Hajnalka Győrffy

Mucosal expression of claudins 2, 3 and 4 in proximal and distal part of duodenum in children with coeliac disease

Duodenal biopsy is an important tool to diagnose coeliac disease (CD); however, the most reliable location of biopsy site is still questionable. Claudins (CLDNs), members of a large family of adherent junction proteins, show characteristic expression pattern in inflammatory disorders; nevertheless, CLDN expression in CD is unknown. This is a comparative study to examine the CLDN 2, 3 and 4 expressions in proximal and distal part of duodenum in children with CD and in controls. Thirty-three children with newly diagnosed CD were enrolled. Fourteen healthy children served as controls. Biopsies from proximal and distal part of duodenum were taken for routine histological analysis. Immunohistochemistry were used to detect CD3+ intraepithelial lymphocytes and CLDN 2, 3 and 4 protein expressions. Macroscopic picture, routine histology and Marsh grade depicted no differences between biopsies taken from proximal or distal part of duodenum. However, CLDN 2 expression was significantly increased in severe form of coeliac disease in bulb and in distal duodenum, and in distal part of non-severe coeliac patients, in comparison to controls. Similar association was found concerning CLDN 3 expression. Expression of CLDN 4 was similar in all groups studied. Both proximal and distal mucosal duodenal biopsies are suitable for diagnosing villous atrophy in patients with CD. Increased expressions of CLDN 2 and 3 suggest structural changes of tight junction in coeliac disease which may be, at least in part, responsible for increased permeability and proliferation observed in coeliac disease.

Intestinal alkaline phosphatase in the colonic mucosa of children with inflammatory bowel disease

AIM To investigate intestinal alkaline phosphatase (iAP) in the intestinal mucosa of children with inflammatory bowel disease (IBD). METHODS Colonic biopsy samples were taken from 15 newly diagnosed IBD patients and from 10 healthy controls. In IBD patients, specimens were obtained both from inflamed and non-inflamed areas. The iAP mRNA and protein expression was determined by reverse transcription-polymerase chain reaction and Western blotting analysis, respectively. Tissue localization of iAP and Toll-like receptor (TLR) 4 was investigated by immunofluorescent staining. RESULTS The iAP protein level in the inflamed mucosa of children with Crohns disease (CD) and ulcerative colitis (UC) was significantly decreased when compared with controls (both P < 0.05). Similarly, we found a significantly decreased level of iAP protein in the inflamed mucosa in CD compared with non-inflamed mucosa in CD (P < 0.05). In addition, the iAP protein level in inflamed colonic mucosa in patients with UC was decreased compared with non-inflamed mucosa in patients with CD (P < 0.05). iAP protein levels in the non-inflamed mucosa of patients with CD were similar to controls. iAP mRNA expression in inflamed colonic mucosa of children with CD and UC was not significantly different from that in non-inflamed colonic mucosa with CD. Expression of iAP mRNA in patients with non-inflamed mucosa and in controls were similar. Co-localization of iAP with TLR4 showed intense staining with a dotted-like pattern. iAP was present in the inflamed and non-inflamed mucosa of patients with CD, UC, and in control biopsy specimens, irrespective of whether it was present in the terminal ileum or in the colon. However, the fluorescent signal of TLR4 was more pronounced in the colon compared with the terminal ileum in all groups studied. CONCLUSION Lower than normal iAP protein levels in inflamed mucosa of IBD patients may indicate a role for iAP in inflammatory lesions in IBD. Based on our results, administration of exogenous iAP enzyme to patients with the active form of IBD may be a therapeutic option.

Zonula Occludens-1, Occludin, and E-cadherin Protein Expression in Biliary Tract Cancers

The incidence of cholangiocarcinomas originating from intrahepatic and extrahepatic bile ducts, as well as of gallbladder carcinoma is increasing worldwide. The malignant transformation of biliary epithelia involves profound alterations of proteins in the intercellular junctions, among others zonula occludens-1 (ZO-1), occludin, and E-cadherin. Each plays important role in the maintenance of epithelial cell polarity and regulation of cell growth and differentiation. Our aim was to investigate ZO-1, occludin, and E-cadherin immunohistochemical reactions in tissue microarray blocks containing 57 normal and 62 neoplastic biliary tract samples. We demonstrated that the tight junction components ZO-1, occludin, and E-cadherin are downregulated in carcinomas arising from various compartments of the biliary tract (normal intrahepatic and extrahepatic bile ducts, gallbladder) as compared with their normal sites of origin. These results were confirmed by discriminant analysis yielding clear separation of the three normal sample groups from carcinomas in the corresponding locations.

Decreased mucosal expression of intestinal alkaline phosphatase in children with coeliac disease

A major function of the enzyme intestinal alkaline phosphatase (iAP) is the detoxification of lipopolysaccharide (LPS), the ligand of Toll-like receptor 4 (TLR4). Hence, iAP has a role in the defence of maintaining intestinal barrier integrity. As intestinal barrier integrity is impaired in coeliac disease (CD), we tested the expression and localization of iAP in duodenal mucosa specimens from children with newly diagnosed CD (n = 10), with CD on gluten-free diet (GFD) (n = 5) and compared to those from ten healthy children. The mRNA and protein expression was determined by RT-PCR and Western blot analysis, respectively. Tissue localization of iAP and TLR4 was determined by immunofluorescence staining. iAP protein expression level was significantly lower than normal in newly diagnosed CD, while it was normalised in children on GFD. iAP and TLR4 colocalized at the epithelial surface of duodenal mucosa in each group of subjects enrolled. The finding of decreased iAP protein levels in newly diagnosed CD is consistent with its role in decreased intestinal barrier integrity. The latter may be the result of decreased LPS-detoxifying ability.

Characteristics of allergic colitis in breast-fed infants in the absence of cow’s milk allergy

AIM To investigate the characteristics of mucosal lesions and their relation to laboratory data and long-term follow up in breast-fed infants with allergic colitis. METHODS In this study 31 breast-fed infants were prospectively evaluated (mean age, 17.4 wk) whose rectal bleeding had not ceased after a maternal elimination diet for cows milk. Thirty-four age-matched and breast-fed infants (mean age, 16.9 wk) with no rectal bleeding were enrolled for laboratory testing as controls. Laboratory findings, colonoscopic and histological characteristics were prospectively evaluated in infants with rectal bleeding. Long-term follow-up with different nutritional regimes (L-amino-acid based formula or breastfeeding) was also included. RESULTS Iron deficiency, peripheral eosinophilia and thrombocytosis were significantly higher in patients with allergic colitis in comparison to controls (8.4 ± 3.2 μmol/L vs 13.7 ± 4.7 μmol/L, P < 0.001; 0.67 ± 0.49 G/L vs 0.33 ± 0.17 G/L, P < 0.001; 474 ± 123 G/L vs 376 ± 89 G/L, P < 0.001, respectively). At colonoscopy, lymphonodular hyperplasia or aphthous ulceration were present in 83% of patients. Twenty-two patients were given L-amino acid-based formula and 8 continued the previous feeding. Time to cessation of rectal bleeding was shorter in the special formula feeding group (mean, 1.4 wk; range, 0.5-3 wk) when compared with the breast-feeding group (mean, 5.3 wk; range, 2-9 wk). Nevertheless, none of the patients exhibited rectal bleeding at the 3-mo visit irrespective of the type of feeding. Peripheral eosinophilia and cessation of rectal bleeding after administration of elemental formula correlated with a higher density of mucosal eosinophils. CONCLUSION Infant hematochezia, after cows milk allergy exclusion, is generally a benign and probably self-limiting disorder despite marked mucosal abnormality. Formula feeding results in shorter time to cessation of rectal bleeding; however, breast-feeding should not be discouraged in long-lasting hematochezia.

Preliminary studies of the effects of vascular adhesion protein-1 inhibitors on experimental corneal neovascularization

Vascular adhesion protein-1 (VAP-1) controls the adhesion of lymphocytes to endothelial cells and is upregulated at sites of inflammation. Moreover, it expresses amine oxidase activity, due to the sequence identity with semicarbazide-sensitive amine oxidase. Recent studies indicate a significant role for VAP-1 in neovascularization, besides its contribution to inflammation. Pathological blood vessel development in severe ocular diseases (such as diabetes, age-related macula degeneration, trauma and infections) might lead to decreased visual acuity and finally to blindness, yet there is no clear consensus as to its appropriate treatment. In the present case study, the effects of two VAP-1 inhibitors on experimentally induced corneal neovascularization in rabbits were compared with the effects of a known inhibitor of angiogenesis, bevacizumab, an anti-vascular endothelial growth factor antibody. In accordance with recent literature data, the results of the preliminary study reported here indicate that the administration of VAP-1 inhibitors is a potentially valuable therapeutic option in the treatment of corneal neovascularization.

[Crohn's disease of the duodenum. Clinical signs, diagnosis, conservative and surgical treatment].

Duodenal localization of Crohns disease is rare, accounting for only 0.5-4% of all cases. Most common complaints are gastric outlet obstruction and weight loss. Histologic findings of endoscopic biopsy are frequently not definitive, making differentiation from other, benign structures complicated. There are also no standard guidelines regarding indications for surgical management. PATIENTS AND METHODS: We reviewed the cases of three surgically managed patients with duodenal Crohns disease at the 1st Department of Surgery, Semmelweis University of Medicine, Budapest, during a 5-year period (2002-2007). All three patients had persistent symptoms of stomach emptying disorder despite medical therapy and had severe weight loss (13-30 kg). In two cases resection of the stenotic duodenum was performed successfully using Billroth II method. Gastro-jejunal bypass was performed in one case, where the descendent duodenum was inflamed. RESULTS: All patients have been asymptomatic since surgery (9-45 months of follow-up) and recovered their earlier bodyweight. The postoperative period was uneventful. CONCLUSIONS: There is indication of surgery in cases of stenosing duodenal Crohns disease, when medical therapy is not successful, but long-standing malnutrition should be treated preoperatively. We found perioperative morbidity to be similar in patients with duodenal Crohns and in those with Crohns disease of other intestinal locations.UNLABELLED Duodenal localization of Crohns disease is rare, accounting for only 0.5-4% of all cases. Most common complaints are gastric outlet obstruction and weight loss. Histologic findings of endoscopic biopsy are frequently not definitive, making differentiation from other, benign structures complicated. There are also no standard guidelines regarding indications for surgical management. PATIENTS AND METHODS We reviewed the cases of three surgically managed patients with duodenal Crohns disease at the 1st Department of Surgery, Semmelweis University of Medicine, Budapest, during a 5-year period (2002-2007). All three patients had persistent symptoms of stomach emptying disorder despite medical therapy and had severe weight loss (13-30 kg). In two cases resection of the stenotic duodenum was performed successfully using Billroth II method. Gastro-jejunal bypass was performed in one case, where the descendent duodenum was inflamed. RESULTS All patients have been asymptomatic since surgery (9-45 months of follow-up) and recovered their earlier bodyweight. The postoperative period was uneventful. CONCLUSIONS There is indication of surgery in cases of stenosing duodenal Crohns disease, when medical therapy is not successful, but long-standing malnutrition should be treated preoperatively. We found perioperative morbidity to be similar in patients with duodenal Crohns and in those with Crohns disease of other intestinal locations.

Expression of PARK7 is increased in celiac disease

Recently, it has been suggested that the gene called Parkinson’s disease 7 (PARK7) might be an upstream activator of hypoxia-inducible factor (HIF)-1α, which plays a major role in sustaining intestinal barrier integrity. Furthermore, PARK7 has been proposed to participate in the Toll-like receptor (TLR)-dependent regulation of the innate immune system. Our aim was to investigate the involvement of PARK7 in the pathogenesis of coeliac disease (CD). Duodenal biopsy specimens were collected from 19 children with untreated CD, five children with treated CD (maintained on gluten-free diet), and ten children with histologically normal duodenal biopsies. PARK7 mRNA expression and protein level were determined by real-time polymerase chain reaction (PCR) and Western blot, respectively. Localization of PARK7 was visualized by immunofluorescence staining. Protein level of PARK7 increased in the duodenal mucosa of children with untreated CD compared to children with treated CD or to control biopsies (p <0.03). We detected intensive PARK7 staining in the epithelial cells and lamina propria of the duodenal mucosa of children with untreated CD compared with that in control biopsies. Our finding that mucosal expression of PARK7 is increased suggests that PARK7 is involved in the pathogenesis of gastrointestinal diseases, notably CD. Our results suggest that PARK7 may alter processes mediated by HIF-1α and TLR4, which supports a role for PARK7 in the maintenance of epithelial barrier integrity, immune homeostasis, or apoptosis.

Peroxisome proliferator-activated receptor-γ and thymic stromal lymphopoietin are involved in the pathophysiology of childhood coeliac disease

Celiac disease (CD) is a chronic autoimmune enteropathy caused by exposure to dietary gluten in genetically predisposed individuals. The transcription factor peroxisome proliferator-activated receptor gamma (PPARγ) was shown to exert protective effects in several immune-mediated disorders. Activation of PPARγ suppressed the expression of thymic stromal lymphopoietin (TSLP), an inducer of proinflammatory cytokines. Since the role of TSLP in gluten-sensitive enteropathy is completely unknown, we investigated the involvement of TSLP and its regulator PPARγ in childhood CD. We collected duodenal biopsy specimens from 19 children with newly diagnosed CD, 6 children with treated CD (gluten-free diet, GFD), and 10 controls. Expression of mRNA and protein levels of PPARγ, TSLP, and TSLP receptor were determined by real-time RT-PCR and Western blot, respectively. Duodenal localization of PPARγ and TSLP was studied by immunohistochemistry. In duodenal mucosa of children with CD, the amount of PPARγ was significantly lower and simultaneously that of TSLP significantly higher compared to controls (p < 0.05). In GFD-treated patients, the levels of PPARγ mRNA and protein were significantly higher while that of TSLP markedly lower compared to newly diagnosed CD (p < 0.05). Immunohistochemistry revealed PPARγ and TSLP expression in lamina propria immune cells and in enterocytes. Low expression of PPARγ and high expression of TSLP in the duodenal mucosa of children with newly diagnosed CD suggest that they are involved in the pathophysiology of CD. We hypothesize that PPARγ may be an inhibitory regulator of TSLP-stimulated inflammatory processes in CD.

The role of intestinal alkaline phosphatase in pediatric inflammatory bowel and celiac diseases

Intestinal alkaline phosphatase enzyme plays a pivotal role in the maintenance of intestinal mucosal barrier integrity with the detoxification capacity of lipopolysaccharide, the ligand of Toll-like receptor 4. The inappropriate immune responses and the damage of the mucosal barrier may contribute to the initiation of inflammatory bowel and celiac diseases. In the inflamed colonic mucosa of children with inflammatory bowel disease and in the duodenal mucosa of newly diagnosed children with celiac disease, the decreased intestinal alkaline phosphatase and increased Toll-like receptor 4 protein expression may generate enhanced lipopolysaccharide activity, which may strengthen tissue damaging processes. The enhancement of intestinal alkaline phosphatase activity in an animal model of colitis and in therapy resistant, adult patients with ulcerative colitis reduced the symptoms of intestinal inflammation. In accordance with these results, the targeted intestinal administration of the enzyme in the two examined disorders may be a supplemental therapeutic option in the future.