Colitis Biopsies Research Articles

Immunomodulatory Effect of Vancomycin on Treg in Pediatric Inflammatory Bowel Disease and Primary Sclerosing Cholangitis

Vancomycin has been shown to affect tumor necrosis factor-alpha (TNF-α) pathways as an immunomodulator; this is thought to be separate from its function as an antibiotic [1]. Previous studies have shown that oral vancomycin (OV) is an effective treatment for concomitant primary sclerosing cholangitis (PSC) and inflammatory bowel disease (IBD) in children [2, 3]. Since both diseases are associated with immune dysfunction, we hypothesized that vancomycin's therapeutic effect in IBD and PSC occurs through immunomodulation. Therefore, we examined the in vivo immunological changes that occur during OV treatment of 14 children with PSC and IBD. Within 3 months of OV administration, peripheral gamma-glutamyl transpeptidase (GGT) and alanine aminotransferase (ALT) concentrations, white blood cell (WBC) counts, and neutrophil counts normalized from elevated levels before treatment. Patients also demonstrated improved biliary imaging studies, liver biopsies and IBD symptoms and biopsies. Additionally, plasma transforming growth factor beta (TGF-β) levels were increased without concurrent shifts in Th1-or Th2-associated cytokine production. Peripheral levels of CD4 + CD25hiCD127lo and CD4 + FoxP3+ regulatory T (Treg) cells also increased in OV-treated PSC + IBD patients compared to pretreatment levels. A unique case study shows that the therapeutic effects of OV in the treatment of PSC + IBD do not always endure after OV discontinuation, with relapse of PSC associated with a decrease in blood Treg levels; subsequent OV retreatment was then associated with a rise in blood Treg levels and normalization of liver function tests (LFTs). Taken together, these studies support immune-related pathophysiology of PSC with IBD, which is responsive to OV.

P461 Analysis of the mucosal microbiota from inflammatory bowel disease biopsy tissues using a custom 16s rRNA based phylogenetic microarray

P461 Analysis of the mucosal microbiota from inflammatory bowel disease biopsy tissues using a custom 16s rRNA based phylogenetic microarray

Variable Impact of CD39 in Experimental Murine Colitis

Dysregulation of immune responses in inflammatory bowel diseases (IBD) results in intestinal inflammation and vascular injury while exacerbating systemic disease. CD39 is an ectonucleotidase, expressed by T regulatory cells and dendritic cells, that hydrolyzes extracellular nucleotides to modify those cellular immune responses implicated in IBD. Genetic polymorphisms of CD39 have been linked to Crohn's disease while gene deletion in mice exacerbates dextran sodium sulphate-induced colitis. The aim of this study was to test how global deletion of CD39 in mice impacts other models of experimental colitis. Colitis was induced in CD39-null and -wt mice, using trinitrobenzene sulfonic acid (TNBS, 125 mg/kg) administered intrarectally. Oxazolone colitis (1.5% oxazolone in 50% alcohol) was induced in comparable groups. Morphology, clinical and molecular parameters, and FACS analyses of lamina propria mononuclear cells (LPMC) were examined in CD39-null mice. CD39 expression was analyzed in human IBD biopsies. Paradoxically, TNBS colitis in CD39-null mice was characterized by improved survival, favorable clinical scores, and decreased MPO activity, when compared to wt mice (P < 0.05). LPMC from TNBS colitis contained significantly increased amounts of T-cells (CD3(+) and CD4(+)) and TNF-α mRNA expression were increased over those in CD39 null mice (P < 0.05). In contrast, oxazolone treated CD39-null and wt mice had comparable outcomes. In both ulcerative colitis and Crohn's disease, CD39 is present at high levels in intestinal tissue biopsies. TNBS colitis was attenuated in CD39-null mice whereas oxazolone-induced colitis was not impacted. Impaired adaptive cellular immune reactivity in the CD39-null environment appears protective in hapten-mediated Th1-type colitis. CD39 is expressed at high levels in clinical IBD tissues.

Down-regulation of p38 mitogen-activated protein kinase activation and proinflammatory cytokine production by mitogen-activated protein kinase inhibitors in inflammatory bowel disease

Crohn's disease and ulcerative colitis are inflammatory bowel diseases (IBD) characterized by chronic relapsing mucosal inflammation. Tumour necrosis factor (TNF)-α, a known agonist of the mitogen-activated protein kinase (MAPK) pathway, is a key cytokine in this process. We aimed first to determine whether p38 MAPK is activated in IBD inflamed mucosa, and then studied the effect of four different p38α inhibitory compounds on MAPK phosphorylation and secretion of proinflammatory cytokines by IBD lamina propria mononuclear cells (LPMCs) and organ culture biopsies. In vivo phospho-p38α and p38α expression was evaluated by immunoblotting on intestinal biopsies from inflamed areas of patients affected by Crohn's disease and ulcerative colitis, and from normal mucosa of sex- and age-matched control subjects. Both mucosal biopsies and isolated LPMCs were incubated with four different p38α selective inhibitory drugs. TNF-α, interleukin (IL)-1β and IL-6 were measured in the organ and cell culture supernatants by enzyme-linked immunosorbent assay. We found higher levels of phospho-p38α in the inflamed mucosa of IBD patients in comparison to controls. All the p38α inhibitory drugs inhibited p38α phosphorylation and secretion of TNF-α, IL-1β and IL-6 from IBD LPMCs and biopsies. Activated p38α MAPK is up-regulated in the inflamed mucosa of patients with IBD. Additionally, all the p38α selective inhibitory drugs significantly down-regulated the activation of the MAPK pathway and the secretion of proinflammatory cytokines.

Mechanisms of Diarrhea

Diarrhea is a symptom common to a wide variety of gastrointestinal illnesses, and is an important public health challenge in underdeveloped regions of the world. Normal intestinal absorption is a complex process. Recent research offers new insights into normal physiology and pathophysiology. The role of the enteric nervous system and neurotransmitters in the pathogenesis of diarrhea in inflammatory bowel disease (IBD) and irritable bowel syndrome (IBS) is being actively investigated. In patients with IBD, ileal and sigmoid biopsies showed altered transepithelial sodium and fluid transport, specifically from decreased expression of the NHE3, NHERF-1, and NHE1 epithelial Na channel. This results in changes in normal intestinal electroneutral NaCl absorption and may be an additional factor contributing to the diarrhea in patients with IBD. Physiologic studies in humans suggest that primary bile acid malabsorption may be caused by an abnormal feedback system resulting in the increased bile salts, which may explain the watery diarrhea. Finally, the role of zinc in treatment of infectious diarrhea led to studies of its effect on intracellular human enterocyte ion secretion. Understanding such basic mechanisms may lead to better and novel therapies for treatment of diarrhea.

Crypt abscess-associated microbiota in inflammatory bowel disease and acute self-limited colitis

To evaluate whether crypt abscesses from inflammatory bowel disease (IBD) patients contain bacteria and to establish their nature. We studied 17 ulcerative colitis patients, 11 Crohn's disease patients, 7 patients with acute self-limited colitis (ASLC) and normal colonic biopsies from 5 subjects who underwent colonoscopy for colon cancer screening. A fluorescent in situ hybridization technique was applied to colonic biopsies to assess the microbiota composition of the crypts and crypt abscesses. Crypts colonized by bacteria were observed in 42.9% and 3.6% of ASLC and IBD patients, respectively (P = 0.019). Crypt abscesses colonized by bacteria were observed in 28.6% and 0.0% of ASLC and IBD patients, respectively (P = 0.035). These results do not support the hypothesis that crypt abscesses in IBD are the result of localized dysbiosis arising from persistence of living bacteria colonizing the crypts.

Graft-Versus-Host Disease (GVHD): A Rare but Lethal Cause of Common Gastrointestinal Symptoms Following Orthotopic Liver Transplantation (OLT)

Purpose: 1) To increase awareness of GVHD as an uncommon but life-threatening etiology of common gastrointestinal symptoms after OLT. 2) To highlight the role of histopathology in augmenting endoscopy as a powerful diagnostic tool. Case: A 45-year-old male with a history of OLT four months earlier for alpha-1 antitrypsin deficiency was admitted to hospital with two days of diarrhea, abdominal pain and vomiting. He was febrile, tachycardic and jaundiced with a benign abdominal exam. Laboratory tests showed pancytopenia, acute renal insufficiency, hyperbilirubinemia and positive stool Clostridium difficile (C. diff.) toxin. Computed tomogram showed diffuse small bowel wall thickening. Treatment for C. diff. infection was initiated. Mycophenolate was discontinued and he was maintained on tacrolimus. He continued to clinically deteriorate and have high stool output despite aggressive treatment of C. diff. infection. Flexible sigmoidoscopy showed pseudomembranous colitis but colon biopsies were non-diagnostic. His course was further complicated by pneumonia, bacteremia, coagulopathy, dialysis-dependent renal failure and upper gastrointestinal bleed. Esophagogastroduo-denoscopy (EGD) showed gastric erythema and friability consistent with possible stress gastritis and normal duodenum. Biopsies were not taken. Due to clinical deterioration, the patient underwent colectomy. Histopathology of the colon revealed possible GVHD. Intraoperative liver biopsy showed cholestasis only. Repeat upper endoscopy to establish the diagnosis showed severely denuded mucosa in the stomach and duodenum. Gastric and small bowel biopsies confirmed GVHD. He was treated with high-dose corticosteroids and extracorporeal plasmapheresis, complicated by line-related sepsis. Treatment was discontinued. The patient expired 45 days following admission. Discussion: Although GVHD is commonly associated with bone marrow transplantation (BMT), it is a rare and life-threatening complication of OLT with an incidence of 0.1% to 2%. The risk is significantly increased when the recipient and donor share a major histocompatibility antigen haplotype. The liver allograft is always spared. The diagnosis is often delayed because other common OLT complications, such as infection, can present with a similar clinical picture. Most experience for treatment of GVHD comes from BMT. However, these therapies have been disappointing in the setting of GVHD after OLT. Conclusions: GVHD is a rare but serious complication of OLT with limited treatment options. High clinical suspicion and obtaining biopsies for histopathologic diagnosis augments the diagnostic yield of endoscopy, particularly in challenging cases that defy usual diagnosis.

Targeting Gut T Cell Ca2+ Release-Activated Ca2+ Channels Inhibits T Cell Cytokine Production and T-Box Transcription Factor T-Bet in Inflammatory Bowel Disease

Prolonged Ca(2+) entry through Ca(2+) release-activated Ca(2+) (CRAC) channels is crucial in activating the Ca(2+)-sensitive transcription factor NFAT, which is responsible for directing T cell proliferation and cytokine gene expression. To establish whether targeting CRAC might counteract intestinal inflammation, we evaluated the in vitro effect of a selective CRAC inhibitor on T cell cytokine production and T-bet expression by lamina propria mononuclear cells (LPMC) and biopsy specimens from inflammatory bowel disease (IBD) patients. The inhibitory activity of the CRAC blocker was investigated through patch-clamp experiments on rat basophilic leukemia cells and fluorometric imaging plate reader intracellular Ca(2+) assays using thapsigargin-stimulated Jurkat T cells and its detailed selectivity profile defined using a range of in vitro radioligand binding and functional assays. Anti-CD3/CD28-stimulated LPMC and biopsy specimens from 51 patients with IBD were cultured with a range of CRAC inhibitor concentrations (0.01-10 microM). IFN-gamma, IL-2, IL-8, and IL-17 were analyzed by ELISA. T-bet was determined by immunoblotting. We found that the CRAC blocker concentration-dependently inhibited CRAC current in rat basophilic leukemia cells and thapsigargin-induced Ca(2+) influx in Jurkat T cells. A concentration-dependent reduction in T-bet expression and production of IFN-gamma, IL-2, IL-17, but not IL-8, was observed in IBD LPMC and biopsy specimens treated with the CRAC inhibitor. In conclusion, we provide evidence that the suppression of CRAC channel function may dampen the increased T cell response in the inflamed gut, thus suggesting a promising role for CRAC inhibitor drugs in the therapeutic management of patients with IBD.

Clonal Expansion of T/NK-Cells during Tyrosine Kinase Inhibitor Dasatinib Therapy

Tyrosine kinase inhibitors (TKIs) targeting the BCR-ABL fusion protein are an effective therapy for Philadelphia chromosome positive (Ph+) leukemia. Dasatinib, a 2nd generation pan-TKI, also inhibits wild-type kinases, which may result in unexpected clinical responses. Recent data suggest that dasatinib has a potent immunosuppressive effect on T- and NK-cells in vitro. In contrast, we have noticed a marked expansion of lymphocytes in blood in a subset of dasatinib patients, but its clinical significance and molecular mechanisms are unclear. In this multicenter study, 19 Ph+ leukemia patients with marked lymphoproliferation in blood while on dasatinib (9 CML CP, 2 CML AP, 2 CML BC, 6 Ph+ALL) were identified. Clonality, immunophenotype, and intracellular signaling was analyzed and related clinical information was collected. In addition, prevalence and prognostic significance of the phenomenon was retrospectively assessed in a Phase II clinical study on 46 Ph+ ALL patients. An abrupt lymphocytosis (peak count range 4–20×109/L) with large granular lymphocyte (LGL) morphology was observed after a median of 3 months from the start of therapy (range 1–8 months). In most patients, LGL lymphocytosis was long-lasting and continued throughout dasatinib therapy, albeit with marked fluctuations in absolute lymphocyte count (Fig. 1a). In all evaluable patients (n=4), lymphocyte counts normalized after drug discontinuation. All patients were previously exposed to imatinib without similar changes in lymphocyte count or morphology. By immunophenotyping, 14 patients had a cytotoxic T-cell and 5 patients a NK-cell phenotype. After initiation of dasatinib therapy, the CD4/CD8 ratio shifted, expression of activation antigens HLA-DR and CD57 increased, expression of homing antigen CD62L decreased and relative numbers of regulatory T-cells were significantly decreased (p<0.001). All patients with T-LGL phenotype had mono/oligoclonal rearrangements in TCR g/d genes. Quantitative allele-specific oligonucleotide PCR analysis from samples before and during dasatinib therapy indicated that LGL lymphocytosis results from expansion of a pre-existing terminal memory cell clone (CD8+CD62Llow). By using single-cell profiling of nodal phosphoproteins (ERK1/2, STAT1, STAT3, STAT5, STAT6) by multi-color FACS, we showed that during dasatinib therapy LGL patients were more responsive to cytokine or growth factor stimuli (IL-2, IL-4, IL-6, IFNs, GM-CSF), when compared to dasatinib patients without lymphocytosis or to healthy controls. This indicates alternative signaling pathway usage not inhibited by dasatinib and/or cytokine hypersensitivity. Dasatinib-related severe adverse effects were common in patients with LGL lymphocytosis. Pleural effusions and/or pulmonary infiltrates were seen in 13/19 patients, modest CMV reactivation in 9/19 patients, colitis in 11/19 patients and long lasting mild fever in 14/19 patients. Accumulation of pheno- and genotypically identical cytotoxic T-cells was also detected in pleural effusion and colitis biopsy samples. Adverse effects were temporally related to dasatinib therapy and emerged after the appearance of LGL lymphocytosis. All patients with relapsed poor prognosis leukemia (Ph+ ALL n=5, blastic phase CML n=2) achieved durable complete molecular responses (CMR) during dasatinib therapy, CMR still ongoing in 3 patients (follow-up 18+ months). In the confirmatory Phase II dasatinib study in Ph+ ALL (START-L), patients with lymphocytosis had superior survival compared to patients without lymphocytosis (Fig. 1b). In conclusion, we propose that by inhibiting immunoregulatory kinases, dasatinib may induce a reversible state of aberrant immune reactivity in a distinct subset of patients and result in anti-leukemia and anti-host responses driven by cytotoxic T/NK LGL cells. Further studies pinpointing the mechanisms, such as the target antigens on the malignant cells and activation pathways are ongoing. [Display omitted]

Regional variation in gene expression in the healthy colon is dysregulated in ulcerative colitis

Objective:To investigate differential intestinal gene expression in patients with ulcerative colitis and in controls.Design:Genome-wide expression study (41 058 expression sequence tags, 215 biopsies).Setting:Western General Hospital, Edinburgh, UK, and Genentech, San...

Glutathione peroxidase 2 and aquaporin 8 as new markers for colonic inflammation in experimental colitis and inflammatory bowel diseases: an important role for H2O2?

Different mouse models of inflammatory bowel diseases (IBD) demonstrate various aspects of the pathophysiology of IBD. We looked for overlapping gene expression profiles in three different mouse models of experimental colitis and analysed whether these overlapping genes are of help to find new genes that could be used as general markers in human IBD. Using Agilent mouse TOX oligonucleotide microarrays, we analysed the gene expression profiles in three widely used models of experimental colitis: 2,4,6-trinitrobenzene sulphonic acid, dextran sodium sulfate and CD4CD45RB transfer and looked for overlapping gene expression in these models. Overlapping genes were analysed using Lightcycler (Roche Diagnostics, Mannheim, Germany) in biopsy materials from human IBD and control tissue. Compared with control mice in dextran sodium sulfate, 2,4,6-trinitrobenzene sulphonic acid and the CD45RB transfer colitis mice five known genes, extracellular proteinase inhibitor (Expi), glutathione peroxidase 2 (Gpx2), mast cell protease 1 (Mcpt1), resistin-like beta (Retnlb) and sulphatase 2 (Sulf2), and two unknown genes were upregulated and the two genes aquaporin 8 (Aqp8) and kallikrein 5 (Klk5) were downregulated in all three models. In human Crohn's disease and ulcerative colitis biopsies, one of the upregulated glutathione peroxidase (Gpx2) and one of the downregulated Aqp8 genes in the mouse models were also differentially expressed in affected colonic tissue of patients with IBD. Experimental mouse models are suitable models for the search of new markers for human IBD. As both Gpx2 and Aqp8 are involved in H2O2 metabolism (Gpx2 as a radical scavenger whereas Aqp8 facilitates its diffusion), upregulation of Gpx2 and downregulation of Aqp8 could be a mechanism to defend against severe oxidative stress and indicate that H2O2 is a universal mediator in the inflammatory process in the colon. This provides a focus on homeostasis of the antioxidant pathway and its importance in IBD.

Pitfalls in the Interpretation of Nonneoplastic Mucosal Biopsies in Inflammatory Bowel Disease

This review provides a summary of common diagnostic problems encountered by both pathologists and gastroenterologists when evaluating patients with diarrhea and in whom inflammatory bowel disease (IBD) is suspected. The two most common forms of IBD, ulcerative colitis (UC) and Crohn's disease (CD), may, in certain settings, show overlapping endoscopic and pathologic features, potentially resulting in diagnostic confusion. For instance, some cases of UC may show unusual CD-like features, such as rectal sparing, discontinuous disease, aphthous ulceration, ileal or extracolonic involvement, and granulomatous inflammation, all of which may be evident in mucosal biopsy specimens. CD may also present as a diffuse, superficial pancolitis with ileal sparing that mimics the endoscopic and histologic appearance of UC. Furthermore, other forms of colitis, such as microscopic colitis, diverticulitis, diversion colitis, and nonsteroidal anti-inflammatory drug (NSAID)-induced colonic injury may also show IBD-like changes in mucosal biopsies. The potential diagnostic pitfalls faced by physicians, as well as features that aid in the distinction among these entities, are discussed in detail in this review.

Differential expression in histologically normal crypts of ulcerative colitis suggests primary crypt disorder

Ulcerative colitis is characterized by crypt infiltration particularly of neutrophils. However, it is not known whether it reflects a primary crypt disorder or a secondary inflammatory response. In this study, we analyzed the expression profiles of histologically normal crypts microdissected from formalin-fixed biopsies of early stage ulcerative colitis. Total RNAs were extracted, amplified, and applied to Affymetrix GeneChip(R) X3P Array. For the control, similar crypts from nonspecific colitis biopsies were applied. A total of 353 (4.3%) and 111 (1.4%) genes were >3 times up-, and down-regulated in ulcerative colitis. Up-regulated genes included FCGBP (Fc fragment of IgG binding protein), cyclophilin A, chemokine (C-X-C motif) ligand 3, and genes associated with lipid metabolism. Down-regulated genes included APOA4 (apolipoprotein A-IV), cylindromatosis, BCL2-like 10, claudin 8, and numerous transcriptional regulators. FCGBP and APOA4 have been implicated in ulcerative colitis previously. Our data show differential expression in the crypt epithelia of ulcerative colitis before active inflammation is initiated, suggesting primary crypt abnormalities that might be implicated in the pathogenesis of ulcerative colitis.

Efficacy and tolerability of oral iron therapy in inflammatory bowel disease: a prospective, comparative trial

In patients with inflammatory bowel disease, oral iron is anecdotally reported to be less effective and less well tolerated than in those without inflammatory bowel disease, and to increase disease activity. To study prospectively the effects of oral iron in patients with and without inflammatory bowel disease. Patients with ulcerative colitis, Crohn's disease and non-inflammatory bowel disease controls, all with iron deficiency anaemia, were assessed with symptom diaries, a quality of life questionnaire (Inflammatory Bowel Disease Questionnaire; inflammatory bowel disease patients only) and blood tests to measure iron repletion, disease activity and antioxidant capacity before and after starting 4 weeks of oral iron. In patients with ulcerative colitis, sigmoidoscopic scoring and rectal biopsies for reactive oxygen metabolite production were performed before and after iron therapy. All groups showed increases in haemoglobin and ferritin. Iron intolerance occurred in about a quarter of patients in each group. Two of 33 (6%) of inflammatory bowel disease patients had a relapse during treatment. Symptoms worsened in ulcerative colitis, but not in Crohn's disease or non-inflammatory bowel disease patients; Inflammatory Bowel Disease Questionnaire scores improved in ulcerative colitis. Laboratory markers of disease activity, sigmoidoscopic scores, histological scores, antioxidant capacity levels and reactive oxygen metabolite production did not change. Oral iron is equally efficacious and well tolerated in inflammatory bowel disease and non-inflammatory bowel disease patients. A tiny minority of inflammatory bowel disease patients relapse in association with use of oral iron therapy.

Comparation of whole genomic expression pattern of ulcerative colitis and Crohn-colitis biopsy samples

Background: The biological interpretation of hundreds of down- and upregulated genes from the gene expression analyses' results was not worked out until present days.

Economic Comparison of Current Endoscopic Practices: Barrett’s Surveillance vs. Ulcerative Colitis Surveillance vs. Biopsy for Sprue vs. Biopsy for Microscopic Colitis

Health care costs are an increasingly important study outcome. Endoscopic practice consumes a large proportion of gastroenterology-related health expenses. An economic comparison of several currently accepted endoscopic practices was performed, ranking them according their cost-effectiveness, as viewed from the payer perspective. The cost-effectiveness of four currently accepted standard endoscopic practices was examined: small bowel biopsy to assess for celiac sprue, colonoscopic biopsy to assess for microscopic colitis, surveillance of Barrett's esophagus, and surveillance of chronic ulcerative colitis (CUC). Parameter estimates were obtained from the published literature. Charges were based on Medicare professional plus facility/technical fees. Performing colonoscopic biopsies for microscopic colitis in the setting of chronic nonbloody diarrhea was the most cost-effective practice ($2447/case detected), while small bowel biopsy for sprue in the setting of a patient with a first-degree relative with sprue ($3042/case detected) or with anemia ($2982/case detected) was also a cost-effective approach. Small bowel biopsy in the setting of diarrhea ($3900/case detected) was less cost-effective, while CUC surveillance ($14,119/detection of dysplasia) and performance of small bowel biopsy in an asymptomatic patient ($15,209/case detected) were clearly the least economical. As efforts are made to reduce the costs of health care, more attention will be focused on the cost-effectiveness of routine endoscopic practices. Although, our findings put endoscopic practices into economic perspective, future perspective, future prospective trials are required to confirm the validity of these findings.

Helicobacter species in human colon biopsies

Sirs, We read with interest the study by Bell et al., who failed to detect DNA of the Helicobacter genus in colon biopsies from subjects with or without inflammatory bowel disease.1 At the XVIth International Workshop on Gastrointestinal Pathology and Helicobacter (Stockholm, September 3–6, 2003), we presented a similar study that came to a partly different conclusion.2 Twenty patients undergoing colonoscopy at Lund University Hospital for clinical reasons were enrolled in our study (15 females and five males; age range, 17–66 years; mean age, 43 years). Of the 20 patients, five had ulcerative colitis, four had Crohn's disease and 11 had other ailments. The study was approved by the Lund University Ethical Committee (permit: LU 345-02) and signed informed consent was obtained from all patients. Biopsies from the proximal and distal colon were collected from each patient in a transport medium (tryptone soy broth, 10% horse serum, 20% glycerol) and transported to the laboratory, where DNA was extracted using the QIAamp DNA Mini Kit (QIAGEN, Hilden, Germany) within 1–2 h. Detection of the Helicobacter genus was performed by a nested 16S rDNA Helicobacter genus-specific polymerase chain reaction (PCR) assay using primers C97, C05 for step one3 and N16S 1F, N16S 2R for step two.4 Positive products, as determined by visualization in gel electrophoresis, were analysed by denaturing gradient gel electrophoresis and sequencing as described elsewhere.5 Positive samples were re-amplified to obtain ∼ 400-bp or ∼ 800-bp amplicons for sequencing, followed by comparison with known DNA sequences using BLASTN 2.2.1.6 PCR displayed a Helicobacter-specific product in five patients (Table 1). One 771-bp sequence clustered with Helicobacter cholecystus (two mismatches, both ‘n’ in Genbank U46129), Helicobacter pametensis (eight mismatches) and Helicobacter sp. B9A Seymour (12 mismatches). One sequence clustered with Helicobacter muridarum (784- of 784-bp, Genbank AF302104) and the closest other sequence was Helicobacter typhlonius (16 mismatches). At least three different species of Helicobacter were detected in our study, two of which (H. cholecystus and H. muridarum), to our knowledge, have not been described previously in humans. Our investigation has so far not found any differences in Helicobacter detection in the two studied groups (inflammatory bowel disease vs. other ailments). Culture showed no growth of Helicobacter spp.; however, culture from biopsies from one patient displayed massive growth of Campylobacter concisus. This isolate came from a male patient (46 years of age) with abdominal pain, bloody stool but normal colonoscopy; that is, no inflammatory bowel disease. Thus, we were able to detect Helicobacter spp. in a few subjects (five of 20 patients), in contrast with the results of Bell et al.,1 but could not demonstrate a difference in Helicobacter spp. detection in colon biopsies in inflammatory bowel disease vs. other ailments, in concordance with the results of Bell et al.1 We find it intriguing, however, that Bell et al. did not detect any Helicobacter spp. in their samples. We believe that this difference between the studies may be explained by the fact that we used a nested PCR, whereas Bell et al. used a single-step PCR.1 Other studies have demonstrated difficulties in detecting Helicobacter spp. DNA in tissue samples (other than gastric biopsy specimens) not using a nested or semi-nested PCR.7, 8 At the XVIth International Workshop on Gastrointestinal Pathology and Helicobacter (Stockholm, September 3–6, 2003), there were several reports on PCR detection of Helicobacter spp. in colon biopsies, supporting the existence of this genus in the human colon.2, 9, 10 Whether gastric or enterohepatic Helicobacter spp. have a role in human enteric disease is still an open question and will surely be addressed by various research groups. This study was supported by grants from the Hedda and John Forssman Foundation, the Royal Physiographic Society in Lund, the Golje Foundation and the Swedish Research Council (16x-04723).

Human beta-defensin 2 but not beta-defensin 1 is expressed preferentially in colonic mucosa of inflammatory bowel disease.

Various antimicrobial peptides such as defensins are part of innate immunity and contribute to the intestinal barrier that may be defective in inflammatory bowel disease (IBD). This study investigated beta-defensin mRNA and peptide expression in the colon from controls and patients with Crohn's disease, ulcerative colitis or unspecific colitis as inflammatory controls. Mucosal mRNA expression was measured by multiplex reverse transcriptase-polymerase chain reaction (RT-PCR) with primers for human beta-defensin 1 (HBD-1) and human beta-defensin 2 (HBD-2) in CaCo-2 cells and in biopsies from 103 patients (33 controls, 24 Crohn's disease patients, 36 ulcerative colitis patients, 10 unspecific colitis patients). Paraffin-embedded tissue from colonic resections was tested for HBD-1 and HBD-2 peptides by immunohistochemistry. HBD-1 mRNA was expressed constitutively whereas HBD-2 was induced by pro-inflammatory cytokines in CaCo-2 cells. HBD-1 mRNA was detectable in 61% of control and Crohn's disease biopsies and 53% of ulcerative colitis biopsies. HBD-2 transcript was expressed differentially, with 18% of control biopsies positive as opposed to 34% in Crohn's disease and 53% in ulcerative colitis. HBD-2 mRNA but not HBD-1 mRNA was expressed preferentially in inflamed areas. Immunohistochemical investigation demonstrated the presence of defensin peptides in colonic epithelium as well as the differential induction in IBD. HBD-1 is expressed constitutively in colonic tissue irrespective of inflammation. HBD-2 is barely present in uninflamed colon but it is induced in inflammation. The lower expression of HBD-2 in Crohn's disease compared with ulcerative colitis indicates different responses of the mucosal innate defence. Defensins may play a crucial role in controlling pathogen invasion in IBD, although the functional significance remains to be established.

Differential levels of granzyme B, regulatory cytokines, and apoptosis in Crohn's disease and ulcerative colitis at first presentation.

The mechanisms of tissue damage in ulcerative colitis and Crohn's disease may reflect disordered humoral or cell-mediated effector mechanisms, respectively. Mucosal biopsies from untreated inflammatory bowel disease patients and normal controls were analysed for the expression of granzyme B, a cytotoxic effector molecule specifically associated with cell-mediated immunity, and for regulatory cytokines. Messenger RNA (mRNA) was analysed by reverse transcription-polymerase chain reaction and enzyme-linked oligonucleotide chemiluminescence assay. Mucosal biopsies were analysed by immunohistochemistry for granzyme B protein and lymphocyte markers and for the presence of apoptotic cells by terminal deoxynucleotidyl transferase end labelling. Granzyme B mRNA was elevated in Crohn's disease, but not in ulcerative colitis or control mucosal biopsies. Granzyme B mRNA levels correlated with interferon gamma mRNA levels in Crohn's disease. Granzyme B was expressed in CD3+, CD8+ T cells in the lamina propria of Crohn's disease mucosa and there were significantly more apoptotic cells in the lamina propria in Crohn's disease. In conclusion, granzyme B-expressing T lymphocytes are present in the focal mucosal lesions of Crohn's disease, together with spatially related apoptotic cell death. These results support the hypothesis that T-cell-mediated cytotoxic effector mechanisms may play a role in Crohn's disease.

Quantitative PCR analysis of TNF-alpha and IL-1 beta mRNA levels in pediatric IBD mucosal biopsies.

Inflammatory bowel disease (IBD) is associated with increased activation of intestinal immune cells, whose overproduction of proinflammatory cytokines such as tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1 beta) is implicated in mediating the sustained inflammatory response. Studies to date have largely reported qualitative differences in cytokine gene expression between IBD and controls. Our aim was to perform quantitative analysis of intestinal mucosal mRNA expression in colonic biopsies from pediatric IBD patients using a competitive polymerase chain reaction. IL-1 beta and TNF-alpha were expressed in all IBD and control biopsies. Compared to controls, IL-1 beta mRNA levels were increased in involved tissue from Crohn's disease (CD) patients, but not in histologically uninvolved CD or in ulcerative colitis (UC) mucosa. IL-1 beta expression in the latter groups were equivalent to those found in tissue from patients with eosinophilic colitis (EOC). Significantly higher levels of IL-1 beta mRNA were found in uninvolved mucosa from CD patients who presented with a relapse of disease activity, as compared to newly diagnosed cases with histological features of CD at an early stage. TNF-alpha mRNA transcripts were also significantly elevated in involved CD mucosa, but not in the other groups. TNF-alpha gene expression in CD-involved tissue decreased with disease duration. Follow-up of the patients revealed that high cytokine expression in uninvolved CD tissue correlated with an early clinical relapse. In conclusion, quantitative determination of proinflammatory cytokine gene expression reveals differences between the type, severity, and clinical course in patients with IBD.