ABSTRACT Objectives: Therapy with broad‐spectrum antibiotics is a common practice for premature infants. This treatment can reduce the biodiversity of the fecal microbiota and may be a factor in the cause of necrotizing enterocolitis. In contrast, probiotic treatment of premature infants reduces the incidence of necrotizing enterocolitis. We hypothesized that 1 mechanism for these observations is the influence of bacteria on postnatal development of the mucosal immune system. Materials and Methods: Expression of immune molecules and microbial sensors was investigated in the postnatal mouse gastrointestinal tract by real‐time polymerase chain reaction. Subsequently, 2‐week‐old specific pathogen‐free and microbial‐reduced (MR; antibiotic treated) mice were compared for immune molecule and microbial sensor expression, mesenteric lymph node T‐cell numbers and activation, intestinal barrier function/permeability, systemic lymphocyte numbers, and T‐cell phenotype commitment. Results: Toll‐like receptor 2, 4, and 5 expression was highest in 2‐week‐old specific pathogen‐free mice, and this expression was decreased in MR mice. There was no difference in intestinal tight‐junctional function, as evaluated by fluorescein isothiocyanate‐dextran uptake, but MR mice had increased bacterial translocation across the intestinal epithelial barrier. MR mice had significantly fewer splenic B cells and mesenteric lymph node CD4+ T cells, but there were normal numbers of splenic T cells. These systemic T cells from MR mice produced more interleukin‐4 and less interferon‐γ and IL‐17, indicative of maintenance of the fetal, T‐helper cell type 2 phenotype. Conclusions: The present study shows that intestinal commensal microbiota have an influence on early postnatal immune development. Determining specific bacteria and/or bacterial ligands critical for this development could provide insight into the mechanisms by which broad‐spectrum antibiotics and/or probiotic therapy influence the development of the mucosal immune system and mucosal‐related diseases.
Summary: There are now many experimental models of inflammatory bowel disease (IBD), most of which are due to induced mutations in mice that result in an impaired homeostasis with the intestinal microbiota. These models can be clustered into several broad categories that, in turn, define the crucial cellular and molecular mechanisms of host microbial interactions in the intestine. The first of these components is innate immunity defined broadly to include both myeloid and epithelial cell mechanisms. A second component is the effector response of the adaptive immune system, which, in most instances, comprises the CD4 + T cell and its relevant cytokines. The third component is regulation, which can involve multiple cell types, but again particularly involves CD4 + T cells. Severe impairment of a single component can result in disease, but many models demonstrate milder defects in more than one component. The same is true for both spontaneous models of IBD, C 3 H/HeJBir and SAMPI/Yit mice. The thesis is advanced that ‘multiple hits’ or defects in these interacting components is required for IBD to occur in both mouse and human.
A 37-week small-for-gestational age (birth weight 1.8 kg) white male infant was referred for evaluation at 2 weeks of age with a history of hyperglycemia and diarrhea. The pregnancy was remarkable for oligohydramnios and intrauterine growth restriction. There was no relevant family history. On day of life 2, the patient developed hyperglycemia (536 mg/dL) necessitating an insulin drip. Initial genetic testing was normal, including chromosomal microarray analysis, as well as negative sequencing results for neonatal diabetes associated genes: KCNJ11, INS, ABCC8, and PDX1. Starting on day of life 13, the baby developed watery diarrhea. Laboratory testing showed a non–anion gap metabolic acidosis, eosinophilia, and low insulin levels. The diarrhea improved with cessation of feeds, and stool electrolytes were consistent with an osmotic diarrhea. He was fed various different formulas (high medium-chain triglyceride oil content and carbohydrate-free formulas) without stool improvement. His fecal elastase was <50 μg/g (normal >200 μg/g). Abdominal ultrasound demonstrated a normal-sized pancreas, although diffusely echogenic consistent with medical pancreatic disease. Pancreatic enzyme replacement did not result in clinical improvement. On repeat testing, fecal elastase was normal (449 μg/g). At 3 weeks of age he developed a diffuse dry erythematous rash. Because stool output increased independent of oral intake, transformation from an osmotic to a secretory diarrhea was suspected. At 5 weeks of age, an upper endoscopy was performed. Biopsies showed villous atrophy and increased cellularity in the lamina propria consistent with autoimmune enteropathy (1) (Fig. 1A and B). Shortly thereafter, thrombocytopenia and Coombs positive hemolytic anemia were noted. With these additional findings, the diagnosis of immune dysregulation, polyendocrinopathy, enteropathy X-linked (IPEX) syndrome was considered (2). Sequencing of the FOXP3 gene revealed a novel mutation in the C-terminal portion of the forkhead DNA-binding domain, resulting in a deletion of 9 nucleotides within the coding region (c.1227_1235delTGAGCTGGA) that is near other known causative mutations (Fig. 2). This deletion causes an amino acid substitution of glutamic acid for aspartic acid at position 409 (p.Asp409Glu) followed by in-frame deletion of 3 additional amino acids (p.Glu410_Glu412del). As a result, few CD25+FOXP3+ regulatory T cells (Tregs) were observed in the CD4+ T-cell population (Fig. 3).FIGURE 1: A, Hematoxylin and eosin staining at ×33 magnification of a biopsied section of small intestine demonstrating villous blunting. B, Depicts a magnified image (×132) of the duodenal lamina propria demonstrating increased mononuclear and neutrophil infiltrates.FIGURE 2: Schematic representation of the FOXP3 protein. The bold arrow indicates the location of the novel mutation in the forkhead DNA-binding domain at amino acid position 409. The narrow arrows represent several previously identified mutations. RD = repressor domain; ZF = zinc finger; LZ = leucine zipper.FIGURE 3: Flow cytometry showing CD25+FOXP3+ regulatory cells in the CD4 T-cell population in peripheral blood. The area within the dashed box shows the difference in expression of FOXP3 in the cells and the decreased percentage of FOXP3-expressing regulatory T cells in the patient compared with the control.After the diagnosis of IPEX was confirmed, immunosuppressive therapy with cyclosporine A was initiated and within 1 week transient improvements in stooling, blood counts, and rash were noted. While waiting for the nutritional status to improve and for identification of a bone marrow donor, he developed cellulitis, recurrent bacteremia, and worsening rash. At 5 months of age he underwent a reduced- intensity allogeneic matched unrelated bone marrow transplant. He had several complications, including veno-occlusive disease, pulmonary hemorrhage, and candidemia, and died on day 9 after transplant. DISCUSSION IPEX is a rare condition with a variable clinical phenotype, likely related to the specific mutation and degree of functional FOXP3 protein expression (2). FOXP3 is a transcription factor whose expression is primarily confined to a subset of T cells (Tregs) that play a primary role in regulation of immune responses (3). Mutation of the FOXP3 gene (with subsequent alterations in protein expression) leads to an unchecked autoimmune response. This results in inflammation affecting multiple organ systems. Although immunosuppressive therapy can alleviate symptoms, hematopoietic stem cell transplantation remains the only known cure for IPEX (4). Our patient developed symptoms early in the postnatal period. In addition to insulin-dependent diabetes from the second day of life, his disease course progressed to multiorgan autoimmunity including enteropathy, cytopenias, and eczema. Unfortunately, he developed severe and recurrent infections, which contributed significantly to his ultimate demise. FOXP3 is a member of a large family of transcriptional regulators that have a highly conserved forkhead DNA-binding domain. To date, approximately 55 gene mutations associated with IPEX have been described, with many of these occurring in the forkhead domain (5). Our patient had a new, previously undescribed FOXP3 mutation affecting the forkhead DNA-binding domain of the protein. The virtual absence of CD25+FOXP3+ Tregs in the CD4+ T-cell population suggests that this mutation leads to a FOXP3 protein that is functionally defective and unable to sustain Treg development. The FOXP3 gene sequences of both parents were normal, suggesting a de novo mutation in our patient. Onset of insulin-dependent diabetes on the second day of life in our patient implies that an autoimmune attack of the pancreas began in utero, well before birth. This suggests that usual physiologic immunoregulatory mechanisms exerted by the placenta to prevent rejection of the fetus by the mother are insufficient to prevent autoimmunity in the baby in the absence of the infant's own Tregs. Diabetes is often, although not always, the first feature to develop in IPEX. Because there are now well over 20 gene causes for neonatal diabetes, the traditional approach has been to proceed with testing of genes based on suggestive symptoms (6). Nevertheless, because cost of gene sequencing continues to fall and comprehensive testing can be done more efficiently, it will become increasingly possible to make an early genetic diagnosis that will allow for prompt recognition and treatment of all syndromic features. Although our patient had severe, early-onset disease characterized by the classic phenotype of IPEX including enteropathy, endocrinopathy, dermatitis, and other autoimmunity, it is important to remember that a significant proportion of IPEX patients have FOXP3 mutations that lead to less severe disease that may not present with the full clinical spectrum of disease (7). We recommend that a clinical suspicion for IPEX be raised in any male patient with diabetes, particularly if they exhibit signs of enteropathy or other organ-specific autoimmunity. In all cases, the criterion standard for confirming a diagnosis of IPEX is FOXP3 gene sequencing.
Cardiovascular associations of Crohn’s Disease (CD) are rare and include valvular heart disease, aortitis and aneurysm. We report an 11 year old with thoracic aortitis and aneurysm found during initial presentation of CD. An 11 year old African American female presented with symptoms concerning for inflammatory bowel disease. Work up revealed leukocytosis, and serum evidence of inflammation. Imaging showed thickening of the distal ileum, suggestive of CD. Surprisingly an aneurysm involving the descending thoracic aorta, with wall thickening, concerning for an impending rupture was also found which was confirmed on an angiogram. Subsequent endoscopy revealed ileocolitis; with no evidence of vasculitis. She was started on infliximab and methotrexate. An elective repair with reconstruction of the aneurysm was performed. The resected aorta revealed neutrophilic infiltrate consistent with inflammation. Work up for primary vasculitis was unrevealing. Her symptoms improved and laboratory markers on the current therapy. Prevalence of EIMs in CD ranges from 10% to 50%. Cardiovascular associations in CD are rare. Adult patients with long standing history of CD may rarely develop aortitis, however, presence of aortitis at diagnosis of CD is not yet reported. Noninfectious aortitis can be managed with steroids, immunosuppressants or surgery. Given the findings of the aneurysm, we performed an elective surgical repair. We chose to avoid steroids because of limited complaints related to her disease and to limit her risk of infection during her upcoming surgery. • IBD and aortitis can present simultaneously, especially CD. • Concomitance of IBD and aortitis is most likely to be related, than a mere coincidence. • Aortitis with limited symptoms can be managed with infliximab and methotrexate without the use of high dose steroids.
To review a case of quintuplets with all babies developing necrotizing enterocolitis.A retrospective study of preterm quintuplets all developing necrotizing enterocolitis. Clinical outcomes were reviewed.Quintuplets were born at 24 weeks gestation. Each baby developed NEC and was treated. One baby died. Currently the remaining 4 infants are on full enteral nutrition.Further studies are needed to better understand this emerging population of multiple birth pregnancy and the frequency of NEC development.
Abstract Frequently fatal, primary hemophagocytic lymphohistiocytosis (HLH) occurs in infancy resulting from homozygous mutations in natural killer (NK) and CD8 T cell cytolytic pathway genes. Secondary HLH presents after infancy and may be associated with heterozygous mutations in HLH genes. We report 2 unrelated teenagers with HLH and an identical heterozygous RAB27A mutation (259 G>C). The contribution of this Rab27A missense (A87P) mutation on NK cell cytotoxicity was studied by cloning it into a lentiviral expression vector prior to introduction into the human NK-92 cell line. NK cell degranulation (CD107a expression), target cell conjugation, and K562 target cell lysis was compared between mutant and wild-type (WT) transduced NK-92 cells. Polarization of granzyme B to the immunologic synapse and interaction of mutant Rab27A (A87P) with Munc13-4 were explored by confocal microscopy and proximity ligation assay (PLA), respectively. Over-expression of the RAB27A mutation had no effect on cell conjugate formation between the NK and target cells but decreased NK cell cytolytic activity and degranulation. Moreover, the mutant Rab27A protein was predicted to disrupt binding to WT Munc13-4 by crystal structure modelling, and decreased interaction of Rab27A (A87P) with WT Munc13-4 was shown by PLA in situ and by co-immunoprecipiation in vitro. Finally, Rab27A (A87P) over-expression in NK-92 cells delayed granzyme B polarization toward the immunologic synapse, as noted by confocal microscopy, with a resulting increase in interferon-γ production, a cytokine responsible for HLH. This heterozygous RAB27A mutation blurs the genetic distinction between primary and secondary HLH by contributing to HLH via a partial dominant-negative effect.
To determine whether serum transaminases at presentation predict the need for dialysis in children with hemolytic uremic syndrome (HUS).Single-center, retrospective chart review of pediatric patients with HUS. Data collected included demographics, clinical and laboratory parameters, and need for dialysis. These factors were compared between two groups: "dialysis" versus "no dialysis." Continuous data were compared using a t test whereas categoric data were compared by the chi-squared test. Multivariate logistic regression was performed on a prior set of variables to determine if serum transaminases independently predict the need for dialysis.A total of 70 children were included in the study, of which, 39 (27%) received dialysis. The no-dialysis group had a higher proportion of white patients compared with the dialysis group (74% dialysis versus 94% no dialysis). The only clinical sign at admission associated with dialysis was reduced urine output (56% versus 16%, P<0.001). Univariate logistic regression identified admission serum creatinine, aspartate transaminase (AST), and alanine transaminase (ALT) to be associated with the need for dialysis. Multivariate logistic regression showed serum AST and ALT to be independent predictors of the need for dialysis, with both improving the performance of the regression model. Sensitivity analysis showed a cutoff of 129 U/L for AST and 83 U/L for ALT with high specificity.Serum transaminases at presentation are independently associated with the subsequent need for dialysis in patients with HUS. Our study suggests that when both serum ALT and AST are normal, the likelihood to need dialysis is very low; alternatively, when both serum ALT and AST are more than two times the upper level of normal, the need for dialysis is very high.
