Camilla Salvestrini

Heparan sulfate and syndecan-1 are essential in maintaining murine and human intestinal epithelial barrier function

Patients with protein-losing enteropathy (PLE) fail to maintain intestinal epithelial barrier function and develop an excessive and potentially fatal efflux of plasma proteins. PLE occurs in ostensibly unrelated diseases, but emerging commonalities in clinical observations recently led us to identify key players in PLE pathogenesis. These include elevated IFN-gamma, TNF-alpha, venous hypertension, and the specific loss of heparan sulfate proteoglycans from the basolateral surface of intestinal epithelial cells during PLE episodes. Here we show that heparan sulfate and syndecan-1, the predominant intestinal epithelial heparan sulfate proteoglycan, are essential in maintaining intestinal epithelial barrier function. Heparan sulfate- or syndecan-1-deficient mice and mice with intestinal-specific loss of heparan sulfate had increased basal protein leakage and were far more susceptible to protein loss induced by combinations of IFN-gamma, TNF-alpha, and increased venous pressure. Similarly, knockdown of syndecan-1 in human epithelial cells resulted in increased basal and cytokine-induced protein leakage. Clinical application of heparin has been known to alleviate PLE in some patients but its unknown mechanism and severe side effects due to its anticoagulant activity limit its usefulness. We demonstrate here that non-anticoagulant 2,3-de-O-sulfated heparin could prevent intestinal protein leakage in syndecan-deficient mice, suggesting that this may be a safe and effective therapy for PLE patients.

Expression of Human Beta-Defensins in Children with Chronic Inflammatory Bowel Disease

Background Human beta-defensins (hBDs) are antimicrobial peptides known to play a major role in intestinal innate host defence. Altered mucosal expression of hBDs has been suggested to be implicated in chronic inflammatory bowel disease pathogenesis. However, little is known about expression of these peptides in children. Methods Intestinal biopsies were obtained from the duodenum (n = 88), terminal ileum (n = 90) and ascending colon (n = 105) of children with Crohns disease (n = 26), ulcerative colitis (n = 11) and healthy controls (n = 16). Quantitative real-time (RT) PCR was performed and absolute mRNA copy numbers analyzed for hBD1-3 as well as inflammatory cytokines IL-8 and TNF-alpha. Results Significant induction of hBD2 and hBD3 was observed in the inflamed terminal ileum and ascending colon of IBD children. In the ascending colon induction of hBD2 was found to be significantly lower in children with Crohns disease compared to ulcerative colitis. A strong correlation was found between inducible defensins hBD2 and 3 and the inflammatory cytokines IL-8 and TNF-alpha, both in the terminal ileum and ascending colon. Conclusion Our study demonstrates distinct changes in hBD expression throughout the intestinal tract of children with IBD, lending further support for their potential role in disease pathogenesis.

Reduced production of sulfated glycosaminoglycans occurs in Zambian children with kwashiorkor but not marasmus

BACKGROUND Kwashiorkor, a form of severe malnutrition with high mortality, is characterized by edema and systemic abnormalities. Although extremely common, its pathophysiology remains poorly understood, and its characteristic physical signs are unexplained. OBJECTIVE Because kwashiorkor can develop in protein-losing enteropathy, which is caused by a loss of enterocyte heparan sulfate proteoglycan (HSPG), and previous observations suggest abnormal sulfated glycosaminoglycan (GAG) metabolism, we examined whether intestinal GAG and HSPG are abnormal in children with kwashiorkor. DESIGN Duodenal biopsy samples collected from Zambian children with marasmus (n = 18), marasmic kwashiorkor (n = 8), and kwashiorkor (n = 15) were examined for expression of HSPG, GAGs, and immunologic markers and compared against reference samples from healthy UK control children. GAG and HSPG expression density and inflammatory cell populations were quantitated by computerized analysis. RESULTS The kwashiorkor group was less wasted and had a lower HIV incidence than did the other groups. All duodenal biopsy samples showed inflammation compared with the histologically uninflamed control samples. Biopsy samples from marasmic children had greater inflammation and greater CD3+ and HLA-DR (human leukocyte antigen DR)-positive cell densities than did samples from children with kwashiorkor. Expression of both HSPG and GAGs was similar between marasmic and well-nourished UK children but was markedly lower in children with kwashiorkor in both the epithelium and lamina propria. Although underglycosylated and undersulfated, epithelial syndecan-1 protein was normally expressed in kwashiorkor, which confirmed that abnormalities arise after core protein synthesis. CONCLUSIONS Intestinal HSPG loss occurs in kwashiorkor, which may precipitate protein-losing enteropathy to cause edema. If occurring systemically, impaired HSPG expression could cause several previously unexplained features of kwashiorkor. We speculate that a genetic predisposition to reduced HSPG biosynthesis may offer a contrasting selective advantage, by both diminishing protein catabolism during transient undernutrition and protecting against specific infectious diseases.

Defining eosinophilic colitis in children: insights from a retrospective case series.

Objectives: Although it is a well-described syndrome in infants, eosinophilic colitis is a loosely defined and poorly understood diagnosis in older children. The aims of this case series were to characterise colonic eosinophilia in children and to determine whether it represents a distinct clinicopathological condition. Methods: We retrospectively reviewed symptomatic children older than 12 months with the principal diagnosis of colonic eosinophilia who presented between January 2000 and February 2007 (n = 38) and a further 10 children whose colonic biopsies were reported as histologically normal. The eosinophil density in all available gastrointestinal biopsies (n = 620) of these children was determined using a validated quantitative morphometric method. Patients were subdivided according to mean colonic eosinophil levels into 3 groups (marked, moderate, or minimal colonic eosinophilia). The following patient information was obtained and compared among patient groups: symptoms prompting endoscopy, atopic history, outcome, serum C-reactive protein and total immunoglobulin E (IgE) levels, erythrocyte sedimentation rate, blood eosinophil count, and endoscopic findings. Results: In all 3 patient groups, there was a colonic gradient of decreasing eosinophil density from caecum to rectum. Upper gastrointestinal tract biopsies did not exhibit eosinophilia. Although a significant association (P = 0.03) between abnormal total IgE levels and moderate or severe colonic eosinophilia was found, there was no significant difference (P > 0.05) in other patient characteristics. Furthermore, follow-up data did not show a consistent relation between eosinophil density and progression of symptoms. Conclusions: We find no association between “eosinophilic colitis,” defined as a histologically demonstrated marked colonic eosinophilia, and symptoms, history of atopy, inflammatory markers, or clinical outcome.

Intestinal alpha‐defensin expression in pediatric inflammatory bowel disease

Background: Reduced alpha‐defensin expression has been reported in the terminal ileum (TI) of adult patients with ileal Crohns disease (CD). However, little is known about alpha‐defensin expression in children with chronic inflammatory bowel disease (IBD). Methods: In all, 283 intestinal biopsies were obtained from children with CD, ulcerative colitis (UC), and healthy controls. Absolute mRNA copy numbers for HD5, HD6, IL‐8, Villin 1, and Tcf‐4 were analyzed by reverse‐transcription polymerase chain reaction (RT‐PCR). HD5 immunostaining was performed on biopsy sections and patients genotyped for NOD2 mutations. Results: Equal expression levels of alpha‐defensins (HD5 and HD6) were found in TI biopsies of children with ileal CD (L1+L3) compared to patients with colonic disease (L2) and healthy controls. In contrast, we found significantly higher levels of alpha‐defensins in the TI of children with UC compared to CD and controls. Reduced expression of Tcf‐4 was observed exclusively in the duodenum and TI of CD patients with L1+L3 phenotype. We demonstrate significantly increased expression of HD5 and HD6 in the inflamed colon of IBD children (UC and CD) attributable to the presence of metaplastic Paneth cells. Conclusions: In this study no difference in alpha‐defensin expression was found in the TI of CD children and controls. However, significant reduction of Tcf‐4 in L1+L3 phenotype suggests that a possibly impaired PC differentiation may lead to altered HD5 and HD6 expression at some stage of disease. Additionally, substantially increased expression of alpha‐defensins in the inflamed colonic mucosa of children with IBD raises the question for their potential involvement in modulating inflammation in these patients. (Inflamm Bowel Dis 2010;)

Desquamative enteropathy and pyloric atresia without skin disease caused by a novel intracellular beta4 integrin mutation

Background: Mutations in α6 or β4 integrins (ITGA6, ITGB4) are known to cause junctional epidermolysis bullosa with pyloric atresia (JEB-PA), often lethal in infancy through skin desquamation. There is 1 report of pyloric atresia associated with a desquamatory enteropathy but without skin disease, of unknown molecular basis. Patients and Methods: We report 2 Kuwaiti siblings with pyloric atresia and life-threatening intestinal desquamation without significant skin abnormality. The older sibling died of intractable diarrhoea, and the younger sibling suffered episodes of massive protein-losing enteropathy, triggered by viral infections, in addition to obstructive uropathy. Mutation analysis was performed for ITGA6 and ITGB4 and expression of ITGA6 and ITGB4 protein was examined in skin and intestinal biopsies. Her serum also was incubated with normal intestine. Results: We identified a novel mutation in ITGB4, with homozygous deletion of a single residue (isoleucine 1314) within the intracellular plectin-binding domain. Expression of ITGA6 and ITGB4 within skin, duodenal, and colonic epithelium was normal or minimally reduced, in contrast to previous reports. Biopsies taken during relapse showed accumulation of immunoglobulin G and C1q within intestinal basement membrane, whereas immunoglobulin G from her serum bound to basement membrane of normal small intestine. Immunomodulatory therapy induced significant improvement following relapses. Conclusions: ITGB4 mutation may induce a desquamative enteropathy in infancy without significant skin disease. A history of pyloric atresia is important in infants with severe chronic diarrhoeal disease and should prompt investigation for JEB-PA associated mutations. Acquired immune responses may exacerbate primary genetic disorders of epithelial adhesion and immunomodulatory therapy may be beneficial.

Feasibility of a finger prick-based self-testing kit in first- and second-degree relatives of children with coeliac disease.

AIM To assess feasibility of a finger prick-based kit as method for self-testing of first and second-degree relatives of coeliac disease (CD) patients. METHODS A total number of 379 subjects were invited to participate in this study, consisting of 197 first-degree and 182 second-degree relatives of CD patients. The self-testing kit (Biocard™) was sent out with included instructions for use. Completed tests were sent back to the study coordinator for assessment. RESULTS One hundred and ninety-six invited relatives carried out the Biocard™ test at home. Amongst these, 70% were children. In 97% of the cases the test was performed correctly. Three tests revealed a positive result, all of which were later confirmed by serology and histology as coeliac disease. CONCLUSION Our study indicates that Biocard™ test is a reliable, easy to use and well-accepted tool for home testing of first- and second-degree relatives of CD patients.

An 18‐month‐old child with seizures and bloody diarrhea

To the Editor: An 18 month-old infant with stupor status was admitted to the local hospital in April 2002. Three months earlier, he had presented with occasional mucus and fresh blood in his stool. One week prior to hospital admission, he begun to experience progressively worsening paleness, fatigue, and bloody diarrhea with mucus, and had an episode of tonicclonic fits of the right arm lasting a few seconds. On hospital admission, a blood analysis revealed the following: hemoglobin concentration, 3.6 mg/dL; platelet count, 748,000/mm; and C-reactive protein concentration, 2.9 mg/dL. A lumbar puncture was performed, revealing normal cerebral spinal fluid. During a blood transfusion, the child experienced a generalized seizure with loss of consciousness and upward deviation of his eyesight. Computerized axial tomography, magnetic resonance (MR) imaging, and MR angiography scans of the brain revealed bilateral hemorrhagic infarctions of the basal ganglia, and thrombosis of the internal cerebral veins, Galeno’s vein, rectum, and left transverse sinus (Fig. 1). Blood flow was preserved in the superior sagittal sinus and the right transverse sinus. The child was then transferred to the pediatric intensive care unit of our hospital where a physical examination revealed a suffering, pale, dehydrated child, who was in opisthotonus without signs of meningism. Some type of encephalitis was initially suspected, and therapy with antibiotics, steroids, acyclovir, aspirin, and phenobarbitone was started. Because of intestinal bleeding, anticoagulants were not administered. As soon as his clinical condition was stabilized, he was transferred to our ward. His neurologic condition improved, but bloody diarrhea with mucus was still present up to 7 to 10 times a day. Prothrombin time, partial thromboplastin time, plasma levels of protein C and protein S, and resistance to activated protein C and antithrombin III were compatible with endothelial distress. The Leyden factor V mutation and the G20210A mutation of factor II were absent, although the patient was etherozygous for the methyltetrahydrofolate reductase (C677T) mutation. The plasma concentrations of homocysteine, vitamin B12, and folate were normal. The results of immunologic tests, such as those for anticardiolipin antibodies and lupus anticoagulants, were normal, but test results for antineutrophil cytoplasmic antibodies with a perinuclear pattern were positive. The findings of a rectosigmoidoscopy with biopsy and histopathologic examination were consistent with the diagnosis of ulcerative colitis (UC). The patient was started on a treatment regimen of broad-spectrum intravenous antibiotics, intravenous methylprednisolone, and total parenteral nutrition. After 9 days of treatment, he was still presenting with bloody diarrhea, and a blood transfusion was required. For this reason, intravenous cyclosporine (3 mg/kg/d) was added to therapy, and then continued orally (7 mg/kg/d) with 6 mercaptopurine. At this stage, an MR imaging scan of the brain and abdomen was performed, and it revealed the complete recanalization of the cerebral vessels, although irregular appearances of the sigmoid colon and rectum were detected (Fig. 1). Despite such intensive medical treatment, the child continued to have bloody diarrhea with slowly declining hemoglobin levels, which required a blood transfusion. Therefore, a total colectomy with ileoanoanastomosis and protective ileostomy was performed 3 months after hospital admission. The macroscopic and histologic appearance of the colon confirmed the diagnosis of UC. During the postoperative course, the patient initially required therapy for severe electrolyte disturbance. His clinical condition then improved, and consequently oral feeding was reintroduced and total parenteral nutrition was stopped. The ileostomy was closed after 1 year. At the 2-year follow-up, the child was growing well. From a neurologic perspective, however, ambulation and speech deficits were present, and he developed postischemic epilepsy. Thromboembolism (TE) is a potentially life-threatening event that is associated with inflammatory bowel disease (IBD). A population-based cohort study has recently shown that IBD patients have a 3-fold increased risk of developing deep venous thrombosis and pulmonary embolism compared with the general population. IBD seems to be a specific risk factor for TE as neither rheumatoid arthritis, another chronic inflammatory conditions, nor celiac disease, another chronic bowel disease, carry an increased risk of TE. IBD paFigure 1. MR imaging scan of the brain (A) and colon (B). A T1-weighted image of the brain showing thrombosis (arrow) of the rectum sinus (A). A T2-expressed sequence showing the irregular shapes of the sigmoid colon and rectum (B).

Matrix Expansion and Syncytial Aggregation of Syndecan-1+ Cells Underpin Villous Atrophy in Coeliac Disease

Background We studied the expression of sulphated glycosaminoglycans (GAGs) in coeliac disease (CD) mucosa, as they are critical determinants of tissue volume, which increases in active disease. We also examined mucosal expression of IL-6, which stimulates excess GAG synthesis in disorders such as Graves ophthalmopathy. Methods We stained archival jejunal biopsies from 5 children with CD at diagnosis, on gluten-free diet and challenge for sulphated GAGs. We then examined duodenal biopsies from 9 children with CD compared to 9 histological normal controls, staining for sulphated GAGs, heparan sulphate proteoglycans (HSPG), short-chain HSPG (Δ-HSPG) and the proteoglycan syndecan-1 (CD138), which is expressed on epithelium and plasma cells. We confirmed findings with a second monoclonal in another 12 coeliac children. We determined mucosal IL-6 expression by immunohistochemistry and PCR in 9 further cases and controls, and used quantitative real time PCR for other Th17 pathway cytokines in an additional 10 cases and controls. Results In CD, HSPG expression was lost in the epithelial compartment but contrastingly maintained within an expanded lamina propria. Within the upper lamina propria, clusters of syndecan-1+ plasma cells formed extensive syncytial sheets, comprising adherent plasma cells, lysed cells with punctate cytoplasmic staining and shed syndecan ectodomains. A dense infiltrate of IL-6+ mononuclear cells was detected in active coeliac disease, also localised to the upper lamina propria, with significantly increased mRNA expression of IL-6 and IL-17A but not IL-23 p19. Conclusions Matrix expansion, through syndecan-1+ cell recruitment and lamina propria GAG increase, underpins villous atrophy in coeliac disease. The syndecan-1+ cell syncytia and excess GAG production recapitulate elements of the invertebrate encapsulation reaction, itself dependent on insect transglutaminase and glutaminated early response proteins. As in other matrix expansion disorders, IL-6 is upregulated and represents a logical target for immunotherapy in patients with coeliac disease refractory to gluten-free diet.

DNA methylation defines regional identity of human intestinal epithelial organoids and undergoes dynamic changes during development

Objective Human intestinal epithelial organoids (IEOs) are increasingly being recognised as a highly promising translational research tool. However, our understanding of their epigenetic molecular characteristics and behaviour in culture remains limited. Design We performed genome-wide DNA methylation and transcriptomic profiling of human IEOs derived from paediatric/adult and fetal small and large bowel as well as matching purified human gut epithelium. Furthermore, organoids were subjected to in vitro differentiation and genome editing using CRISPR/Cas9 technology. Results We discovered stable epigenetic signatures which define regional differences in gut epithelial function, including induction of segment-specific genes during cellular differentiation. Established DNA methylation profiles were independent of cellular environment since organoids retained their regional DNA methylation over prolonged culture periods. In contrast to paediatric and adult organoids, fetal gut-derived organoids showed distinct dynamic changes of DNA methylation and gene expression in culture, indicative of an in vitro maturation. By applying CRISPR/Cas9 genome editing to fetal organoids, we demonstrate that this process is partly regulated by TET1, an enzyme involved in the DNA demethylation process. Lastly, generating IEOs from a child diagnosed with gastric heterotopia revealed persistent and distinct disease-associated DNA methylation differences, highlighting the use of organoids as disease-specific research models. Conclusions Our study demonstrates striking similarities of epigenetic signatures in mucosa-derived IEOs with matching primary epithelium. Moreover, these results suggest that intestinal stem cell-intrinsic DNA methylation patterns establish and maintain regional gut specification and are involved in early epithelial development and disease.