfibrosis byenhancing fatty acidb-oxidation in the mitochondria. Therefore, miR-873-5p inhibitor emerges as a potential tool for NASH treatment.Conclusion:GNMT participates in the regulation of metabolic pathways and mitochondrial functionality through the regulation of Complex II activityin the electron transport chain. In NAFLD, GNMT is repressed by miR-873-5p and its targeting arises as a valuable therapeutic option for treatment

miR-873-5p targets mitochondrial GNMT-Complex II interface contributing to non-alcoholic fatty liver disease

Objective: Non-alcoholic fatly liver disease (NAFLD) is a complex pathology in which several dysfunctions, including alterations in metabolic pathways, mitochondrial functionality and unbalanced lipid import/export, lead to lipid accumulation and progression to inflammation and fibrosis. The enzyme glycine N-methyltransferase (GNMT), the most important enzyme implicated in S-adenosylmethionine catabolism in the liver, is downregulated during NAFLD progression. We have studied the mechanism involved in GNMT downregulation by its repressor microRNA miR-873-5p and the metabolic pathways affected in NAFLD as well as the benefit of recovery GNMT expression. Methods: miR-873-5p and GNMT expression were evaluated in liver biopsies of NAFLD/NASH patients. Different in vitro and in vivo NAFLD murine models were used to assess miR-873-5p/GNMT involvement in fatty liver progression through targeting of the miR-873-5p as NAFLD therapy. Results: We describe a new function of GNMT as an essential regulator of Complex II activity in the electron transport chain in the mitochondria. In NAFLD, GNMT expression is controlled by miR-873-5p in the hepatocytes, leading to disruptions in mitochondria! functionality in a preclinical murine non-alcoholic steatohepatitis (NASH) model. Upregulation of miR-873-5p is shown in the liver of NAFLD/NASH patients, correlating with hepatic GNMT depletion. Importantly, NASH therapies based on anti-miR-873-5p resolve lipid accumulation, inflammation and fibrosis by enhancing fatty acid beta-oxidation in the mitochondria. Therefore, miR-873-5p inhibitor emerges as a potential tool for NASH treatment. Conclusion: GNMT participates in the regulation of metabolic pathways and mitochondria! functionality through the regulation of Complex II activity in the electron transport chain. In NAFLD, GNMT is repressed by miR-873-5p and its targeting arises as a valuable therapeutic option for treatment. (C) 2019 The Authors. Published by Elsevier GmbH.This work was supported by grants from NIH (US Department of Health and Human services)-R01AT001576 (to S.C.L., J.M.M., and M.L.M.-C.), Ministerio de Economia, Industria y Competitividad: SAF2017-87301-R (to M.L.M.-C.), SAF2015-64352-R (to P.A.), Gobierno Vasco-Departamento de Salud 2013111114 (to M.L.M.-C.), Gobierno Vasco-Departamento de Educacion IT-336-10 (to PA), BIOEF (Basque Foundation for Innovation and Health Research: EiTB Maratoia BIO15/CA/016/BD (M.L.M.-C.), ELKARTEK 2016, Departamento de Industria del Gobierno Vasco (to M.L.M.-C), Asociacion Espanola contra el Cancer (to T.C.D., P.F.-T. and M.L.M.-C.), Mitotherapeutix (to M.L.M.-C.), Daniel Alagille award from EASL (to T.C.D), Fundacion Cientifica de la Asociacion Espanola Contra el Cancer (AECC Scientific Foundation) Rare Tumor Calls 2017 (to M.L.M.-C.), La Caixa Foundation Program (to M.L.M.-C.), Ayudas Fundacion BBVA a Equipos de Investigacion Cientifica 2019 (to M.L.M.-C.). Ciberehd_ISCIII_MINECO is funded by the Instituto de Salud Carlos III. We thank this work produced with the support of a 2017 Leonardo Grant for Researchers and Cultural Creators, BBVA Foundation (to M.V.R.). This work was supported by Fonds National de la Recherche Luxembourg and the Deutsche Forschungsgemeinschaft (C12/BM/3975937, FL/997/7-1, Inter "HepmiRSTAT", to I.B. and F.L.). We thank MINECO for the Severo Ochoa Excellence Accreditation (SEV2016-0644)

Mortality from gastrointestinal congenital anomalies at 264 hospitals in 74 low-income, middle-income, and high-income countries: a multicentre, international, prospective cohort study

Summary Background Congenital anomalies are the fifth leading cause of mortality in children younger than 5 years globally. Many gastrointestinal congenital anomalies are fatal without timely access to neonatal surgical care, but few studies have been done on these conditions in low-income and middle-income countries (LMICs). We compared outcomes of the seven most common gastrointestinal congenital anomalies in low-income, middle-income, and high-income countries globally, and identified factors associated with mortality. Methods We did a multicentre, international prospective cohort study of patients younger than 16 years, presenting to hospital for the first time with oesophageal atresia, congenital diaphragmatic hernia, intestinal atresia, gastroschisis, exomphalos, anorectal malformation, and Hirschsprung’s disease. Recruitment was of consecutive patients for a minimum of 1 month between October, 2018, and April, 2019. We collected data on patient demographics, clinical status, interventions, and outcomes using the REDCap platform. Patients were followed up for 30 days after primary intervention, or 30 days after admission if they did not receive an intervention. The primary outcome was all-cause, in-hospital mortality for all conditions combined and each condition individually, stratified by country income status. We did a complete case analysis. Findings We included 3849 patients with 3975 study conditions (560 with oesophageal atresia, 448 with congenital diaphragmatic hernia, 681 with intestinal atresia, 453 with gastroschisis, 325 with exomphalos, 991 with anorectal malformation, and 517 with Hirschsprung’s disease) from 264 hospitals (89 in high-income countries, 166 in middleincome countries, and nine in low-income countries) in 74 countries. Of the 3849 patients, 2231 (58·0%) were male. Median gestational age at birth was 38 weeks (IQR 36–39) and median bodyweight at presentation was 2·8 kg (2·3–3·3). Mortality among all patients was 37 (39·8%) of 93 in low-income countries, 583 (20·4%) of 2860 in middle-income countries, and 50 (5·6%) of 896 in high-income countries (p<0·0001 between all country income groups). Gastroschisis had the greatest difference in mortality between country income strata (nine [90·0%] of ten in lowincome countries, 97 [31·9%] of 304 in middle-income countries, and two [1·4%] of 139 in high-income countries; p≤0·0001 between all country income groups). Factors significantly associated with higher mortality for all patients combined included country income status (low-income vs high-income countries, risk ratio 2·78 [95% CI 1·88–4·11], p<0·0001; middle-income vs high-income countries, 2·11 [1·59–2·79], p<0·0001), sepsis at presentation (1·20 [1·04–1·40], p=0·016), higher American Society of Anesthesiologists (ASA) score at primary intervention (ASA 4–5 vs ASA 1–2, 1·82 [1·40–2·35], p<0·0001; ASA 3 vs ASA 1–2, 1·58, [1·30–1·92], p<0·0001]), surgical safety checklist not used (1·39 [1·02–1·90], p=0·035), and ventilation or parenteral nutrition unavailable when needed (ventilation 1·96, [1·41–2·71], p=0·0001; parenteral nutrition 1·35, [1·05–1·74], p=0·018). Administration of parenteral nutrition (0·61, [0·47–0·79], p=0·0002) and use of a peripherally inserted central catheter (0·65 [0·50–0·86], p=0·0024) or percutaneous central line (0·69 [0·48–1·00], p=0·049) were associated with lower mortality. Interpretation Unacceptable differences in mortality exist for gastrointestinal congenital anomalies between lowincome, middle-income, and high-income countries. Improving access to quality neonatal surgical care in LMICs will be vital to achieve Sustainable Development Goal 3.2 of ending preventable deaths in neonates and children younger than 5 years by 2030