Background T follicular helper (Tfh) cells underpin T-cell dependent humoral immunity and the success of most vaccines. Tfh cells also contribute to human immune disorders such as autoimmunity, immunodeficiency and malignancy. Understanding the molecular requirements for the generation and function of Tfh cells will provide strategies for targeting these cells to modulate their behavior in the setting of these immunological abnormalities. Objective To determine the signaling pathways and cellular interactions required for the development and function of Tfh cells in humans. Methods Human primary immunodeficiencies (PIDs) resulting from monogenic mutations provide a unique opportunity to assess the requirement for particular molecules in regulating human lymphocyte function. Circulating Tfh (cTfh) cell subsets, memory B cells and serum Ig levels were quantified and functionally assessed in healthy controls as well as patients with PIDs resulting from mutations in STAT3, STAT1, TYK2, IL21, IL21R, IL10R, IFNGR1/2, IL12RB1, CD40LG, NEMO, ICOS or BTK. Results Loss-of function (LOF) mutations in STAT3, IL10R, CD40LG, NEMO, ICOS or BTK reduced cTfh frequencies. STAT3, IL21/R LOF and STAT1 gain-of function mutations skewed cTfh differentiation towards a phenotype characterized by over-expression of IFNγ and programmed death -1 (PD-1). IFNγ inhibited cTfh function in vitro and in vivo, corroborated by hypergammaglobulinemia in patients with IFNGR1/2, STAT1 and IL12RB1 LOF mutations. Conclusion Specific mutations impact the quantity and quality of cTfh cells, highlighting the need to assess Tfh cells in patients by multiple criteria, including phenotype and function. Furthermore, IFNγ functions in vivo to restrain Tfh-induced B cell differentiation. These findings shed new light on Tfh biology and the integrated signaling pathways required for their generation, maintenance and effector function, and explain compromised humoral immunity in some PIDs.

Background & AimsCongenital diarrheal disorders are rare inherited intestinal disorders characterized by intractable, sometimes life-threatening, diarrhea and nutrient malabsorption; some have been associated with mutations in diacylglycerol-acyltransferase 1 (DGAT1), which catalyzes formation of triacylglycerol from diacylglycerol and acyl-CoA. We investigated the mechanisms by which DGAT1 deficiency contributes to intestinal failure using patient-derived organoids.MethodsWe collected blood samples from 10 patients, from 6 unrelated pedigrees, who presented with early-onset severe diarrhea and/or vomiting, hypoalbuminemia, and/or (fatal) protein-losing enteropathy with intestinal failure; we performed next-generation sequencing analysis of DNA from 8 patients. Organoids were generated from duodenal biopsies from 3 patients and 3 healthy individuals (controls). Caco-2 cells and patient-derived dermal fibroblasts were transfected or transduced with vectors that express full-length or mutant forms of DGAT1 or full-length DGAT2. We performed CRISPR/Cas9-guided disruption of DGAT1 in control intestinal organoids. Cells and organoids were analyzed by immunoblot, immunofluorescence, flow cytometry, chromatography, quantitative real-time polymerase chain reaction, and for the activity of caspases 3 and 7.ResultsIn the 10 patients, we identified 5 bi-allelic loss-of-function mutations in DGAT1. In patient-derived fibroblasts and organoids, the mutations reduced expression of DGAT1 protein and altered triacylglycerol metabolism, resulting in decreased lipid droplet formation after oleic acid addition. Expression of full-length DGAT2 in patient-derived fibroblasts restored formation of lipid droplets. Organoids derived from patients with DGAT1 mutations were more susceptible to lipid-induced cell death than control organoids.ConclusionsWe identified a large cohort of patients with congenital diarrheal disorders with mutations in DGAT1 that reduced expression of its product; dermal fibroblasts and intestinal organoids derived from these patients had altered lipid metabolism and were susceptible to lipid-induced cell death. Expression of full-length wildtype DGAT1 or DGAT2 restored normal lipid metabolism in these cells. These findings indicate the importance of DGAT1 in fat metabolism and lipotoxicity in the intestinal epithelium. A fat-free diet might serve as the first line of therapy for patients with reduced DGAT1 expression. It is important to identify genetic variants associated with congenital diarrheal disorders for proper diagnosis and selection of treatment strategies.

PurposeBiallelic mutations in SCYL1 were recently identified to cause a syndromal disorder characterized by peripheral neuropathy, cerebellar atrophy, ataxia and recurrent episodes of liver failure. The occurrence of SCYL1 deficiency among patients with previously undetermined infantile cholestasis or acute liver failure has not been studied; furthermore, little is known regarding the hepatic phenotype.MethodsWe aimed at identifying patients with SCYL1 variants within an exome sequencing study of individuals with infantile cholestasis or acute liver failure of unknown etiology. Deep clinical and biochemical phenotyping plus analysis of liver biopsies and functional studies on fibroblasts were performed.ResultsSeven patients from five families with biallelic SCYL1 variants were identified. The main clinical phenotype was recurrent low γ-glutamyl-transferase (GGT) Cholestasis or Acute Liver Failure with onset in infancy And a variable Neurological phenotype of later onset (CALFAN syndrome). Liver crises were triggered by febrile infections and were transient, but fibrosis developed. Functional studies emphasize that SCYL1 deficiency is linked to impaired intracellular trafficking.ConclusionSCYL1 deficiency can cause recurrent low GGT cholestatic liver dysfunction in conjunction with a variable neurological phenotype. Similar to NBAS deficiency, it’s a member of the emerging group of congenital disorders of intracellular trafficking causing hepatopathy.

ABSTRACT. Objectives. Celiac disease (CD), a common cause of malabsorption, is known to be associated with disorders of the skeleton, but there are conflicting data about the effect of diet on bone metabolism. The aims of this study were to investigate the prevalence of osteopenia; to identify the relationship between bone mineral density (BMD), serum calcium, and parathyroid hormone levels; and to determine the effect of glutenfree diet on BMD in children with celiac disease.Design. The study included 32 patients with CD (group 1) and 82 healthy controls (group 2). The patients with CD were evaluated under 2 subgroups, ie, 16 patients with recent diagnosis (group 1a) and 16 patients who follow their diet strictly (group 1b). BMD values and concentrations of calcium, phosphorus, alkaline phosphatase, and intact parathyroid hormone were determined on entry to the study and at 12 months in celiac patients. These values were compared with those of healthy control participants.Results. BMD and bone mineral content values in patients with recent diagnosis were found to be significantly lower than the control group. The BMD values in patients with recent diagnosis were significantly increased after a gluten-free diet for 1 year. Osteopenia was found more commonly in patients with recent diagnosis than patients in whom a gluten-free diet had been instituted. At 1-year follow-up, osteopenia was not resolved with the gluten-free diet, and this was especially true in patients without gastrointestinal manifestation. In patients with recent diagnosis (group 1a), the mean calcium level was found to be lower than the patients who follow their diet strictly (group 1b). There was a positive correlation between calcium level and BMD and bone mineral content.Conclusions. BMD is almost invariably low in newly diagnosed celiac patients in childhood. We therefore recommend that BMD should be evaluated in patients with CD. Strict gluten avoidance promoted a significant increase in BMD. However, values still remained markedly low after 1 year of follow-up in some patients. These patients should be followed for longer periods of time with yearly BMD evaluation, as 1 year of diet therapy was found to be insufficient for osteopenia to be resolved. Pediatrics 2001;108(5). URL: http://www. pediatrics.org/cgi/content/full/108/5/e89; bone mineral density, celiac disease, gluten-free diet, osteopenia, osteoporosis, childhood.

No abstract

Purpose The clinical features of patients with celiac disease (CD) are variable. In the present study, clinical and laboratory features of 109 patients with CD were retrospectively evaluated. Materials and Methods In all cases, diagnosis of CD was made by European Society for Paediatric Gastroenterology, Hepatology and Nutrition (ESPGHAN) criteria and clinical and laboratory findings, including hematological and biochemical analyses, immunoglobulin levels, autoantibodies [antinucler antibody (ANA), antidouble stranded DNA (dsDNA), antimitochondrial antibody (AMA), anti-smooth muscle antibody (ASMA), liver kidney antibody (LKM-1), anti thyroid peroxidase (TPO), anti thyroglobulin (Tg)], bone mineral density (BMD), and electroencephalogram were evaluated. The type of CD was recorded. Results Of 109 patients with CD, 66 (60.6%) were classical type, 41 (37.6%) were atypical type and 2 (1.8%) were silent type. The mean age was 8.81 ± 4.63 years and the most common symptom was diarrhea (53.2%) followed by failure to thrive, short stature, and abdominal pain. Paleness (40.4%), underweight (34.8%), and short stature (31.2%) were the most common findings. Iron deficinecy anemia (81.6%), zinc deficiency (64.1%), prolonged prothrombin time (35.8%), and elevated transaminase levels (24.7%) were the most common laboratory findings. Eight percent of patients had at least 1 autoantibody, and 28 of 52 patients had low BMD. Four of 38 patients had abnormalty in electroencephalograms. The prevalance of selective immunoglobulin (Ig) A deficiency was 9.1%. Histocompatibility antigen HLA-DQ and/or DQ8 genotypes were found in 91% of patients. Abdominal distention, iron deficiency, prolonged prothrombine time, hypoalbuminemia, and elevated transaminase levels were more significantly frequent in the classical type than atypical type ( p < 0.005). Conclusion Although classical CD was seen in most patients in the present study, clinical variability of the condition should be kept in mind.

We describe a patient with a novel missense mutation in LRBA who presented with IBD-like symptoms at early age, illustrating that LRBA deficiency should be considered in the differential diagnosis for IBD(-like) disease even in the absence of overt immunodeficiency.

In the near future, the burden of CD will increase tremendously. Few Mediterranean countries are able to face this expanding epidemic alone.