Immunomodulatory role of Parkinson's disease 7 in inflammatory bowel disease.

Recently the role of Parkinson's disease 7 (PARK7) was studied in gastrointestinal diseases, however, the complex role of PARK7 in the intestinal inflammation is still not completely clear. Expression and localization of PARK7 were determined in the colon biopsies of children with inflammatory bowel disease (IBD), in the colon of dextran sodium sulphate (DSS) treated mice and in HT-29 colonic epithelial cells treated with interleukin (IL)-17, hydrogen peroxide (H2O2), tumor necrosis factor (TNF)-α, transforming growth factor (TGF)-ß or lipopolysaccharide (LPS). Effect of PARK7 on the synthesis of IBD related cytokines was determined using PARK7 gene silenced HT-29 cells and 3,4,5-trimethoxy-N-(4-(8-methylimidazo(1,2-a)pyridine-2-yl)phenyl)benzamide (Comp23)-compound increasing PARK7 activity-treated mice with DSS-colitis. PARK7 expression was higher in the mucosa of children with Crohn's disease compared to that of controls. While H2O2 and IL-17 treatment increased, LPS, TNF-α or TGF-ß treatment decreased the PARK7 synthesis of HT-29 cells. PARK7 gene silencing influenced the synthesis of IL1B, IL6, TNFA and TGFB1 in vitro. Comp23 treatment attenuated the ex vivo permeability of colonic sacs, the clinical symptoms, and mucosal expression of Tgfb1, Il1b, Il6 and Il10 of DSS-treated mice. Our study revealed the role of PARK7 in the regulation of IBD-related inflammation in vitro and in vivo, suggesting its importance as a future therapeutic target.

Interleukin-24 regulates mucosal remodeling in inflammatory bowel diseases.

BACKGROUND: Recently, increased interleukin (IL)-24 expression has been demonstrated in the colon biopsies of adult patients with inflammatory bowel disease (IBD). However, the role of IL-24 in the pathomechanism of IBD is still largely unknown. METHODS: Presence of IL-24 was determined in the samples of children with IBD and in the colon of dextran sodium sulfate (DSS) treated mice. Effect of inflammatory factors on IL24 expression was determined in peripheral blood (PBMCs) and lamina propria mononuclear cells (LPMCs). Also, the impact of IL-24 was investigated on HT-29 epithelial cells and CCD-18Co colon fibroblasts. Expression of tissue remodeling related genes was investigated in the colon of wild type (WT) mice locally treated with IL-24 and in the colon of DSS treated WT and Il20rb knock out (KO) mice. RESULTS: Increased amount of IL-24 was demonstrated in the serum and colon samples of children with IBD and DSS treated mice compared to that of controls. IL-1ß, LPS or H2O2 treatment increased the expression of IL24 in PBMCs and LPMCs. IL-24 treatment resulted in increased amount of TGF-ß and PDGF-B in HT-29 cells and enhanced the expression of extracellular matrix (ECM)-related genes and the motility of CCD-18Co cells. Similarly, local IL-24 treatment increased the colonic Tgfb1 and Pdgfb expression of WT mice. Moreover, expression of pro-fibrotic Tgfb1 and Pdgfb were lower in the colon of DSS treated Il20rb KO compared to that of WT mice. The disease activity index of colitis was less severe in DSS treated Il20rb KO compared to WT mice. CONCLUSION: Our study suggest that IL-24 may play a significant role in the mucosal remodeling of patients with IBD by promoting pro-fibrotic processes.

Role of microRNA-223 in the regulation of poly(ADP-ribose) polymerase in pediatric patients with Crohn's disease.

OBJECTIVES: Crohn's disease (CD) is a multifactorial disease, characterized by oxidant-induced tissue injury with a possible activation of poly(ADP-ribose) polymerase (PARP)-1. MicroRNAs (miRs) can offer a potential link between the genetic susceptibility, environmental and immunologic factors in the pathogenesis of CD. Previously, PARP-1 was identified as a direct target gene of miR-223 in an epithelial cell line. Our aim was to examine PARP activation and miR-223 expression in colonic biopsies of pediatric CD. To support our in vivo findings, the effect of lipopolysaccharide (LPS) on same parameters was examined in HT-29 colonic epithelial cell line. METHODS: Colonic biopsies were taken from patients with macroscopically inflamed and intact mucosa with CD and controls. LPS treated HT-29 cells served as our in vitro model. To analyze the PARP-1 expression real-time PCR, Western blot and immunohistochemical analyses were used. PARP-1 enzymatic activity was assessed on the basis of poly(ADP-ribosyl)ated proteins. Expression of miR-223 was examined by real-time PCR. RESULTS: PARP-1 mRNA and miR-223 expression was significantly elevated, however, the amount of PARP-1 protein and poly(ADP-ribose) was reduced in pediatric CD compared to controls. LPS incubation did not affect the expression of PARP-1 mRNA, however, decreased miR-223 expression, and enhanced PARP-1 activity. CONCLUSIONS: In our study, we showed that the expression of miR-223 is up-regulated and poly(ADP-ribosyl)ation is reduced in pediatric patients with CD. Moreover, we confirmed their opposite change in LPS treated epithelial cells, too. These data suggest that the hypofunctionality of PARP-1 may play a potential role in the pathomechanism of CD.

Specific MicroRNA Pattern in Colon Tissue of Young Children with Eosinophilic Colitis.

Eosinophilic colitis (EC) is a common cause of haematochezia in infants and young children. The exact pathomechanism is not understood, and the diagnosis is challenging. The role of microRNAs as key class of regulators of mRNA expression and translation in patients with EC has not been explored. Therefore, the aim of the present study was to explore the miRNA profile in EC with respect to eosinophilic inflammation. Patients enrolled in the study (n = 10) had persistent rectal bleeding, and did not respond to elimination dietary treatment. High-throughput microRNA sequencing was carried out on colonic biopsy specimens of children with EC (EC: n = 4) and controls (C: n = 4) as a preliminary screening of the miRNA profile. Based on the next-generation sequencing (NGS) results and literature data, a potentially relevant panel of miRNAs were selected for further measurements by real-time reverse transcription (RT)-PCR (EC: n = 14, C: n = 10). Validation by RT-PCR resulted in significantly altered expression of miR-21, -31, -99b, -125a, -146a, -184, -221, -223, and -559 compared to controls (p ≤ 0.05). Elevation in miR-21, -99b, -146a, -221, and -223 showed statistically significant correlation to the extent of tissue eosinophilia. Based on our results, we conclude that the dysregulated miRNAs have a potential role in the regulation of apoptosis by targeting Protein kinase B/Mechanistic target of rapamycin (AKT/mTOR)-related pathways in inflammation by modulating Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB)-related signalling and eosinophil cell recruitment and activation, mainly by regulating the expression of the chemoattractant eotaxin and the adhesion molecule CD44. Our results could serve as a basis for further extended research exploring the pathomechanism of EC.

Selective measurement of α smooth muscle actin: why ß-actin can not be used as a housekeeping gene when tissue fibrosis occurs.

BACKGROUND: Prevalence of fibroproliferative diseases, including chronic kidney disease is rapidly increasing and has become a major public health problem worldwide. Fibroproliferative diseases are characterized by increased expression of α smooth muscle actin (α-SMA) that belongs to the family of the six conserved actin isoforms showing high degree homology. The aim of the present study was to develop real-time PCRs that clearly discriminate α-SMA and ß-actin from other actin isoforms. RESULTS: Real-time PCRs using self-designed mouse, human and rat specific α-SMA or ß-actin primer pairs resulted in the specific amplification of the artificial DNA templates corresponding to mouse, human or rat α-SMA or ß-actin, however ß-actin showed cross-reaction with the housekeeping γ-cyto-actin. We have shown that the use of improperly designed literary primer pairs significantly affects the results of PCRs measuring mRNA expression of α-SMA or ß-actin in the kidney of mice underwent UUO. CONCLUSION: We developed a set of carefully designed primer pairs and PCR conditions to selectively determine the expression of mouse, human or rat α-SMA and ß-actin isoforms. We demonstrated the importance of primer specificity in experiments where the results are normalized to the expression of ß-actin especially when fibrosis and thus increased expression of α-SMA is occur.

Microarray Analysis Reveals Increased Expression of Matrix Metalloproteases and Cytokines of Interleukin-20 Subfamily in the Kidneys of Neonate Rats Underwent Unilateral Ureteral Obstruction: A Potential Role of IL-24 in the Regulation of Inflammation and Tissue Remodeling.

BACKGROUND/AIMS: Congenital obstructive nephropathy (CON) is the main cause of pediatric chronic kidney diseases leading to renal fibrosis. High morbidity and limited treatment opportunities of CON urge the better understanding of the underlying molecular mechanisms. METHODS: To identify the differentially expressed genes, microarray analysis was performed on the kidney samples of neonatal rats underwent unilateral ureteral obstruction (UUO). Microarray results were then validated by real-time RT-PCR and bioinformatics analysis was carried out to identify the relevant genes, functional groups and pathways involved in the pathomechanism of CON. Renal expression of matrix metalloproteinase (MMP)-12 and interleukin (IL)-24 were evaluated by real-time RT-PCR, flow cytometry and immunohistochemical analysis. Effect of the main profibrotic factors on the expression of MMP-12 and IL-24 was investigated on HK-2 and HEK-293 cell lines. Finally, the effect of IL-24 treatment on the expression of pro-inflammatory cytokines and MMPs were tested in vitro. RESULTS: Microarray analysis revealed 880 transcripts showing >2.0-fold change following UUO, enriched mainly in immune response related processes. The most up-regulated genes were MMPs and members of IL-20 cytokine subfamily, including MMP-3, MMP-7, MMP-12, IL-19 and IL-24. We found that while TGF-ß treatment inhibits the expression of MMP-12 and IL-24, H2O2 or PDGF-B treatment induce the epithelial expression of MMP-12. We demonstrated that IL-24 treatment decreases the expression of IL-6 and MMP-3 in the renal epithelial cells. CONCLUSIONS: This study provides an extensive view of UUO induced changes in the gene expression profile of the developing kidney and describes novel molecules, which may play significant role in the pathomechanism of CON.

Altered mucosal expression of microRNAs in pediatric patients with inflammatory bowel disease.

INTRODUCTION: MicroRNAs (miRs) came recently into focus as promising novel research targets offering new insights into the pathogenesis of inflammatory bowel diseases (IBD). AIMS: The aim of our study was to identify a pediatric IBD (pIBD) characteristic miR profile serving as potential Crohn's disease (CD) and ulcerative colitis (UC) specific diagnostic pattern and to further analyze the related target genes. METHODS: Small RNA sequencing was performed on inflamed and intact colonic biopsies of CD, and control patients. Selected miRs were further investigated by RT-PCR, complemented with an UC group, in order to address the differential diagnostic potential of miRs in the two IBD subtypes. To analyze network connection of differentially expressed miRs and their target genes MiRTarBase database and previous transcriptome sequencing data from pediatric patient groups were used. RESULTS: Sequencing analysis identified 170 miRs with altered expression. RT-PCR analysis revealed altered expression of miR-31, -125a, -142-3p, and -146a discriminating between the inflamed mucosa of CD and UC. In the intact mucosa of CD patients the expression of miR-18a, -20a, -21, -31, -99a, -99b, -100, -125a, -126, -142-5p, -146a, -185, -204, -221, and -223 was elevated compared to the controls. The expression of miR-20a, -204 and -221 was elevated exclusively in the intact region of CD patients compared to the controls. Enrichment analysis identified main IBD-related functional groups. CONCLUSIONS: We demonstrated a characteristic colonic miR pattern in pIBD that could facilitate deeper understanding of the pathomechanism of IBD and may serve as a diagnostic tool.

Increased duodenal expression of miR-146a and -155 in pediatric Crohn's disease.

AIM: To evaluate the role of microRNA (miR)-146a, -155 and -122 in the duodenal mucosa of pediatric patients with Crohn's disease (CD) and the effect of transforming growth factor-ß (TGF-ß) on these miRs in duodenal epithelial and fibroblast cells. METHODS: Formalin-fixed, paraffin-embedded biopsies derived from the macroscopically inflamed (CD inflamed: n = 10) and intact (CD intact: n = 10) duodenal mucosa of pediatric CD patients and control children (C: n = 10) were examined. Expression of miR-146a, -155 and -122 was determined by real-time polymerase-chain reaction (PCR). The expression of the above miRs was investigated in recombinant human TGF-ß (1 nmol/L, 24 h) or vehicle treated small intestinal epithelial cells (CCL-241) and primary duodenal fibroblast cells derived from healthy children as well. RESULTS: Expression of miR-146a was significantly higher in the inflamed duodenal mucosa compared to the intact duodenal mucosa of children with CD (CD inflamed: 3.21 ± 0.50 vs CD intact: 0.62 ± 0.26, P ≤ 0.01) and to the control group (CD inflamed: 3.21 ± 0.50 vs C: 1.00 ± 0.33, P ≤ 0.05). The expression of miR-155 was significantly increased in the inflamed region of the duodenum compared to the control group (CD inflamed: 4.87 ± 1.02 vs CONTROL: 1.00 ± 0.40, P ≤ 0.001). The expression of miR-122 was unchanged in the inflamed or intact mucosa of CD patients compared to controls. TGF-ß treatment significantly decreased the expression of miR-155 in small intestinal epithelial cells (TGF-ß: 0.7 ± 0.083 vs CONTROL: 1 ± 0.09, P ≤ 0.05) and also the expression of miR-146a (TGF-ß: 0.67 ± 0.04 vs CONTROL: 1 ± 0.15, P ≤ 0.01) and miR-155 (TGF-ß: 0.72 ± 0.09 vs CONTROL: 1 ± 0.06, P ≤ 0.05) in primary duodenal fibroblasts compared to corresponding vehicle treated controls. TGF-ß treatment did not influence the expression of miR-122. CONCLUSION: The elevated expression of miR-146a and -155 in the inflamed duodenal mucosa of CD patients suggests the role of these miRs in the pathomechanism of inflammatory bowel disease. Anti-inflammatory TGF-ß plays an important role in the regulation of the expression of these miRs.

Sex differences in renin response and changes of capillary diameters after renal ischemia/reperfusion injury.

Activation of the RAS has a crucial role in the progression of ischemia/reperfusion-associated CAD. The regulation of RAS differs in the two genders. However, the extent of gender differences and locations of renin production have not been revealed yet. We investigated in vivo the local renin production in the two genders during ischemia/reperfusion injury. In male and female Wistar rats, renal ischemia was induced followed by a reperfusion period of two, eight, 16, 24, or 48 h. We applied flow cytometry to measure renin content and multiphoton imaging to visualize renin granules and changes of peritubular diameters in vivo during ischemia/reperfusion. Renin content decreased in CD in the first eight h of reperfusion; however, after 16 h, its amount increased. In males, the production of renin was more pronounced, and the duration of vasoconstriction was longer with a subsequent phase of vessel hyperdilation compared to females. Renal ischemia/reperfusion injury induces renin response not only in the JGA, but also in the CD segment. Renin production is more explicit in males than in females which, via increased angiotensin II production, might explain the different dynamism of renal vessel regulation between the two genders.

Role of Altered Expression of miR-146a, miR-155, and miR-122 in Pediatric Patients with Inflammatory Bowel Disease.

BACKGROUND: Evidence suggests the central role of tumor necrosis factor (TNF)-α in the pathomechanism of inflammatory bowel disease (IBD); however, its effect on epigenetic factors, including small non-coding microRNAs (miRs), is less known. Our present aim was the comparative investigation of the expression of TNF-α and immune response-related miRs in children with Crohn's disease (CD) and ulcerative colitis (UC). METHODS: Fresh-frozen (FF) and formalin-fixed, paraffin-embedded (FFPE) biopsies were used to analyze the expression of miR-146a, -155, -122, and TNF-α by real-time reverse transcription polymerase chain reaction in macroscopically inflamed (CD: 12 FFPE and 24 FF; UC: 10 FF) and intact (CD: 12 FFPE; 14 FF) colonic biopsies of children with IBD and controls (16 FFPE; 23 FF). The expression of miR-146a, -155, and -122 was also determined in TNF-α-treated HT-29 colonic epithelial cells. RESULTS: Increased expression of TNF-α was observed in the colonic mucosa of children with CD and UC in comparison with controls. Expression of miR-146a and -155 was higher in the inflamed mucosa of children with CD and UC than in the intact mucosa. Expression of miR-122 elevated in the macroscopically intact colonic regions of CD compared with controls and patients with UC. In HT-29 cells, TNF-α treatment increased the expression of miR-146a and -155, but not that of miR-122. CONCLUSIONS: Our results showed altered expression of miR-146a, -155, and -122 in the colonic mucosa of children with IBD and in TNF-α-treated colonic epithelial cells. Our data suggest the TNF-α-related involvement of these miRs in the pathogenesis of IBD.

Calcineurin-inhibition Results in Upregulation of Local Renin and Subsequent Vascular Endothelial Growth Factor Production in Renal Collecting Ducts.

BACKGROUND: Tacrolimus (Tac) and Cyclosporine A (CyA) calcineurin inhibitors (CNIs) are 2 effective immunosuppressants which are essential to prevent allograft rejection. Calcineurin inhibitors are known to be nephrotoxic. However, the precise mechanism of nephrotoxicity is not fully understood. In this study, we investigated the in vivo effects of CNIs on the local renal renin-angiotensin system in the collecting duct (CD). METHODS: Three-week-old mice were treated with either vehicle, CyA (2 mg/kg per day), Tac (0.075 mg/kg per day), CyA + Aliskiren (25 mg/kg per day), or Tac + Aliskiren for 3 weeks. Serum creatinine was measured. Renin and vascular endothelial growth factor (VEGF) contents in CD were evaluated with flow cytometry and multiphoton microscopy. The diameter of vessels was assessed with multiphoton microscopy, and the amount of renal collagen was determined by real-time polymerase chain reaction and Masson staining. RESULTS: The elevated level of serum creatinine in CNI groups was abolished by Aliskiren. Flow cytometric analysis found elevated renin content in principal cells, which was prevented by Aliskiren. This result was further confirmed with multiphoton microscopy. The VEGF content in CD correlated with reduced capillary diameter and with the formation of fibrotic islands. CONCLUSIONS: Calcineurin inhibitors induce production of renin in the CD that may contribute to decreased renal blood flow. In turn, CD responds with increased VEGF production, resulting in disproportional vessel growth, further worsening the local hypoxia and striped fibrosis surrounding the CDs. Aliskiren, a direct renin inhibitor blocks these effects and improves CNI-induced nephropathy by decreasing renin production in the CDs. Our data suggest that Aliskiren may be used for the prevention of CNI nephrotoxicity.

Fibrosis Related Inflammatory Mediators: Role of the IL-10 Cytokine Family.

Importance of chronic fibroproliferative diseases (FDs) including pulmonary fibrosis, chronic kidney diseases, inflammatory bowel disease, and cardiovascular or liver fibrosis is rapidly increasing and they have become a major public health problem. According to some estimates about 45% of all deaths are attributed to FDs in the developed world. Independently of their etiology the common hallmark of FDs is chronic inflammation. Infiltrating immune cells, endothelial, epithelial, and other resident cells of the injured organ release an orchestra of inflammatory mediators, which stimulate the proliferation and excessive extracellular matrix (ECM) production of myofibroblasts, the effector cells of organ fibrosis. Abnormal amount of ECM disturbs the original organ architecture leading to the decline of function. Although our knowledge is rapidly expanding, we still have neither a diagnostic tool to detect nor a drug to specifically target fibrosis. Therefore, there is an urgent need for the more comprehensive understanding of the pathomechanism of fibrosis and development of novel diagnostic and therapeutic strategies. In the present review we provide an overview of the common key mediators of organ fibrosis highlighting the role of interleukin-10 (IL-10) cytokine family members (IL-10, IL-19, IL-20, IL-22, IL-24, and IL-26), which recently came into focus as tissue remodeling-related inflammatory cytokines.

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.

Involvement of heat shock proteins in gluten-sensitive enteropathy.

Gluten-sensitive enteropathy, also known as coeliac disease (CD), is an autoimmune disorder occurring in genetically susceptible individuals that damages the small intestine and interferes with the absorption of other nutrients. As it is triggered by dietary gluten and related prolamins present in wheat, rye and barley, the accepted treatment for CD is a strict gluten-free diet. However, a complete exclusion of gluten-containing cereals from the diet is often difficult, and new therapeutic strategies are urgently needed. A class of proteins that have already emerged as drug targets for other autoimmune diseases are the heat shock proteins (HSPs), which are highly conserved stress-induced chaperones that protect cells against harmful extracellular factors. HSPs are expressed in several tissues, including the gastrointestinal tract, and their levels are significantly increased under stress circumstances. HSPs exert immunomodulatory effects, and also play a crucial role in the maintenance of epithelial cell structure and function, as they are responsible for adequate protein folding, influence the degradation of proteins and cell repair processes after damage, and modulate cell signalling, cell proliferation and apoptosis. The present review discusses the involvement of HSPs in the pathophysiology of CD. Furthermore, HSPs may represent a useful therapeutic target for the treatment of CD due to the cytoprotective, immunomodulatory, and anti-apoptotic effects in the intestinal mucosal barrier.

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.

[The role of intestinal alkaline phosphatase in pediatric inflammatory bowel and celiac diseases]. / Az intestinalis alkalikus foszfatáz szerepe gyermekkori gyulladásos bélbetegségben és coeliakiában.

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.

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 Crohn's 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.

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.

Increased heat shock protein 72 expression in celiac disease.

BACKGROUND AND OBJECTIVES: Heat shock protein (HSP) 72, a known chaperone, has potential epithelial barrier protecting, antiapoptotic, and immune system regulatory effects; therefore, our aim was to study its involvement in the pathology of celiac disease (CD). PATIENTS AND METHODS: Duodenal biopsy specimens were collected from children with untreated and treated CD and from controls. mRNA expression, protein level, and localization of HSP72 were determined. RESULTS: Elevated HSP72 mRNA expression and higher protein levels were found in the duodenal mucosa of children with untreated CD as well as in children with treated CD compared with those in controls. In the duodenal mucosa of children with treated CD, HSP72 mRNA expression was decreased and HSP72 protein levels were lower than those in children with untreated CD. We detected intensive HSP72 staining in the villous enterocytes and immune cells of the lamina propria in the duodenal villi of children with untreated CD compared with that in controls. CONCLUSIONS: The increased expression and altered localization of HSP72 in CD indicate that HSP72 should have a role in protection against gliadin-induced cytotoxicity. HSP72 may exert antiapoptotic effect and contribute to preservation of intestinal epithelial barrier integrity. Moreover, HSP72 as a ligand of TLR2 and TLR4 may promote innate immune responses and warn the cells of the potential injury.