GATA4 controls regionalization of tissue immunity and commensal-driven immunopathology.

There is growing recognition that regionalization of bacterial colonization and immunity along the intestinal tract has an important role in health and disease. Yet, the mechanisms underlying intestinal regionalization and its dysregulation in disease are not well understood. This study found that regional epithelial expression of the transcription factor GATA4 controls bacterial colonization and inflammatory tissue immunity in the proximal small intestine by regulating retinol metabolism and luminal IgA. Furthermore, in mice without jejunal GATA4 expression, the commensal segmented filamentous bacteria promoted pathogenic inflammatory immune responses that disrupted barrier function and increased mortality upon Citrobacter rodentium infection. In celiac disease patients, low GATA4 expression was associated with metabolic alterations, mucosal Actinobacillus, and increased IL-17 immunity. Taken together, these results reveal broad impacts of GATA4-regulated intestinal regionalization on bacterial colonization and tissue immunity, highlighting an elaborate interdependence of intestinal metabolism, immunity, and microbiota in homeostasis and disease.

IL-15, gluten and HLA-DQ8 drive tissue destruction in coeliac disease.

Coeliac disease is a complex, polygenic inflammatory enteropathy caused by exposure to dietary gluten that occurs in a subset of genetically susceptible individuals who express either the HLA-DQ8 or HLA-DQ2 haplotypes1,2. The need to develop non-dietary treatments is now widely recognized3, but no pathophysiologically relevant gluten- and HLA-dependent preclinical model exists. Furthermore, although studies in humans have led to major advances in our understanding of the pathogenesis of coeliac disease4, the respective roles of disease-predisposing HLA molecules, and of adaptive and innate immunity in the development of tissue damage, have not been directly demonstrated. Here we describe a mouse model that reproduces the overexpression of interleukin-15 (IL-15) in the gut epithelium and lamina propria that is characteristic of active coeliac disease, expresses the predisposing HLA-DQ8 molecule, and develops villous atrophy after ingestion of gluten. Overexpression of IL-15 in both the epithelium and the lamina propria is required for the development of villous atrophy, which demonstrates the location-dependent central role of IL-15 in the pathogenesis of coeliac disease. In addition, CD4+ T cells and HLA-DQ8 have a crucial role in the licensing of cytotoxic T cells to mediate intestinal epithelial cell lysis. We also demonstrate a role for the cytokine interferon-γ (IFNγ) and the enzyme transglutaminase 2 (TG2) in tissue destruction. By reflecting the complex interaction between gluten, genetics and IL-15-driven tissue inflammation, this mouse model provides the opportunity to both increase our understanding of coeliac disease, and develop new therapeutic strategies.

How Future Pharmacologic Therapies for Celiac Disease Will Complement the Gluten-Free Diet.

The only proven treatment for celiac disease is adherence to a strict, lifelong, gluten-free diet. However, complete dietary gluten avoidance is challenging and a substantial number of patients do not respond fully, clinically, or histologically, despite their best efforts. As celiac disease is common and its central pathophysiology is well elucidated, it has become attractive for drug development to address the limitations of dietary treatment. Most efforts address nonresponsive celiac disease, defined as continued symptoms and/or signs of disease activity despite a gluten-free diet, and the more severe forms of refractory celiac disease, types I and II. An increasing spectrum of therapeutic approaches target defined mechanisms in celiac disease pathogenesis and some have advanced to current phase 2 and 3 clinical studies. We discuss these approaches in terms of potential efficiency, practicability, safety, and need, as defined by patients, regulatory authorities, health care providers, and payors.

High Cardiac Troponin Levels in Infants with Acute SARS-CoV-2 Infection: A Prospective Comparative Study.

OBJECTIVE: To investigate the specific role of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in inducing elevation of marker of myocardial injury in infants with acute coronavirus disease 2019 (COVID-19). STUDY DESIGN: A prospective, multicentric 3-arm comparative study (March 2020 through March 2022) enrolling 152 infants hospitalized for COVID-19, 79 children with acute infections other than SARS-CoV-2, and 71 healthy controls. Determination of high-sensitivity cardiac troponin (hs-cTn) levels was the primary outcome. RESULTS: The proportion of children with hs-cTn values above the upper limit of normal (44 [28.9%]), as well as with a 3-fold increased value (20 [13.2%]) were significantly higher in the COVID-19 group than those in both control groups. The risk of presenting a 3-fold increased hs-cTn value was higher in children with SARS-CoV-2 infection compared with either healthy children (OR, 5.23; 95% CI, 1.19-23.02) or those with other infections (OR, 11.89; 95% CI, 1.56-89.79). In children with COVID-19, hs-cTn elevation was associated with neither clinical nor biochemical characteristics, nor perinatal risk factors, but with an age of <3 months (P < .001). After adjustment for age, sex, and underlying clinical conditions, elevated hs-cTn was independently associated with COVID-19 in a multivariable regression model. All children showed a progressive reduction of hs-cTn until normalization over time, without clinical, ECG, or echocardiographic manifestations up to 1 year of follow-up. CONCLUSIONS: Infants with acute SARS-CoV-2 infection may show a subclinical and transient alteration of myocardial injury markers, especially in the first months of life. hs-cTn levels normalized during follow-up and were not associated with cardiac functional impairment; nevertheless, long-term consequences are unknown and should be followed carefully.

ESPGHAN Position Paper on Management and Follow-up of Children and Adolescents With Celiac Disease.

OBJECTIVES: To gather the current evidence and to offer recommendations for follow-up and management. METHODS: The Special Interest Group on Celiac Diseases of the European Society of Paediatric Gastroenterology Hepatology and Nutrition formulated ten questions considered to be essential for follow-up care. A literature search (January 2010-March 2020) was performed in PubMed or Medline. Relevant publications were identified and potentially eligible studies were assessed. Statements and recommendations were developed and discussed by all coauthors. Recommendations were voted upon: joint agreement was set as at least 85%. RESULTS: Publications (n = 2775) were identified and 164 were included. Using evidence or expert opinion, 37 recommendations were formulated on: The need to perform follow-up, its frequency and what should be assessed, how to assess adherence to the gluten-free diet, when to expect catch-up growth, how to treat anemia, how to approach persistent high serum levels of antibodies against tissue-transglutaminase, the indication to perform biopsies, assessment of quality of life, management of children with unclear diagnosis for which a gluten-challenge is indicated, children with associated type 1 diabetes or IgA deficiency, cases of potential celiac disease, which professionals should perform follow-up, how to improve the communication to patients and their parents/caregivers and transition from pediatric to adult health care. CONCLUSIONS: We offer recommendations to improve follow-up of children and adolescents with celiac disease and highlight gaps that should be investigated to further improve management.

Identification of a γc Receptor Antagonist That Prevents Reprogramming of Human Tissue-resident Cytotoxic T Cells by IL15 and IL21.

BACKGROUND & AIMS: Gamma chain (γc) cytokines (interleukin [IL]2, IL4, IL7, IL9, IL15, and IL21) signal via a common γc receptor. IL2 regulates the immune response, whereas IL21 and IL15 contribute to development of autoimmune disorders, including celiac disease. We investigated whether BNZ-2, a peptide designed to inhibit IL15 and IL21, blocks these cytokines selectively and its effects on intraepithelial cytotoxic T cells. METHODS: We obtained duodenal biopsies from 9 patients with potential celiac disease (positive results from tests for anti-TG2 but no villous atrophy), 30 patients with untreated celiac disease (with villous atrophy), and 5 patients with treated celiac disease (on a gluten-free diet), as well as 43 individuals without celiac disease (controls). We stimulated primary intestinal intraepithelial CD8+ T-cell lines, or CD8+ T cells directly isolated from intestinal biopsies, with γc cytokines in presence or absence of BNZ-2. Cells were analyzed by immunoblots, flow cytometry, or RNA-sequencing analysis for phosphorylation of signaling molecules, gene expression profiles, proliferation, and levels of granzyme B. RESULTS: Duodenal tissues from patients with untreated celiac disease had increased levels of messenger RNAs encoding IL15 receptor subunit alpha (IL15RA) and IL21 compared with tissues from patients with potential celiac disease and controls. Activation of intraepithelial cytotoxic T cells with IL15 or IL21 induced separate signaling pathways; incubation of the cells with IL15 and IL21 cooperatively increased their transcriptional activity, proliferation, and cytolytic properties. BNZ-2 specifically inhibited the effects of IL15 and IL21, but not of other γc cytokines. CONCLUSIONS: We found increased expression of IL15RA and IL21 in duodenal tissues from patients with untreated celiac disease compared with controls. IL15 and IL21 cooperatively activated intestinal intraepithelial cytotoxic T cells. In particular, they increased their transcriptional activity, proliferation, and cytolytic activity. The peptide BNZ-2 blocked these effects, but not those of other γc cytokines, including IL2. BNZ-2 might be used to prevent cytotoxic T-cell-mediated tissue damage in complex immune disorders exhibiting upregulation of IL15 and IL21.

Pediatric Celiac Disease Patients Show Alterations of Dendritic Cell Shape and Actin Rearrangement.

Celiac disease (CD) is a frequent intestinal inflammatory disease occurring in genetically susceptible individuals upon gluten ingestion. Recent studies point to a role in CD for genes involved in cell shape, adhesion and actin rearrangements, including a Rho family regulator, Rho GTPase-activating protein 31 (ARHGAP31). In this study, we investigated the morphology and actin cytoskeletons of peripheral monocyte-derived dendritic cells (DCs) from children with CD and controls when in contact with a physiological substrate, fibronectin. DCs were generated from peripheral blood monocytes of pediatric CD patients and controls. After adhesion on fibronectin, DCs showed a higher number of protrusions and a more elongated shape in CD patients compared with controls, as assessed by immunofluorescence actin staining, transmitted light staining and video time-lapse microscopy. These alterations did not depend on active intestinal inflammation associated with gluten consumption and were specific to CD, since they were not found in subjects affected by other intestinal inflammatory conditions. The elongated morphology was not a result of differences in DC activation or maturation status, and did not depend on the human leukocyte antigen (HLA)-DQ2 haplotype. Notably, we found that ARH-GAP31 mRNA levels were decreased while RhoA-GTP activity was increased in CD DCs, pointing to an impairment of the Rho pathway in CD cells. Accordingly, Rho inhibition was able to prevent the cytoskeleton rearrangements leading to the elongated morphology of celiac DCs upon adhesion on fibronectin, confirming the role of this pathway in the observed phenotype. In conclusion, adhesion on fibronectin discriminated CD from the controls' DCs, revealing a gluten-independent CD-specific cellular phenotype related to DC shape and regulated by RhoA activity.

Progression of Celiac Disease in Children With Antibodies Against Tissue Transglutaminase and Normal Duodenal Architecture.

BACKGROUND & AIMS: Potential celiac disease is characterized by positive results from serologic tests for tissue transglutaminase antibodies (anti-TG2) but normal duodenal architecture (Marsh stages 0-1). There is controversy over the best way to manage these patients. We investigated risk factors associated with the development of villous atrophy in children with potential celiac disease. METHODS: We performed a prospective study of 280 children (ages 2-18 years) in Italy with suspected celiac disease, followed for up to 12 years (range, 18-150 months; median 60 months). The subjects had 2 consecutive positive results from tests for anti-TG2, tested positive for the endomysial antibody (anti-EMA), had total serum levels of immunoglobulin A in the normal range, normal duodenal architecture (Marsh stages 0-1) in 5 biopsies, and HLA DQ2- or DQ8-positive haplotypes. The children underwent serologic tests and clinical analyses every 6 months and a small bowel biopsy was taken every 2 years. A total of 210 patients of the original cohort were assessed at the 9-year follow-up evaluation. We performed multivariate analyses of clinical, genetic, and histologic data to identify factors associated with progression to villous atrophy. RESULTS: During the follow-up period, 42 (15%) of 280 children developed villous atrophy, whereas 89 (32%) children no longer tested positive for anti-TG2 or anti-EMA. The cumulative incidence of progression to villous atrophy was 43% at 12 years. In multivariate analysis, the baseline factors most strongly associated with development of villous atrophy were numbers of γδ intraepithelial lymphocyte cells followed by age and homozygosity for the HLA DQB1*02. In discriminant analysis, these baseline factors identified 80% of the children who developed baseline atrophy. CONCLUSIONS: In a long-term study of 280 children with suspected celiac disease (based on anti-TG2 and anti-EMA) on gluten-containing diets, the cumulative incidence of progression to villous atrophy was 43% over a 12-year period. We identified factors that can be used to identify children at highest risk for villous atrophy. This approach might be used to determine whether children with suspected celiac disease should immediately start a gluten-free diet or be monitored on their regular diet.

The Effect of Gluten-free Diet on Clinical Symptoms and the Intestinal Mucosa of Patients With Potential Celiac Disease.

In this prospective study, we evaluated the effect of gluten-free diet (GFD) in a cohort of 65 children with potential celiac disease. Patients received GFD for signs/symptoms (N = 47) or parents' choice (N = 18). Most frequent signs/symptoms were low body mass index (36%), recurrent abdominal pain (34%), and diarrhea (19%). Of the 35/47 patients followed-up on GFD, only 54% (19/35) showed a complete clinical response. In 9 of 65 patients an intestinal biopsy was also performed after at least 1 year of GFD. No significant differences were observed in terms of Marsh grade (P = 0.33), lamina propria CD25+ cells (P = 0.80), CD3+ (P = 0.9), and γδ+ (P = 0.59) intraepithelial lymphocytes density and intestinal anti-TG2 deposits (P = 0.60). In conclusion, caution is necessary before attributing all symptoms to gluten in this condition.

Does infant feeding modulate the manifestation of celiac disease and type 1 diabetes?

PURPOSE OF REVIEW: The review aims to critically discuss the role of infant feeding in the development of celiac disease and type 1 diabetes (T1D). RECENT FINDINGS: Prospective observational and randomized interventional studies show that breastfeeding (BF) or BF during gluten introduction does not reduce the risk of developing CD, but high gluten consumption before age 2 years increased the risk in Swedish children.Despite evidence from retrospective studies, prospective trials failed to find a protective effect of breastfeeding against the risk of T1D development. Nevertheless, breastfeeding at the time of cereal introduction decreases this risk. There is some evidence demonstrating that early exposure to sugar-sweetened beverages increases the risk of T1D in childhood, whereas the timing of gluten introduction, except if introduced very early, does not affect it. SUMMARY: Breastfeeding and/or timing of gluten introduction does not influence celiac disease risk. Breastfeeding at the time of cereal introduction might be protective against T1D. The introduction of certain solid foods at an early age may be associated with the risk of T1D.

Distinct and Synergistic Contributions of Epithelial Stress and Adaptive Immunity to Functions of Intraepithelial Killer Cells and Active Celiac Disease.

BACKGROUND & AIMS: The mechanisms of tissue destruction during progression of celiac disease are poorly defined. It is not clear how tissue stress and adaptive immunity contribute to the activation of intraepithelial cytotoxic T cells and the development of villous atrophy. We analyzed epithelial cells and intraepithelial cytotoxic T cells in family members of patients with celiac disease, who were without any signs of adaptive antigluten immunity, and in potential celiac disease patients, who have antibodies against tissue transglutaminase 2 in the absence of villous atrophy. METHODS: We collected blood and intestinal biopsy specimens from 268 patients at tertiary medical centers in the United States and Italy from 2004 to 2012. All subjects had normal small intestinal histology. Study groups included healthy individuals with no family history of celiac disease or antibodies against tissue transglutaminase 2 (controls), healthy family members of patients with celiac disease, and potential celiac disease patients. Intraepithelial cytotoxic T cells were isolated and levels of inhibitory and activating natural killer (NK) cells were measured by flow cytometry. Levels of heat shock protein (HSP) and interleukin 15 were measured by immunohistochemistry, and ultrastructural alterations in intestinal epithelial cells (IECs) were assessed by electron microscopy. RESULTS: IECs from subjects with a family history of celiac disease, but not from subjects who already had immunity to gluten, expressed higher levels of HS27, HSP70, and interleukin-15 than controls; their IECs also had ultrastructural alterations. Intraepithelial cytotoxic T cells from relatives of patients with celiac disease expressed higher levels of activating NK receptors than cells from controls, although at lower levels than patients with active celiac disease, and without loss of inhibitory receptors for NK cells. Intraepithelial cytotoxic T cells from potential celiac disease patients failed to up-regulate activating NK receptors. CONCLUSIONS: A significant subset of healthy family members of patients with celiac disease with normal intestinal architecture had epithelial alterations, detectable by immunohistochemistry and electron microscopy. The adaptive immune response to gluten appears to act in synergy with epithelial stress to allow intraepithelial cytotoxic T cells to kill epithelial cells and induce villous atrophy in patients with active celiac disease.

In the Intestinal Mucosa of Children With Potential Celiac Disease IL-21 and IL-17A are Less Expressed than in the Active Disease.

OBJECTIVES: Potential celiac disease (CD) patients are at an increased risk to developing CD as indicated by positive CD-associated serology. We investigated in duodenal mucosa of such patients the presence of both IL-21 and IL-17A and the role of gliadin peptides and IL-15 in their expression. METHODS: Duodenal biopsies from 76 active CD, 90 potential CD, and 58 control patients were analyzed for IL-21 and/or IL-17A production by quantitative real-time PCR, immunohistochemistry, flow cytometry, and ELISA. The presence of IL-21 receptor was investigated by western blot. Potential CD duodenal fragments were cultured with gliadin peptides (PTG) and/or IL-15 and the expression/production of IL-21 and IL-17A assessed by quantitative real-time PCR and by immunohistochemistry. RESULTS: In potential CD, IL-21 was lower than in active CD, in terms of RNA expression (P<0.01), density of lamina propria (LP) IL-21(+) cells (P<0.05), and protein secretion (P<0.05). Also, IL-21R was weakly detectable in potential CD. Several LP cell types produced IL-21 in CD. In potential CD, CD4(+)IL-21(+) cells increased after PMA-ionomycin stimulation and co-produced IFN-γ but not IL-17A. After 24 hours of culture stimulation with PTG, IL-21-producing cells increased but not the ones producing IL-17A. This increase was further enhanced by the addition of IL-15 to culture medium. CONCLUSIONS: In potential CD, IL-21 is less expressed than in active CD; however, IL-21-producing cells are present and prone to respond after specific stimuli. This suggests a key role of IL-21 in the progression of mucosal damage in CD.

Metagenomics Reveals Dysbiosis and a Potentially Pathogenic N. flavescens Strain in Duodenum of Adult Celiac Patients.

OBJECTIVES: Celiac disease (CD)-associated duodenal dysbiosis has not yet been clearly defined, and the mechanisms by which CD-associated dysbiosis could concur to CD development or exacerbation are unknown. In this study, we analyzed the duodenal microbiome of CD patients. METHODS: The microbiome was evaluated in duodenal biopsy samples of 20 adult patients with active CD, 6 CD patients on a gluten-free diet, and 15 controls by DNA sequencing of 16S ribosomal RNA libraries. Bacterial species were cultured, isolated and identified by mass spectrometry. Isolated bacterial species were used to infect CaCo-2 cells, and to stimulate normal duodenal explants and cultured human and murine dendritic cells (DCs). Inflammatory markers and cytokines were evaluated by immunofluorescence and ELISA, respectively. RESULTS: Proteobacteria was the most abundant and Firmicutes and Actinobacteria the least abundant phyla in the microbiome profiles of active CD patients. Members of the Neisseria genus (Betaproteobacteria class) were significantly more abundant in active CD patients than in the other two groups (P=0.03). Neisseria flavescens (CD-Nf) was the most abundant Neisseria species in active CD duodenum. Whole-genome sequencing of CD-Nf and control-Nf showed genetic diversity of the iron acquisition systems and of some hemoglobin-related genes. CD-Nf was able to escape the lysosomal compartment in CaCo-2 cells and to induce an inflammatory response in DCs and in ex-vivo mucosal explants. CONCLUSIONS: Marked dysbiosis and an abundance of a peculiar CD-Nf strain characterize the duodenal microbiome in active CD patients thus suggesting that the CD-associated microbiota could contribute to the many inflammatory signals in this disorder.

Mulibrey nanism: Two novel mutations in a child identified by Array CGH and DNA sequencing.

In childhood, several rare genetic diseases have overlapping symptoms and signs, including those regarding growth alterations, thus the differential diagnosis is sometimes difficult. The proband, aged 3 years, was suspected to have Silver-Russel syndrome because of intrauterine growth retardation, postnatal growth retardation, typical facial dysmorphic features, macrocephaly, body asymmetry, and bilateral fifth finger clinodactyly. Other features were left atrial and ventricular enlargement and patent foramen ovale. Total X-ray skeleton showed hypoplasia of the twelfth rib bilaterally and of the coccyx, slender long bones with thick cortex, and narrow medullary channels. The genetic investigation did not confirm Silver-Russel syndrome. At the age of 5 the patient developed an additional sign: hepatomegaly. Array CGH revealed a 147 kb deletion (involving TRIM 37 and SKA2 genes) on one allele of chromosome 17, inherited from his mother. These results suggested Mulibrey nanism. The clinical features were found to fit this hypothesis. Sequencing of the TRIM 37 gene showed a single base change at a splicing locus, inherited from his father that provoked a truncated protein. The combined use of Array CGH and DNA sequencing confirmed diagnosis of Mulibrey nanism. The large deletion involving the SKA2 gene, along with the increased frequency of malignant tumours in mulibrey patients, suggests closed monitoring for cancer of our patient and his mother. Array CGH should be performed as first tier test in all infants with multiple anomalies. The clinician should reconsider the clinical features when the genetics suggests this. © 2016 Wiley Periodicals, Inc.

Insulin-dependent diabetes induced by pancreatic beta cell expression of IL-15 and IL-15Rα.

Increased serum levels of IL-15 are reported in type 1 diabetes (T1D). Here we report elevated serum soluble IL-15Rα levels in human T1D. To investigate the role of IL-15/IL-15Rα in the pathogenesis of T1D, we generated double transgenic mice with pancreatic ß-cell expression of IL-15 and IL-15Rα. The mice developed hyperglycemia, marked mononuclear cell infiltration, ß-cell destruction, and anti-insulin autoantibodies that mimic early human T1D. The diabetes in this model was reversed by inhibiting IL-15 signaling with anti-IL2/IL15Rß (anti-CD122), which blocks IL-15 transpresentation. Furthermore, the diabetes could be reversed by administration of the Janus kinase 2/3 inhibitor tofacitinib, which blocks IL-15 signaling. In an alternative diabetes model, nonobese diabetic mice, IL15/IL-15Rα expression was increased in islet cells in the prediabetic stage, and inhibition of IL-15 signaling with anti-CD122 at the prediabetic stage delayed diabetes development. In support of the view that these observations reflect the conditions in humans, we demonstrated pancreatic islet expression of both IL-15 and IL-15Rα in human T1D. Taken together our data suggest that disordered IL-15 and IL-15Rα may be involved in T1D pathogenesis and the IL-15/IL15Rα system and its signaling pathway may be rational therapeutic targets for early T1D.

Does gluten intake influence the development of celiac disease-associated complications?

Celiac disease (CD) is regarded as the most common autoimmune enteropathy in western countries. Epidemiological studies indicate that approximately 1:100 individuals may present with histologically proven CD. CD develops in genetically predisposed subjects after gluten ingestion. It usually subsides after gluten is withdrawn from their diet. Gluten is the only known environmental factor that affects the progression/regression of the intestinal villous atrophy, which is the hallmark of this disease. CD generally follows a benign course after gluten elimination. However, it is also associated with the development of other autoimmune disorders or of intestinal malignancies. The issue of whether such complications, sometimes of significant clinical and prognostic impact, are or are not the result of ongoing gluten ingestion, is an important one that has been investigated over the recent years with conflicting results. In terms of practical implications, the presence of a positive correlation between gluten intake and the development of severe complications would lead to the need for early diagnosis and mass screening. The lack of such correlation would instead suggest a less aggressive diagnostic strategy. This review aims at critically summarizing the evidence supporting either hypothesis.

Celiac disease and autoimmunity.

Celiac disease (CD) has a multifactorial etiology with complex genetics and frequently occurs in association with other autoimmune disorders. Even though triggered by a dietary antigen, it shows many autoimmune features, the most peculiar being the presence of high titers of anti-tissue transglutaminase 2 autoantibodies, produced in the small intestinal mucosa since the early stages of the disease. More than 60% of CD-associated susceptibility loci are shared with at least another autoimmune condition, suggesting common pathogenic mechanisms. In particular, recognition of peptides by HLA molecules, posttranslational modifications required for optimal peptide binding and immune mechanisms leading to tissue damage have been found in CD as well as in other autoimmune diseases. This review briefly summarizes the main autoimmune features of CD, underlining the similarities with other autoimmune disorders, in particular with type 1 diabetes mellitus. Furthermore, the role of gluten and microbiome in driving autoimmunity is discussed.

Combined RNAscope and immunohistochemistry staining on duodenal paraffin sections as a new tool to reveal cytolytic potential of intraepithelial lymphocytes.

Immunohistochemistry (IHC) is a consolidated technique for the identification of surface and cytoplasmic antigens in cells or tissue sections using specific antibodies, yet simultaneous detection of two markers on the same cell may be difficult to achieve. Here we develop a protocol to perform a double staining using RNAscope, a new in-situ hybridization (ISH) technology, to visualize perforin transcripts, and classical IHC to visualize either CD8 or TcRγδ positive intraepithelial lymphocytes (IELs) in small intestinal paraffin sections of celiac disease (CD) patients. This double assay will allow to investigate the cytotoxic properties of two subsets of IELs in different stages of CD, thus contributing to understand the events leading to tissue destruction and healing.

Increased Relapse Rate During COVID-19 Lockdown in an Italian Cohort of Children With Juvenile Idiopathic Arthritis.

OBJECTIVE: Changes of routine disease management associated with COVID-19 lockdown might have potentially affected the clinical course of juvenile idiopathic arthritis (JIA). The aim of our study was to assess the rate of disease flare before and during COVID-19 lockdown to investigate its impact on disease course in children with JIA. METHODS: A single-center retrospective study was conducted, including patients presenting with inactive JIA between September 1, 2018 and March 9, 2019 (group A) and between September 1, 2019 and March 9, 2020 (group B). For each patient, demographic and clinical data were collected. The rate of JIA flare from March 10, 2019 to June 30, 2019 for group A and from March 10, 2020 to June 30, 2020 for group B was compared. RESULTS: Group A included 126 patients, and group B 124 patients. Statistical analysis did not show significant differences among the 2 cohorts with respect to age, sex, age at JIA onset, JIA subtype, co-occurrence of uveitis, antinuclear antibody positivity, and past or ongoing medications. The rate of disease flare during lockdown at the time of the first COVID-19 pandemic wave was significantly higher in comparison to the previous year (16.9% versus 6.3%; P = 0.009). CONCLUSION: Our study showed that COVID-19 lockdown was associated with a higher rate of joint inflammation in children with JIA. This finding has a considerable clinical implication, as restrictive measures may be necessary in order to contain pandemics. Our data highlight the need for rearrangement in the home and health care management of children with JIA during lockdowns.