Autoantibodies toward ATP4A and ATP4B subunits of gastric proton pump H+,K+-ATPase are reliable serological pre-endoscopic markers of corpus atrophic gastritis

INTRODUCTION: Noninvasive assessment of corpus atrophic gastritis (CAG), a condition at increased risk of gastric cancer, is based on the measurement of pepsinogens, gastrin, and Helicobacter pylori antibodies. Parietal cell autoantibodies (PCAs) against the gastric proton pump (ATP4) are potential serological biomarkers of CAG. The purpose of this study was to compare the diagnostic performance of PCA and pepsinogen I tests in patients with clinical suspicion of CAG with the histopathological evaluation of gastric biopsies as reference standard. METHODS: A prospective case-finding study was performed on 218 naive adult patients (131 women, median age 65 years) who underwent gastric biopsies to confirm/exclude CAG. Patients with histopathological CAG were defined as cases, conversely as controls. Autoantibodies against the individual alpha (ATP4A) and beta (ATP4B) subunits of ATP4 were measured by luciferase immunoprecipitation, and global PCA and pepsinogen I by enzyme-linked immunosorbent assay. RESULTS: Histopathology classified 107 subjects (49%) as cases (CAG+, autoimmune 81.2%, and multifocal extensive 18.8%) and 111 subjects (51%) as controls (CAG−). In cases, ATP4A, ATP4B, and PCA titers were increased compared with controls, whereas pepsinogen I was reduced (P < 0.0001 for all). ATP4B, ATP4A, and pepsinogen I tests showed sensitivities of 77%, 75%, and 73% and specificities of 88%, 88%, and 80%, respectively. The receiver operating characteristic (ROC) area under the ROC curve (AUC) of these serological biomarkers confirmed their ability to discriminate cases from controls (ATP4B = 0.838, ATP4A = 0.826, pepsinogen I = 0.775, and PCA = 0.805), whereas the partial ROC-pAUC90 analysis showed that the ATP4B test had the best diagnostic performance (P = 0.008 vs ATP4; P = 0.0002 vs pepsinogen I). The presence of autoimmune or extensive gastritis was not significantly different between ATP4B positive or negative cases (P = 0.217). DISCUSSION: PCAs are promising serological biomarkers for the identification of CAG in high-risk individuals, particularly in an autoimmune pattern but also in an extensive-multifocal atrophy pattern

The first 142 amino acids of glutamate decarboxylase do not contribute to epitopes recognized by autoantibodies associated with Type 1 diabetes

Human adenovirus (HAdV) E1B-55K is a multifunctional regulator of productive viral replication and oncogenic transformation in nonpermissive mammalian cells. These functions depend on E1B-55K's posttranslational modification with the SUMO protein and its binding to HAdV E4orf6. Both early viral proteins recruit specific host factors to form an E3 ubiquitin ligase complex that targets antiviral host substrates for proteasomal degradation. Recently, we reported that the PML-NB associated factor Daxx represses efficient HAdV productive infection and is proteasomally degraded via a SUMO-E1B-55K-dependent, E4orf6-independent pathway, the details of which remained to be established. RNF4, a cellular SUMO-targeted ubiquitin ligase (STUbL), induces ubiquitinylation of specific SUMOy lated proteins and plays an essential role during DNA repair. Here, we show that E1B-55K recruits RNF4 to the insoluble nuclear matrix fraction of the infected cell to support RNF4/Daxx association, promoting Daxx PTM and thus inhibiting this antiviral factor. Removing RNF4 from infected cells using RNA interference resulted in blocking the proper establishment of viral replication centers and significantly diminished viral gene expression. These results provide a model for how HAdV antagonize the antiviral host responses by exploiting the functional capacity of cellular STUbLs. Thus, RNF4 and its STUbL function represent a positive factor during lytic infection and a novel candidate for future therapeutic antiviral intervention strategies.IMPORTANCE Daxx is a PML-NB-associated transcription factor that was recently shown to repress efficient HAdV productive infection. To counteract this antiviral measurement during infection, Daxx is degraded via a novel pathway including viral E1B-55K and host proteasomes. This virus-mediated degradation is independent of the classical HAdV E3 ubiquitin ligase complex, which is essential during viral infection to target other host antiviral substrates. To maintain a productive viral life cycle, HAdV E1B-55K early viral protein inhibits the chromatin-remodeling factor Daxx in a SUMO-dependent manner. In addition, viral E1B-55K protein recruits the STUbL RNF4 and sequesters it into the insoluble fraction of the infected cell. E1B-55K promotes complex formation between RNF4-and E1B-55K-targeted Daxx protein, supporting Daxx posttranslational modification prior to functional inhibition. Hence, RNF4 represents a novel host factor that is beneficial for HAdV gene expression by supporting Daxx counteraction. In this regard, RNF4 and other STUbL proteins might represent novel targets for therapeutic intervention

A novel LIPS assay for insulin autoantibodies

Insulin autoantibodies (IAA) are often the first marker of autoimmunity detected in children in the preclinical phase of type 1 diabetes (T1D). Currently, the vast majority of laboratories adopt the radiobinding micro-assay (RBA) for measuring IAA. Our aim was to replace RBA with a novel non-radioactive IAA Luciferase Immuno Precipitation System (LIPS) assay with improved performance. We developed (pro)insulin antigens with alternative placements of a NanoLuc (TM) luciferase reporter (NLuc). Performance in LIPS was evaluated by testing sera from new onset T1D (n = 80), blood donors (n = 123), schoolchildren (n = 186), first-degree relatives (FDRs) from the Bart's Oxford family study (n = 53) and from the Belgian Diabetes Registry (n = 136), coded sera from the Islet Autoantibody Standardization Program (IASP) (T1D n = 50, blood donors n = 90). IAA LIPS based on B chain-NLuc proinsulin or B chain-NLuc insulin, in which NLuc was fused at the C-terminus of the insulin B chain, required only 2 mu L of serum and a short incubation time, showed high concordance with RBA (Spearman r = 0.866 and 0.833, respectively), high assay performance (B chain-NLuc proinsulin ROC-AUC = 0.894 and B chain-NLuc insulin ROC-AUC = 0.916), and an adjusted sensitivity at 95% specificity ranking on par with the best assays submitted to the two most recent IASP workshops. In FDRs, the IAA LIPS showed improved discrimination of progressors to T1D compared to RBA. We established a novel high-performance non-radioactive IAA LIPS that might replace the current gold standard RBA and find wide application in the study of the IAA response in T1D

A/H1N1 hemagglutinin antibodies show comparable affinity in vaccine-related Narcolepsy type 1 and control and are unlikely to contribute to pathogenesis

An increased incidence of narcolepsy type 1 (NT1) was observed in Scandinavia following the 2009–2010 influenza Pandemrix vaccination. The association between NT1 and HLA-DQB1*06:02:01 supported the view of the vaccine as an etiological agent. A/H1N1 hemagglutinin (HA) is the main antigenic determinant of the host neutralization antibody response. Using two different immunoassays, the Luciferase Immunoprecipitation System (LIPS) and Radiobinding Assay (RBA), we investigated HA antibody levels and affinity in an exploratory and in a confirmatory cohort of Swedish NT1 patients and healthy controls vaccinated with Pandemrix. HA antibodies were increased in NT1 patients compared to controls in the exploratory (LIPS p = 0.0295, RBA p = 0.0369) but not in the confirmatory cohort (LIPS p = 0.55, RBA p = 0.625). HA antibody affinity, assessed by competition with Pandemrix vaccine, was comparable between patients and controls (LIPS: 48 vs. 39 ng/ml, p = 0.81; RBA: 472 vs. 491 ng/ml, p = 0.65). The LIPS assay also detected higher HA antibody titres as associated with HLA-DQB1*06:02:01 (p = 0.02). Our study shows that following Pandemrix vaccination, HA antibodies levels and affinity were comparable NT1 patients and controls and suggests that HA antibodies are unlikely to play a role in NT1 pathogenesis

Impaired hydration status in acutely admitted older patients: prevalence and impact on mortality

Background: impaired hydration is common in the older people, however studies of its effects on outcome in the acute setting are limited. Objectives: to assess (i) the prevalence of impaired hydration, (ii) its relationship with laboratory markers of altered hydration and with (iii) short- and long-term mortality. Design: retrospective cohort study. Setting: University Hospital-Internal Medicine Department. Subjects: a total of 5,113 older patients consecutively acutely admitted from October 2015 to July 2016. Methods: according to calculated serum osmolarity at admission hydration status was stratified in: low osmolarity (300 mmol/L). Relationships with serum sodium, potassium, glucose, urea, estimated glomerular filtration rate (eGFR), haematocrit, urea/creatinine ratio (Urea/Cr) and urine specific gravity (USG) were determined. Charlson Comorbidity Index, Modified Early Warning Score, Glasgow Prognostic Score, Norton score and Nutritional Risk Screening-2002 were calculated. Results: current and impending dehydration, euhydration and low-osmolarity were detected in 51.7, 17.1, 28.5 and 2.7% of the patients, respectively. Osmolarity correlated with urea (r = 0.846). Associations with serum sodium, creatinine, eGFR and urea/Cr were low but significant, being negligible that with USG and haematocrit. Serum sodium and urea increased in the transition from low- to high-osmolarity (P < 0.001 in all pairwise comparisons). In multivariate modelling current dehydration, functional dependence, clinical instability and high nutritional risk were associated (P < 0.001) with reduced short- and long-term survival. Conclusions: impaired hydration is common in older people acutely admitted to medical care and is associated with poor outcome. Early assessment of calculated serum osmolarity is mandatory to target dehydration and hypoosmolar disorders

Gender-sex differences in autoimmune atrophic gastritis

Gender-sex differences in autoimmune diseases are gaining increasing attention due to their effects on prevalence and clinical features. Data on gender-sex differences in autoimmune atrophic gastritis (AAG), a chronic not-self-limiting inflammatory condition characterized by corpus-oxyntic mucosa atrophy sparing the antrum, are lacking. This study aimed to assess possible gender-sex differences of clinical, serological, histological, and genetic features in AAG patients. Cross-sectional study on 435 patients with histological-AAG, stratified according to female-male gender. In subsets of patients, serum gastric-autoantibodies against intrinsic-factor (IFA) and parietal-cells (PCA) by luminescent-immunoprecipitation-system (LIPS) (n = 81) and of HLA-DRB1-genotyping (n = 89) were available and stratified according to sex. Female AAG-patients were preponderant: 69.2%vs30.8%, P &lt; 0.0001(ratio 2.2:1). Females were more frequently PCA and/or IFA-positive than males (90.9%vs73.1%, P = 0.0361). HLA-DRB1*06-alleles were significantly more frequent in females [30%vs4%, P = 0.01, OR 10.1(95%CI 1.3–80.4); HLA-DRB1*04-alleles were more frequent and HLA-DRB1*03 and *05-alleles less frequent in females without reaching statistical significance. At logistic regression, iron-deficiency-anemia [OR 3.6(95%CI 1.9–7.0)], body-mass-index &lt;25m2/kg [OR 3.1(95%CI 1.7–5.6)], autoimmune-thyroid-disease [OR 2.5(95%CI 1.4–4.5), and dyspepsia [OR 2.4(95%CI 1.4–4.3) were significantly associated to females. Body-mass-index&gt;25m2/kg [OR 3.2(95%CI1.8–5.6)], absence of autoimmune-thyroid-disease [OR 2.3(95%CI 1.3–4.2)] and dyspepsia [OR 2.1(95%CI 1.2–3.7)], smoking habit [OR 1.8(95%CI 1.1–3.1)], and pernicious-anemia [OR 1.7(95%CI 1.0–3.0)], were significantly associated to males. AAG was preponderant in women who showed stronger autoimmune serological responsiveness and different HLA-DRB1 association. AAG showed differential clinical profiles in female and male patients occurring mainly in normal weight, dyspeptic women with iron-deficiency anemia and autoimmune thyroid disease, but in overweight male smokers with pernicious anemia. Stratification for sex and gender should be considered in future genetic, immunological, and clinical studies on autoimmune atrophic gastritis

Intrinsic factor autoantibodies by luminescent immuno-precipitation system in patients with corpus atrophic gastritis

Background: Corpus atrophic gastritis (CAG) may lead to intrinsic factor (IF) deficiency and vitamin B12 malabsorption. Intrinsic factor autoantibodies (IFA) are considered markers of pernicious anemia, but their clinical utility in CAG has not been evaluated. This study aimed to assess IFA in CAG patients and controls using a luciferase immunoprecipitation system (LIPS). Methods: Recombinant nanoluciferase-tagged IF secreted from transfected Expi293F cells was used as antigen in an IFA-LIPS assay. IFA IgG were measured in sera from subjects undergoing gastroscopy and biopsy (updated Sydney system) mainly for anemia (57%) or dyspepsia (34%). This cohort comprised 105 patients with histologically-proven-CAG (cases: median age 64 years, 68% females) and 110 subjects with suspected CAG that were histologically negative (controls: median age 67 years, 54% females). Cut-off values were selected by Q-Qplot analysis (negative: &lt;2.5 arbitrary units). Results: IFA levels were higher in cases than in controls (Mann-Whitney:p &lt; 10 5). The ROC-AUC was 0.67 (95% CI 0.60–0.73, p &lt; 0.0001). The IFA LIPS sensitivity and specificity for CAG were 32% (95% CI 24–42) and 95% (95% CI 90–99). This diagnostic performance remained similar after stratification for the presence/absence of anemia, dyspepsia or vitamin B12 deficiency. IFA levels were higher in females compared with males (p = 0.0127). In females aged &lt;65 years, IFA-positives were more prevalent than in males (43.5% vs 6.6%, p = 0.011). Conclusions: The IFA-LIPS assay discriminated between CAG patients and controls showing a good specificity (95%) at the cost of sensitivity (32%). IFA-positivity occurred independently from anemia and vitamin B12 deficiency, but was more frequent in younger females. IFA testing should be considered in patients at high clinical suspicion of CAG

A novel, high-performance, low-volume, rapid luciferase immunoprecipitation system (LIPS) assay to detect autoantibodies to zinc transporter 8

Background: Zinc transporter 8 autoantibodies (ZnT8A) are thought to appear close to type 1 diabetes (T1D) onset and can identify high-risk multiple (≥2) autoantibody positive individuals. Radiobinding assays (RBA) are widely used for ZnT8A measurement but have limited sustainability. We sought to develop a novel, high-performance, non-radioactive luciferase immunoprecipitation system (LIPS) assay to replace RBA.Methods:A custom dual C-terminal ZnT8 (aa268-369; R325/W325) heterodimeric antigen, tagged with a Nanoluciferase (TM) (Nluc-ZnT8) reporter, and LIPS assay was developed. Assay performance was evaluated by testing sera from new onset T1D (n=573), healthy schoolchildren (n=521), and selected first-degree relatives (FDRs) from the Bart’s Oxford family study (n=617; 164 progressed to diabetes). Results:In new-onset T1D, ZnT8A levels by LIPS strongly correlated with RBA [Spearman’s r=0.89; p&lt;0.0001), and positivity was highly concordant (94.3%). At a high specificity (95%), LIPS and RBA had comparable assay performance [LIPS pROC-AUC(95) 0.032 (95% CI: 0.029-0.036); RBA pROC-AUC(95) 0.031 (95% CI: 0.028-0.034); p=0.376]. Overall, FDRs found positive by LIPS or RBA had a comparable 20-year diabetes risk (52.6% and 59.7%, respectively), but LIPS positivity further stratified T1D risk in FDRs positive for at least one other islet autoantibody detected by RBA (p=0.0346).Conclusion:This novel, high-performance, cheaper, quicker, higher throughput, low blood volume Nluc-ZnT8 LIPS assay is a safe, non-radioactive alternative to RBA with enhanced sensitivity and ability to discriminate T1D progressors. This method offers an advanced approach to current strategies to screen the general population for T1D risk for immunotherapy trials and to reduce rates of diabetic ketoacidosis at diagnosis.<br/

Immunoprecipitation System (LIPS) for detection of autoantibodies against ATP4A and ATP4B subunits of gastric proton pump H+,K+-ATPase in atrophic body gastritis patients

OBJECTIVES: Circulating autoantibodies targeting the H+/K+-ATPase proton pump of gastric parietal cells are considered markers of autoimmune gastritis, whose diagnostic accuracy in atrophic body gastritis, the pathological lesion of autoimmune gastritis, remains unknown. This study aimed to assess autoantibodies against ATP4A and ATP4B subunits of parietal cells H+, K+-ATPase in atrophic body gastritis patients and controls. METHODS: One-hundred and four cases with atrophic body gastritis and 205 controls were assessed for serological autoantibodies specific for ATP4A or ATP4B subunits using luminescent immunoprecipitation system (LIPS). Recombinant luciferase-reporter-fused-antigens were expressed by in vitro transcription-translation (ATP4A) or after transfection in Expi293F cells (ATP4B), incubated with test sera, and immune complexes recovered using protein-A-sepharose. LIPS assays were compared with a commercial enzyme immunoassay (EIA) for parietal cell autoantibodies. RESULTS: ATP4A and ATP4B autoantibody titers were higher in cases compared to controls (P&lt;0.0001). The area under the receiver-operating characteristic curve was 0.98 (95% CI 0.965-0.996) for ATP4A, and 0.99 (95% CI 0.979-1.000) for ATP4B, both higher as compared with that of EIA: 0.86 (95% CI 0.809-0.896), P&lt;0.0001. Sensitivity-specificity were 100-89% for ATP4A and 100-90% for ATP4B assay. Compared with LIPS, EIA for parietal cell autoantibodies showed a lower sensitivity (72%, P&lt;0.0001) at a similar specificity (92%, P=0.558). CONCLUSIONS: Positivity to both, ATP4A and ATP4B autoantibodies is closely associated with atrophic body gastritis. Both assays had the highest sensitivity, at the cost of diagnostic accuracy (89 and 90% specificity), outperforming traditional EIA. Once validated, these LIPS assays should be valuable screening tools for detecting biomarkers of damaged atrophic oxyntic mucosa