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 NanoluciferaseTM (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 < 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.

Islet Autoantibody Standardization Program: interlaboratory comparison of insulin autoantibody assay performance in 2018 and 2020 workshops.

AIMS/HYPOTHESIS: The Islet Autoantibody Standardization Program (IASP) aims to improve the performance of immunoassays measuring autoantibodies in type 1 diabetes and the concordance of results across laboratories. IASP organises international workshops distributing anonymised serum samples to participating laboratories and centralises the collection and analysis of results. In this report, we describe the results of assays measuring IAA submitted to the IASP 2018 and 2020 workshops. METHODS: The IASP distributed uniquely coded sera from individuals with new-onset type 1 diabetes, multiple islet autoantibody-positive individuals, and diabetes-free blood donors in both 2018 and 2020. Serial dilutions of the anti-insulin mouse monoclonal antibody HUI-018 were also included. Sensitivity, specificity, area under the receiver operating characteristic curve (ROC-AUC), partial ROC-AUC at 95% specificity (pAUC95) and concordance of qualitative/quantitative results were compared across assays. RESULTS: Results from 45 IAA assays of seven different formats and from 37 IAA assays of six different formats were submitted to the IASP in 2018 and 2020, respectively. The median ROC-AUC was 0.736 (IQR 0.617-0.803) and 0.790 (IQR 0.730-0.836), while the median pAUC95 was 0.016 (IQR 0.004-0.021) and 0.023 (IQR 0.014-0.026) in the 2018 and 2020 workshops, respectively. Assays largely differed in AUC (IASP 2018 range 0.232-0.874; IASP 2020 range 0.379-0.924) and pAUC95 (IASP 2018 and IASP 2020 range 0-0.032). CONCLUSIONS/INTERPRETATION: Assay formats submitted to this study showed heterogeneous performance. Despite the high variability across laboratories, the in-house radiobinding assay (RBA) remains the gold standard for IAA measurement. However, novel non-radioactive IAA immunoassays showed a good performance and, if further improved, might be considered valid alternatives to RBAs.

Genetic determinants of type 1 diabetes in individuals with weak evidence of islet autoimmunity at disease onset.

AIMS/HYPOTHESIS: Islet autoantibodies (AAbs) are detected in >90% of individuals with clinically suspected type 1 diabetes at disease onset. A single AAb, sometimes at low titre, is often detected in some individuals, making their diagnosis uncertain. Type 1 diabetes genetic risk scores (GRS) are a useful tool for discriminating polygenic autoimmune type 1 diabetes from other types of diabetes, particularly the monogenic forms, but testing is not routinely performed in the clinic. Here, we used a type 1 diabetes GRS to screen for monogenic diabetes in individuals with weak evidence of autoimmunity, i.e. with a single AAb at disease onset. METHODS: In a pilot study, we genetically screened 142 individuals with suspected type 1 diabetes, 42 of whom were AAb-negative, 27 of whom had a single AAb (single AAb-positive) and 73 of whom had multiple AAbs (multiple AAb-positive) at disease onset. Next-generation sequencing (NGS) was performed in 41 AAb-negative participants, 26 single AAb-positive participants and 60 multiple AAb-positive participants using an analysis pipeline of more than 200 diabetes-associated genes. RESULTS: The type 1 diabetes GRS was significantly lower in AAb-negative individuals than in those with a single and multiple AAbs. Pathogenetic class 4/5 variants in MODY or monogenic diabetes genes were identified in 15/41 (36.6%) AAb-negative individuals, while class 3 variants of unknown significance were identified in 17/41 (41.5%). Residual C-peptide levels at diagnosis were higher in individuals with mutations compared to those without pathogenetic variants. Class 3 variants of unknown significance were found in 11/26 (42.3%) single AAb-positive individuals, and pathogenetic class 4/5 variants were present in 2/26 (7.7%) single AAb-positive individuals. No pathogenetic class 4/5 variants were identified in multiple AAb-positive individuals, but class 3 variants of unknown significance were identified in 19/60 (31.7%) patients. Several patients across the three groups had more than one class 3 variant. CONCLUSIONS/INTERPRETATION: These findings provide insights into the genetic makeup of patients who show weak evidence of autoimmunity at disease onset. Absence of islet AAbs or the presence of a single AAb together with a low type 1 diabetes GRS may be indicative of a monogenic form of diabetes, and use of NGS may improve the accuracy of diagnosis.

The Immunological Profile of SARS-CoV-2 Infection in Children Is Linked to Clinical Severity and Age.

Coronavirus disease 19 (COVID-19) is clinically less severe in children, even if the wide variety and degree of severity of symptoms reported in children pose a still-unresolved challenge for clinicians. We performed an in-depth analysis of the immunological profiles of 18 hospitalized SARS-CoV-2-infected children, whose results were compared to those obtained from 13 age- and sex-matched healthy controls (HC). The patients were categorized as paucisymptomatic/moderate (55.6%) or severe/critical (44.5%) according to established diagnostic criteria and further stratified into the categories of infants (1-12 months), children (1-12 years), and adolescents (>12 years). We assessed SARS-CoV-2-specific RBD antibodies (Ab), neutralizing antibodies (nAb), and circulating cytokines/chemokines in the plasma, and the SARS-CoV-2-specific immune response was measured in PBMCs by gene expression and secretome analyses. Our results showed peculiar circulating cytokine/chemokine profiles among patients sharing a similar clinical phenotype. A cluster of patients consisting of infants with severe symptoms presented hyperinflammatory profiles, together with extremely polarized antibody profiles. In a second cluster consisting of paucisymptomatic patients, a less pronounced increase in the level of inflammatory cytokines, together with an association between the selected cytokines and humoral responses, was observed. A third cluster, again consisting of paucisymptomatic patients, showed a circulating cytokine/chemokine profile which overlapped with that of the HC. The SARS-CoV-2-stimulated production of pro-inflammatory proteins, T lymphocyte activation, and migration-specific proteins, were significantly increased in SARS-CoV-2-infected children compared to the HC. Our findings suggest that immune response activation in the course of SARS-CoV-2 infection in children is directly correlated with clinical severity and, to a lesser extent, age.

Seasonal Betacoronavirus Antibodies' Expansion Post-BNT161b2 Vaccination Associates with Reduced SARS-CoV-2 VoC Neutralization.

SARS-CoV-2 vaccination is known to induce antibodies that recognize also variants of concerns (VoCs) of the virus. However, epidemiological and laboratory evidences indicate that these antibodies have a reduced neutralization ability against VoCs. We studied binding and neutralizing antibodies against the Spike protein domains and subunits of the Wuhan-Hu-1 virus and its alpha, beta, delta VoCs and of seasonal betacoronaviruses (HKU1 and OC43) in a cohort of 31 health care workers prospectively followed post-vaccination with BNT162b2-Comirnaty. The study of sequential samples collected up to 64 days post-vaccination showed that serological assays measuring IgG against Wuhan-Hu-1 antigens were a poor proxy for VoC neutralization. In addition, in subjects who had asymptomatic or mild COVID-19 prior to vaccination, the loss of nAbs following disease could be rapid and accompanied by post-vaccination antibody levels similar to those of naïve vaccinees. Interestingly, in health care workers naïve for SARS-CoV-2 infection, vaccination induced a rapid and transient reactivation of pre-existing seasonal coronaviruses IgG responses that was associated with a subsequent reduced ability to neutralize alpha and beta VoCs.

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 (<275 mmol/L), euhydration (275-295 mmol/L), impending (296-300 mmol/L) and current dehydration (>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.

Stage-related implications of community-acquired pressure injuries for the acute medical inpatients.

AIMS: To analyse the prevalence of any-stage pressure injuries at hospital admission and their impact on short-, mid- and late-term mortality. Patient characteristics associated with pressure injuries and the impact on hospital costs were also investigated. BACKGROUND: In medical patients acutely admitted to hospital, no study analysed the presence of pre-existing pressure injuries and the related short- and long-term mortality according to the overall stages of severity thereof. DESIGN: Retrospective cohort study following the STROBE guideline. METHODS: In a population of 7217 acute medical inpatients, the presence and staging of pressure injuries were assessed at hospital admission. The impact of pressure injury on 30-, 180- and 365-day mortality was analysed by multivariate Cox regression models. RESULTS: The prevalence of community-acquired pressure injuries was 14.9% (stage-1: 8.1%; stage-2: 3.5%; stage-3: 1.6%; stage-4: 1.1%; unstageable: 0.5%). Hemiplegia/paraplegia, anaemia, poor functional status, high nutritional risk, clinical instability and systemic inflammatory response, but not hydration status, were found to be associated with the occurrence of stage-2-and-above pressure injuries. An increasing difference was found in Diagnosis-Related Groups (DRG) weight according to pressure injury stages. A distinct and progressively increasing risk-of-death for any-stage pressure injury was shown after 365-days. A significantly increased mortality risk for all considered time intervals was found for unstageable and stage-4 pressure injuries. CONCLUSIONS: In acute medical inpatients, the presence of community-acquired pressure injuries is part of a multidimensional clinical complexity. The presence and staging of pressure injuries have an independent dramatic impact on of early-to-late mortality and hospital costs. RELEVANCE TO CLINICAL PRACTICE: This study documented as community-acquired pressure injuries are highly prevalent and represents an independent predictor of outcomes in strict dependence of the progression of thereof stage. The presence of community-acquired pressure injuries should be interpreted as a critical marker of frailty and increased vulnerability.

Antibody response to multiple antigens of SARS-CoV-2 in patients with diabetes: an observational cohort study.

AIMS/HYPOTHESIS: The aim of the study was to characterise the humoral response against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in patients with diabetes. Demonstrating the ability to mount an appropriate antibody response in the presence of hyperglycaemia is relevant for the comprehension of mechanisms related to the observed worse clinical outcome of coronavirus disease 2019 (COVID-19) pneumonia in patients with diabetes and for the development of any future vaccination campaign to prevent SARS-CoV-2 infection. METHODS: Using a highly specific and sensitive measurement of antibodies by fluid-phase luciferase immunoprecipitation assays, we characterised the IgG, IgM and IgA response against multiple antigens of SARS-CoV-2 in a cohort of 509 patients with documented diagnosis of COVID-19, prospectively followed at our institution. We analysed clinical outcomes and antibody titres according to the presence of hyperglycaemia, i.e., either diagnosed or undiagnosed diabetes, at the time of, or during, hospitalisation. RESULTS: Among patients with confirmed COVID-19, 139 (27.3%) had diabetes: 90 (17.7%) had diabetes diagnosed prior to the hospital admission (comorbid diabetes) while 49 (9.6%) had diabetes diagnosed at the time of admission (newly diagnosed). Diabetes was associated with increased levels of inflammatory biomarkers and hypercoagulopathy, as well as leucocytosis and neutrophilia. Diabetes was independently associated with risk of death (HR 2.32 [95% CI 1.44, 3.75], p = 0.001), even after adjustment for age, sex and other relevant comorbidities. Moreover, a strong association between higher glucose levels and risk of death was documented irrespective of diabetes diagnosis (HR 1.14 × 1.1 mmol/l [95% CI 1.08, 1.21], p < 0.001). The humoral response against SARS-CoV-2 in patients with diabetes was present and superimposable, as for timing and antibody titres, to that of non-diabetic patients, with marginal differences, and was not influenced by glucose levels. Of the measured antibody responses, positivity for IgG against the SARS-CoV-2 spike receptor-binding domain (RBD) was predictive of survival rate, both in the presence or absence of diabetes. CONCLUSIONS/INTERPRETATION: The observed increased severity and mortality risk of COVID-19 pneumonia in patients with hyperglycaemia was not the result of an impaired humoral response against SARS-CoV-2. RBD IgG positivity was associated with a remarkable protective effect, allowing for a cautious optimism about the efficacy of future vaccines against SARs-COV-2 in people with diabetes. Graphical abstract.

Gene expression analysis of embryonic pancreas development master regulators and terminal cell fate markers in resected pancreatic cancer: A correlation with clinical outcome.

BACKGROUND: Despite the recent introduction of new drugs and the development of innovative multi-target treatments, the prognosis of pancreatic ductal adenocarcinoma (PDAC) remains very poor. Even when PDAC is resectable, the rate of local or widespread disease recurrence remains particularly high. Currently, reliable prognostic biomarkers of recurrence are lacking. We decided to explore the potential usefulness of pancreatic developmental regulators as biomarkers of PDAC relapse. METHODS: We analyzed by quantitative real-time PCR the mRNA of selected factors involved either in pancreatic organogenesis (ISL1, NEUROD1, NGN3, NKX2.2, NKX6.1, PAX4, PAX6, PDX1 and PTF1α) or associated with terminally committed pancreatic cells (CHGA, CHGB, GAD2, GCG, HNF6α, INS, KRT19, SYP) in 17 PDAC cell lines and in frozen tumor samples from 41 PDAC patients. RESULTS: High baseline levels of the ISL1, KRT19, PAX6 and PDX1 mRNAs in PDAC cell lines, were risk factors for time-dependent xenograft appearance after subcutaneous injection in CD1-Nude mice. Consistently, in human PDAC samples, high levels of KRT19 mRNA were associated with reduced overall survival and earlier recurrence. Higher levels of PDX1 or PAX6 mRNAs were instead associated with a higher frequency of local recurrence. CONCLUSIONS: Our findings suggest that selected factors associated with pancreas development or its terminal differentiation might be implicated in mechanisms of PDAC progression and/or metastatic spread and that the measurement of their mRNA in tumors might be potentially used to improve patient prognostic stratification and prediction of the relapse site.

The long noncoding RNA SUMO1P3 as urinary biomarker for monitoring bladder cancer progression.

Introduction: Urothelial Bladder Cancer (BC) is the ninth most common cancer worldwide. It is classified into Non Muscle Invasive (NMIBC) and Muscle Invasive Bladder Cancer (MIBC), which are characterized by frequent recurrences and progression rate, respectively. The diagnosis and monitoring are obtained through invasive methods as cystoscopy and post-surgery biopsies. Thus, a panel of biomarkers able to discriminate BC based on grading or staging represents a significant step forward in the patients' workup. In this perspective, long non-coding RNAs (lncRNAs) are emerged as reliable candidates as potential biomarker given their specific and regulated expression. In the present work we propose two lncRNAs, the Small Ubiquitin Modifier 1 pseudogene 3 (SUMO1P3), a poorly characterized pseudogene, and the Urothelial Carcinoma Associated 1 (UCA1) as candidates to monitor the BC progression. Methods: This study was a retrospective trial enrolling NMIBC and MIBC patients undergoing surgical intervention: the expression of the lncRNA SUMO1P3 and UCA1 was evaluated in urine from 113 subjects (cases and controls). The receiver operating characteristic curve analysis was used to evaluate the performance of single or combined biomarkers in discriminating cases from controls. Results: SUMO1P3 and UCA1 expression in urine was able to significantly discriminate low grade NMIBC, healthy control and benign prostatic hyperplasia subjects versus high grade NMIBC and MIBC patients. We also demonstrated that miR-320a, which binds SUMO1P3, was reduced in high grade NMIBC and MIBC patients and the SUMO1P3/miR-320a ratio was used to differentiate cases versus controls, showing a statistically significant power. Finally, we provided an automated method of RNA extraction coupled to ddPCR analysis in a perspective of clinical application. Discussion: We have shown that the lncRNA SUMO1P3 is increased in urine from patients with high grade NMIBC and MIBC and that it is likely to be good candidate to predict bladder cancer progression if used alone or in combination with UCA1 or with miRNA320a.

Improved Specificity of Glutamate Decarboxylase 65 Autoantibody Measurement Using Luciferase-Based Immunoprecipitation System Assays.

Autoantibodies to glutamate decarboxylase (GADA) are widely used in the prediction and classification of type 1 diabetes. GADA radiobinding assays (RBAs) using N-terminally truncated antigens offer improved specificity, but radioisotopes limit the high-throughput potential for population screening. Luciferase-based immunoprecipitation system (LIPS) assays are sensitive and specific alternatives to RBAs with the potential to improve risk stratification. The performance of assays using the Nanoluc luciferase (Nluc)-conjugated GAD65 constructs, Nluc-GAD65(96-585) and full length Nluc-GAD65(1-585), were evaluated in 434 well-characterized serum samples from patients with recent-onset type 1 diabetes and first-degree relatives. Nonradioactive, high-throughput LIPS assays are quicker and require less serum than RBAs. Of 171 relatives previously tested single autoantibody positive for autoantibodies to full-length GAD65 by RBA but had not progressed to diabetes, fewer retested positive by LIPS using either truncated (n = 72) or full-length (n = 111) antigen. The Nluc-GAD65(96-585) truncation demonstrated the highest specificity in LIPS assays overall, but in contrast to RBA, N-terminus truncations did not result in a significant increase in disease-specificity compared with the full-length antigen. This suggests that binding of nonspecific antibodies is affected by the conformational changes resulting from addition of the Nluc antigen. Nluc-GAD65(96-585) LIPS assays offer low-blood-volume, high-specificity GADA tests for screening and diagnostics.

Directed self-assembly of a xenogeneic vascularized endocrine pancreas for type 1 diabetes.

Intrahepatic islet transplantation is the standard cell therapy for ß cell replacement. However, the shortage of organ donors and an unsatisfactory engraftment limit its application to a selected patients with type 1 diabetes. There is an urgent need to identify alternative strategies based on an unlimited source of insulin producing cells and innovative scaffolds to foster cell interaction and integration to orchestrate physiological endocrine function. We previously proposed the use of decellularized lung as a scaffold for ß cell replacement with the final goal of engineering a vascularized endocrine organ. Here, we prototyped this technology with the integration of neonatal porcine islet and healthy subject-derived blood outgrowth endothelial cells to engineer a xenogeneic vascularized endocrine pancreas. We validated ex vivo cell integration and function, its engraftment and performance in a preclinical model of diabetes. Results showed that this technology not only is able to foster neonatal pig islet maturation in vitro, but also to perform in vivo immediately upon transplantation and for over 18 weeks, compared to normal performance within 8 weeks in various state of the art preclinical models. Given the recent progress in donor pig genetic engineering, this technology may enable the assembly of immune-protected functional endocrine organs.

Case Report: Favorable outcome of allogeneic hematopoietic stem cell transplantation in SARSCoV2 positive recipient, risk-benefit balance between infection and leukemia.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) in SARS-CoV-2 positive candidates is usually delayed until the clinical resolution of the infection's symptoms and a negative nasopharyngeal molecular test. However, prolonged SARS-CoV-2 positivity has been frequently observed in haematological malignancies, thus representing a challenge for the timing of transplant procedures. Here, we report on the case of a 34-year-old patient with recent pauci-symptomatic COVID-19 undergoing transplant for high-risk acute B-lymphoblastic leukemia before achieving viral clearance. Shortly before their scheduled allogeneic HSCT from a matched unrelated donor, the patient developed mild Omicron BA.5 infection receiving nirmatrelvir/ritonavir with fever resolution within 72 hours. Twenty-three days after COVID-19 diagnosis, because of increasing minimal residual disease values in the context of high-risk refractory leukemia and clinical resolution of SARS-2-CoV infection with reduction of viral load at surveillance nasopharyngeal swabs, it was decided not to delay further allo-HSCT. During myelo-ablative conditioning, the nasopharyngeal SARS-CoV-2 viral load increased while the patient remained asymptomatic. Consequently, two days before the transplant, intra-muscular tixagevimab/cilgavimab 300/300 mg and a 3-day course of intravenous remdesivir were administered. During the pre-engraftment phase, veno-occlusive disease (VOD) occurred at day +13, requiring defibrotide treatment to obtain a slow but complete recovery. The post-engraftment phase was characterized by mild COVID-19 at day +23 (cough, rhino-conjunctivitis, fever) that spontaneously resolved, achieving viral clearance at day +28. At day +32, she experienced grade I acute graft-versus host disease (a-GVHD, skin grade II) treated with steroids and photo-apheresis, without further complications during follow-up until day +180. Addressing the issue of allo-HSCT timing in patients recovering from SARS-CoV-2 infection with high-risk malignant diseases is challenging because of 1] the high risk of COVID-19 clinical progression, 2] the impact of transplant delay on leukemia prognosis and 3] the occurrence of endothelial complications such as VOD, a-GVHD, and transplant associated thrombotic micro-angiopathy. Our report describes the favourable outcome of allo-HSCT in a recipient with active SARS-CoV2 infection and high-risk leukemia thanks to timely anti-SARS-CoV-2 preventive therapies and prompt management of transplant-related complications.

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 < 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 <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>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.

A Case Study to Dissect Immunity to SARS-CoV-2 in a Neonate Nonhuman Primate Model.

Most children are less severely affected by coronavirus-induced disease 2019 (COVID-19) than adults, and thus more difficult to study progressively. Here, we provide a neonatal nonhuman primate (NHP) deep analysis of early immune responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in blood and mucosal tissues. In addition, we provide a comparison with SARS-CoV-2-infected adult NHP. Infection of the neonate resulted in a mild disease compared with adult NHPs that develop, in most cases, moderate lung lesions. In concomitance with the viral RNA load increase, we observed the development of an early innate response in the blood, as demonstrated by RNA sequencing, flow cytometry, and cytokine longitudinal data analyses. This response included the presence of an antiviral type-I IFN gene signature, a persistent and lasting NKT cell population, a balanced peripheral and mucosal IFN-γ/IL-10 cytokine response, and an increase in B cells that was accompanied with anti-SARS-CoV-2 antibody response. Viral kinetics and immune responses coincided with changes in the microbiota profile composition in the pharyngeal and rectal mucosae. In the mother, viral RNA loads were close to the quantification limit, despite the very close contact with SARS-CoV-2-exposed neonate. This pilot study demonstrates that neonatal NHPs are a relevant model for pediatric SARS-CoV-2 infection, permitting insights into the early steps of anti-SARS-CoV-2 immune responses in infants.

Natural history of type 1 diabetes on an immunodysregulatory background with genetic alteration in B-cell activating factor receptor: A case report.

The immunological events leading to type 1 diabetes (T1D) are complex and heterogeneous, underscoring the necessity to study rare cases to improve our understanding. Here, we report the case of a 16-year-old patient who showed glycosuria during a regular checkup. Upon further evaluation, stage 2 T1D, autoimmune thrombocytopenic purpura (AITP), and common variable immunodeficiency (CVID) were diagnosed. The patient underwent low carb diet, losing > 8 kg, and was placed on Ig replacement therapy. Anti-CD20 monoclonal antibody (Rituximab, RTX) was administered 2 years after diagnosis to treat peripheral polyneuropathy, whereas an atypical mycobacteriosis manifested 4 years after diagnosis and was managed with prolonged antibiotic treatment. In the fifth year of monitoring, the patient progressed to insulin dependency despite ZnT8A autoantibody resolution and IA-2A and GADA autoantibody decline. The patient had low T1D genetic risk score (GRS = 0.22817) and absence of human leukocyte antigen (HLA) DR3/DR4-DQ8. Genetic analysis identified the monoallelic mutation H159Y in TNFRSF13C, a gene encoding B-cell activating factor receptor (BAFFR). Significant reduced blood B-cell numbers and BAFFR levels were observed in line with a dysregulation in BAFF-BAFFR signaling. The elevated frequency of PD-1+ dysfunctional Tfh cells composed predominantly by Th1 phenotype was observed at disease onset and during follow-up. This case report describes a patient progressing to T1D on a BAFFR-mediated immunodysregulatory background, suggesting a role of BAFF-BAFFR signaling in islet-specific tolerance and T1D progression.

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.

COVID-19 in Solid Organ Transplant Recipient: Exploring Cumulative Incidence, Seroprevalence and Risk Factors for Disease Severity.

BACKGROUND: Solid organ transplant (SOT) recipients may be at increased risk for severe disease and mortality from COVID-19 because of immunosuppression and prolonged end-stage organ disease. As a transplant center serving a diverse patient population, we report the cumulative incidence and outcomes of SARS-CoV-2 infection in our cohort of SOT recipients. METHODS: We prospectively included in this observational study SOT recipients with a functioning kidney (n = 201), pancreas ± kidney (n = 66) or islet transplant (n = 24), attending outpatient regular follow-up at the San Raffaele Hospital from February 2020 to April 2021. Antibodies to SARS-CoV-2 were tested in all patients by a luciferase immunoprecipitation system assay. RESULTS: Of the 291 SOT recipients, 30 (10.3%) tested positive for SARS-CoV-2 during the study period and prevalence was not different among different transplants. The SARS-CoV-2 antibody frequency was around 2.6-fold higher than the incidence of cases who tested positive for SARS-CoV-2 RT-PCR. As for the WHO COVID-19 severity classification, 19 (63.3%) SOT recipients were mild, nine (30%) were moderate, and two were critical and died yielding a crude mortality rate in our patient population of 6.7%. Kidney transplant (OR 12.9 (1.1-150) p = 0.041) was associated with an increased risk for moderate/critical disease, while statin therapy (OR 0.116 (0.015-0.926) p = 0.042) and pancreas/islet transplant (OR 0.077 (0.007-0.906) p = 0.041) were protective. CONCLUSIONS: The incidence of SARS-CoV-2 infection in SOT recipients may be higher than previously described. Due to the relative high crude mortality, symptomatic SOT recipients must be considered at high risk in case of SARS-CoV-2 infection.

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-Q-plot analysis (negative: <2.5 arbitrary units). RESULTS: IFA levels were higher in cases than in controls (Mann-Whitney:p < 10-5). The ROC-AUC was 0.67 (95%CI 0.60-0.73, p < 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 <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.