Proteomic mapping identifies serum marker signatures associated with MIS-C specific hyperinflammation and cardiovascular manifestation.

Multisystem inflammatory syndrome in children (MIS-C) shares several clinical and immunological features with Kawasaki Disease (KD) and pediatric hyperinflammation, but the immuno-phenotypic overlap among these clinical mimics is still incompletely understood. Here we analyzed serum samples from treatment-naïve patients with MIS-C (n = 31) and KD (n = 11), pediatric hyperinflammation (n = 13) and healthy controls (HC, n = 10) by proximity extension assay (PEA) to profile 184 blood biomarkers. Collectively, immunophenotypic overlap between MIS-C and hyperinflammation exceeds overlap with KD. Overexpression of IL-17A in MIS-C and KD could best separate these conditions from hyperinflammatory conditions, while those were hallmarked by overabundance of adenosin deaminase and IL-18. Depletion in serum TNF-related subfamily member 9 (TNFRSF9) and apoptosis inducing ligand (TRAIL) linked with cardiovascular manifestations and myocarditis in MIS-C. Altogether, our analysis highlights important differences in molecular marker signatures also across different MIS-C and KD cohorts and suggests several previously unidentified molecular associations in context of cardiovascular inflammation.

Calculating the fraction of Kawasaki disease potentially attributable to seasonal pathogens: a time series analysis.

Background: Kawasaki disease is an acute, febrile, systemic vasculitis of children that primarily affects medium-sized blood vessels with a tropism for the coronary arteries. Although the etiological factors remain unknown, infections have been suggested as the trigger of Kawasaki disease. We sought to calculate the fraction of Kawasaki disease potentially attributable to seasonal infections. Methods: This cohort study used a population-based time series analysis from the French hospitalisation database (Programme de Médicalisation des Systèmes d'Information), which includes all inpatients admitted to any public or private hospital in France. We included all children aged 0-17 years hospitalised for Kawasaki disease in France over 13 years. The monthly incidence of Kawasaki disease per 10,000 children over time was analysed by a quasi-Poisson regression model. The model accounted for seasonality by using harmonic terms (a pair of sines and cosines with 12-month periods). The circulation of eight common seasonal pathogens (adenovirus, influenza, metapneumovirus, Mycoplasma pneumoniae, norovirus, rhinovirus, rotavirus, respiratory syncytial virus, and Streptococcus pneumonia) over the same period was included in the model to analyse the fraction of Kawasaki disease potentially attributable to each pathogen. Infections were identified on the basis of polymerase chain reaction or rapid antigen testing in hospital laboratories. Findings: Between Jan 1, 2007, and Dec 31, 2019, we included 10,337 children with Kawasaki disease and 442,762 children with the selected infectious diseases. In the Kawasaki disease cohort, the median age [IQR] was 2 [0-4] years, 6164 [59.6%] were boys. Adenovirus infection was potentially responsible for 24.4% [21.5-27.8] (p < 0.001) of Kawasaki diseases, Norovirus for 6.7% [1.3-11.2] (p = 0.002), and RSV 4.6% [1.2-7.8] (p = 0.022). Sensitivity analyses found similar results. Interpretation: This cohort study of data from a comprehensive national hospitalisation database indicated that approximately 35% of Kawasaki diseases was potentially attributable to seasonal infections. Funding: None.

Crohn's Disease: Is the Cold Chain Hypothesis Still Hot?

Crohn's disease [CD] is an inflammatory bowel disease of unknown aetiology. During recent decades, significant technological advances led to development of -omic datasets allowing a detailed description of the disease. Unfortunately these have not, to date, resolved the question of the aetiology of CD. Thus, it may be necessary to [re]consider hypothesis-driven approaches to resolve the aetiology of CD. According to the cold chain hypothesis, the development of industrial and domestic refrigeration has led to frequent exposure of human populations to bacteria capable of growing in the cold. These bacteria, at low levels of exposure, particularly those of the genus Yersinia, are believed to be capable of inducing exacerbated inflammation of the intestine in genetically predisposed subjects. We discuss the consistency of this working hypothesis in light of recent data from epidemiological, clinical, pathological, microbiological, and molecular studies.

Persistent osteoarticular pain in children: early clinical and laboratory findings suggestive of acute lymphoblastic leukemia (a multicenter case-control study of 147 patients).

BACKGROUND: The aim of this study was to identify early clinical and laboratory features that distinguish acute lymphoblastic leukemia (ALL) from juvenile idiopathic arthritis (JIA) in children presenting with persistent bone or joint pain for at least 1 month. METHODS: We performed a multicenter case-control study and reviewed medical records of children who initially presented with bone or joint pain lasting for at least 1 month, all of whom were given a secondary diagnosis of JIA or ALL, in four French University Hospitals. Each patient with ALL was paired by age with two children with JIA. Logistic regression was used to compare clinical and laboratory data from the two groups. RESULTS: Forty-nine children with ALL and 98 with JIA were included. The single most important feature distinguishing ALL from JIA was the presence of hepatomegaly, splenomegaly or lymphadenopathy; at least one of these manifestations was present in 37 cases with ALL, but only in 2 controls with JIA, for an odds ratio (OR) of 154 [95%CI: 30-793] (regression coefficient: 5.0). If the presence of these findings is missed or disregarded, multivariate analyses showed that non-articular bone pain and/or general symptoms (asthenia, anorexia or weight loss) (regression coefficient: 4.8, OR 124 [95%CI: 11.4-236]), neutrophils < 2 × 109/L (regression coefficient: 3.9, OR 50 [95%CI: 4.3-58]), and platelets < 300 × 109/L (regression coefficient: 2.6, OR 14 [95%CI: 2.3-83.9]) were associated with the presence of ALL (area under the ROC curve: 0.96 [95%CI: 0.93-0.99]). CONCLUSIONS: Based on our findings we propose the following preliminary decision tree to be tested in prospective studies: in children presenting with at least 1 month of osteoarticular pain and no obvious ALL in peripheral smear, perform a bone marrow examination if hepatomegaly, splenomegaly or lymphadenopathy is present. If these manifestations are absent, perform a bone marrow examination if there is fever or elevated inflammatory markers associated with non-articular bone pain, general symptoms (asthenia, anorexia or weight loss), neutrophils < 2 × 109/L or platelets < 300 × 109/L.

Early Arthritis Is Associated With Failure of Immunosuppressive Drugs and Severe Pediatric Crohn's Disease Evolution.

Background: Crohn's disease (CD) is a chronic relapsing inflammatory disease. To optimize therapeutic decision making, it is essential to identify parameters that allow early prediction of a severe disease course. The aim of this study was to assess the link between arthritis and medium-term therapeutic failure in pediatric CD. Methods: We conducted a population-based cohort study with prospectively collected electronic data. To be included, patients must be younger than 17 years and have a confirmed CD diagnosed between 2005 and 2014. The primary outcome was the percentage of patients with at least 1 therapeutic failure of immunosuppressive drugs during the 2 years after the CD diagnosis, with a propensity score analysis. Results: We included 272 patients with CD. The median age was 12.1 years (interquartile [10.1-14.2]). Sixty-five patients (23.9%) developed arthritis, which predominantly occurred during the first year after CD diagnosis. We found a highly significant association between arthritis and therapeutic failure of immunosuppressive drugs after 2 years (OR = 6.9; 95% confidence interval [CI], 2.7-18.0; P < 0.0001; propensity score matching analysis). Arthritis was also significantly associated with introduction of biotherapy due to luminal disease 2 years after diagnosis (OR = 3.2, 95% CI, 1.8-6.0; P = 0.0001). Similar results were obtained after 4 years, and arthritis was significantly associated with a higher number of hospitalizations for luminal flare-up or complications after 4 years (OR = 2.2; 95% CI, 1.2-3.9; P = 0.007). Conclusions: Arthritis was strongly associated with medium-term therapeutic failure of pediatric CD. Occurrence of arthritis early in the disease may justify closer follow-up visits or specific therapeutic management.

Yersinia pseudotuberculosis disrupts intestinal barrier integrity through hematopoietic TLR-2 signaling.

Intestinal barrier function requires intricate cooperation between intestinal epithelial cells and immune cells. Enteropathogens are able to invade the intestinal lymphoid tissue known as Peyer's patches (PPs) and disrupt the integrity of the intestinal barrier. However, the underlying molecular mechanisms of this process are poorly understood. In mice infected with Yersinia pseudotuberculosis, we found that PP barrier dysfunction is dependent on the Yersinia virulence plasmid and the expression of TLR-2 by hematopoietic cells, but not by intestinal epithelial cells. Upon TLR-2 stimulation, Y. pseudotuberculosis-infected monocytes activated caspase-1 and produced IL-1ß. In turn, IL-1ß increased NF-κB and myosin light chain kinase activation in intestinal epithelial cells, thus disrupting the intestinal barrier by opening the tight junctions. Therefore, Y. pseudotuberculosis subverts intestinal barrier function by altering the interplay between immune and epithelial cells during infection.

Muckle-Wells syndrome and male hypofertility: a case series.

OBJECTIVES: Muckle-Wells syndrome (MWS) is a rare autoinflammatory disorder associated with NLRP3 gene mutations, which cause excessive caspase-1 activation and processing of interleukin (IL)-1ß and IL-18. Here we investigated whether MWS disease may be associated with impaired fertility in male patients. METHODS: Medical records of all male MWS patients with NLRP3 mutations followed in our tertiary center for inherited autoinflammatory diseases were reviewed retrospectively for data indicating fertility problems. RESULTS: Six of 9 patients were unable to have children despite regular sexual activity during at least 2 years; 3 succeeded in having children through in vitro fertilization. Infertility was the main reason for divorce in 1 patient. Spermiogram analyses were available in 8 of the 9 patients. Oligozoospermia was observed in 5 patients and azoospermia in 3 patients. In 2 patients, treatment with IL-1-targeting drugs for 6 and 12 months, respectively, had a moderate or no effect on spermatozoa counts. In 2 patients testosterone levels were low and testosterone treatment significantly increased spermatozoa counts in 1 of them. CONCLUSIONS: MWS may be associated with subfertility and infertility in male patients. Consequently, sexual health and fertility should be assessed systematically in adolescent and adult male patients. Additional studies are required to establish the frequency of subfertility in male MWS patients, to understand when subfertility occurs in the disease natural history, and, finally, to investigate whether early management with IL-1-targeting drugs, or testosterone treatment or early sperm cryo-conservation may help to allow procreation.

Nod2 regulates the host response towards microflora by modulating T cell function and epithelial permeability in mouse Peyer's patches.

Nucleotide oligomerisation domain 2 (NOD2) mutations are associated with susceptibility to Crohn's disease and graft-versus-host disease, two human disorders related with dysfunctions of Peyer's patches (PPs). In Nod2(-/-) mice transcellular permeability and bacterial translocation are increased in PPs. In this study, we show that both anti-CD4(+) and anti-interferon gamma (anti-IFNgamma) monoclonal antibodies abrogate this phenotype and reduce the expression of tumour necrosis factor (TNF) receptor 2 and the long isoform of myosin light chain kinase, thus demonstrating that immune T cells influence the epithelial functions. In turn, intraperitoneal injection of ML-7 (a myosin light chain kinase inhibitor) normalises the values of CD4(+) T cells, IFNgamma and TNFalpha. This reciprocal cross-talk is under the control of the gut microflora as shown by the normalisation of all parameters after antibiotic treatment. Toll-like receptor 2 (TLR2) and TLR4 expression were increased in Nod2(-/-) mice under basal conditions and TLR2 and TLR4 agonists induced an increased transcellular permeability in Nod2(+/+) mice. Muramyldipeptide (a Nod2 agonist) or ML-7 was able to reverse this phenomenon. It thus appears that Nod2 modulates the cross-talk between CD4(+) T cells and the epithelium recovering PP and that it downregulates the pro-inflammatory effect driven by the ileal microflora, likely by inhibiting the TLR pathways.

Crohn's disease and early exposure to domestic refrigeration.

BACKGROUND: Environmental risk factors playing a causative role in Crohn's Disease (CD) remain largely unknown. Recently, it has been suggested that refrigerated food could be involved in disease development. We thus conducted a pilot case control study to explore the association of CD with the exposure to domestic refrigeration in childhood. METHODOLOGY/PRINCIPAL FINDINGS: Using a standard questionnaire we interviewed 199 CD cases and 207 age-matched patients with irritable bowel syndrome (IBS) as controls. Cases and controls were followed by the same gastroenterologists of tertiary referral clinics in Tehran, Iran. The questionnaire focused on the date of the first acquisition of home refrigerator and freezer. Data were analysed by a multivariate logistic model. The current age was in average 34 years in CD cases and the percentage of females in the case and control groups were respectively 48.3% and 63.7%. Patients were exposed earlier than controls to the refrigerator (X2 = 9.9, df = 3, P = 0.04) and refrigerator exposure at birth was found to be a risk factor for CD (OR = 2.08 (95% CI: 1.01-4.29), P = 0.05). Comparable results were obtained looking for the exposure to freezer at home. Finally, among the other recorded items reflecting the hygiene and comfort at home, we also found personal television, car and washing machine associated with CD. CONCLUSION: This study supports the opinion that CD is associated with exposure to domestic refrigeration, among other household factors, during childhood.

Nod2 mediates susceptibility to Yersinia pseudotuberculosis in mice.

Nucleotide oligomerisation domain 2 (NOD2) is a component of the innate immunity known to be involved in the homeostasis of Peyer patches (PPs) in mice. However, little is known about its role during gut infection in vivo. Yersinia pseudotuberculosis is an enteropathogen causing gastroenteritis, adenolymphitis and septicaemia which is able to invade its host through PPs. We investigated the role of Nod2 during Y. pseudotuberculosis infection. Death was delayed in Nod2 deleted and Crohn's disease associated Nod2 mutated mice orogastrically inoculated with Y. pseudotuberculosis. In PPs, the local immune response was characterized by a higher KC level and a more intense infiltration by neutrophils and macrophages. The apoptotic and bacterial cell counts were decreased. Finally, Nod2 deleted mice had a lower systemic bacterial dissemination and less damage of the haematopoeitic organs. This resistance phenotype was lost in case of intraperitoneal infection. We concluded that Nod2 contributes to the susceptibility to Y. pseudotuberculosis in mice.

CARD15/NOD2 is required for Peyer's patches homeostasis in mice.

BACKGROUND: CARD15/NOD2 mutations are associated with susceptibility to Crohn's Disease (CD) and Graft Versus Host Disease (GVHD). CD and GVHD are suspected to be related with the dysfunction of Peyer's patches (PP) and isolated lymphoid follicles (LFs). Using a new mouse model invalidated for Card15/Nod2 (KO), we thus analysed the impact of the gene in these lymphoid formations together with the development of experimental colitis. METHODOLOGY/PRINCIPAL FINDINGS: At weeks 4, 12 and 52, the numbers of PPs and LFs were higher in KO mice while no difference was observed at birth. At weeks 4 and 12, the size and cellular composition of PPs were analysed by flow cytometry and immunohistochemistry. PPs of KO mice were larger with an increased proportion of M cells and CD4(+) T-cells. KO mice were also characterised by higher concentrations of TNFalpha, IFNgamma, IL12 and IL4 measured by ELISA. In contrast, little differences were found in the PP-free ileum and the spleen of KO mice. By using chamber experiments, we found that this PP phenotype is associated with an increased of both paracellular permeability and yeast/bacterial translocation. Finally, KO mice were more susceptible to the colitis induced by TNBS. CONCLUSIONS: Card15/Nod2 deficiency induces an abnormal development and function of the PPs characterised by an exaggerated immune response and an increased permeability. These observations provide a comprehensive link between the molecular defect and the Human CARD15/NOD2 associated disorders: CD and GVHD.

Ileal involvement is age dependent in pediatric Crohn's disease.

BACKGROUND: Lymphoid follicles (LFs) have been suggested to play a role at the early stage of Crohn's disease (CD) lesions. In the small bowel, LFs are grouped, forming Peyer's patches, which develop early in fetal life, grow in size and number until puberty, and undergo involution. In contrast, colonic LFs are isolated and undergo little change during life. As a result, if LFs play a role in the occurrence of CD lesions, the distribution of ileal and colonic lesions is expected to be altered in small children. METHODS: Medical records of 2 independent French (n = 136) and Swedish (n = 55) cohorts of consecutive pediatric CD were reviewed. Disease sites and age of onset were recorded, and the age-dependent probability to develop ileal lesions was computed. The CARD15/NOD2 genotype was also analyzed when available (n = 99). RESULTS: The curves of disease occurrence were significantly different in case of CD with or without ileal lesions (P < 0.0001). At the age of 8 years, the probability (95% confidence interval) of small bowel involvement was 0.19 (0.07-0.39). It increased until 16 years of age to 0.61 (0.54-0.68). It was slightly higher in patients carrying 1 or more CARD15/NOD2 mutations [0.75 (0.55-0.89)] than in wild-type patients [0.46 (0.34-0.58)]. CARD15 mutations also influenced the age of onset of ileal disease (P < 0.02). CONCLUSIONS: In children, ileal CD lesions are delayed compared with colonic lesions. This observation is in agreement with the previously proposed hypothesis of a pathophysiological role of Peyer's patches in ileal CD. The rarity of small bowel lesions should be a warning to be cautious when classifying chronic colitis in small children.