Colonic microbiota can promote rapid local improvement of murine colitis by thioguanine independently of T lymphocytes and host metabolism.

OBJECTIVE: Mercaptopurine (MP) and pro-drug azathioprine are 'first-line' oral therapies for maintaining remission in IBD. It is believed that their pharmacodynamic action is due to a slow cumulative decrease in activated lymphocytes homing to inflamed gut. We examined the role of host metabolism, lymphocytes and microbiome for the amelioration of colitis by the related thioguanine (TG). DESIGN: C57Bl/6 mice with or without specific genes altered to elucidate mechanisms responsible for TG's actions were treated daily with oral or intrarectal TG, MP or water. Disease activity was scored daily. At sacrifice, colonic histology, cytokine message, caecal luminal and mucosal microbiomes were analysed. RESULTS: Oral and intrarectal TG but not MP rapidly ameliorated spontaneous chronic colitis in Winnie mice (point mutation in Muc2 secretory mucin). TG ameliorated dextran sodium sulfate-induced chronic colitis in wild-type (WT) mice and in mice lacking T and B lymphocytes. Remarkably, colitis improved without immunosuppressive effects in the absence of host hypoxanthine (guanine) phosphoribosyltransferase (Hprt)-mediated conversion of TG to active drug, the thioguanine nucleotides (TGN). Colonic bacteria converted TG and less so MP to TGN, consistent with intestinal bacterial conversion of TG to so reduce inflammation in the mice lacking host Hprt. TG rapidly induced autophagic flux in epithelial, macrophage and WT but not Hprt-/- fibroblast cell lines and augmented epithelial intracellular bacterial killing. CONCLUSIONS: Treatment by TG is not necessarily dependent on the adaptive immune system. TG is a more efficacious treatment than MP in Winnie spontaneous colitis. Rapid local bacterial conversion of TG correlated with decreased intestinal inflammation and immune activation.

Angiotensinogen and transforming growth factor beta1: novel genes in the pathogenesis of Crohn's disease.

BACKGROUND: Angiotensin peptides may act locally as cytokines in several organ systems with elevated mucosal levels present in Crohn's disease. A variant in the angiotensinogen gene promoter results in increased peptide production, while transforming growth factor beta1 (TGFbeta1) codon 25 variants demonstrate variable peptide production, predisposing to fibrosis in several organs. AIMS: Conduct an Australian-based analysis of the angiotensinogen-6 variant in two independent inflammatory bowel disease (IBD) cohorts, and examine the role of angiotensinogen-6 and TGFbeta1 codon 25 variants in shaping Crohn's disease phenotype. METHODS: IBD Patients (Crohn's disease = 347, ulcerative colitis = 147) and CD families (n = 148) from two cohorts, together with 185 healthy controls were genotyped for angiotensinogen-6. Genotype-phenotype analyses were performed for both angiotensinogen-6 and TGFbeta1 codon 25. RESULTS: Angiotensinogen-6 AA genotype was significantly associated with Crohn's disease (p = 0.007, OR = 2.38, CI = 1.32-4.32) in cohort 1, but not in the smaller cohort 2 (p = 0.19). The association remained significant when the two cohorts were combined (p = 0.008), and in a TDT family analysis (p = 0.03). TGF 1 codon 25 was associated with stricturing Crohn's disease (p = 0.01, OR = 2.63, CI = 1.16-5.88) and a shorter time to intestinal resection (p = 0.06). CONCLUSIONS: The association of the angiotensinogen-6 variant with Crohn's disease supports a potential role for angiotensin-converting enzyme inhibitors and angiotensin II receptor antagonists in disease treatment.

MUC13 protects colorectal cancer cells from death by activating the NF-κB pathway and is a potential therapeutic target.

MUC13 is a transmembrane mucin glycoprotein that is over produced by many cancers, although its functions are not fully understood. Nuclear factor-κB (NF-κB) is a key transcription factor promoting cancer cell survival, but therapeutically targeting this pathway has proved difficult because NF-κB has pleiotropic functions. Here, we report that MUC13 prevents colorectal cancer cell death by promoting two distinct pathways of NF-kB activation, consequently upregulating BCL-XL. MUC13 promoted tumor necrosis factor (TNF)-induced NF-κB activation by interacting with TNFR1 and the E3 ligase, cIAP1, to increase ubiquitination of RIPK1. MUC13 also promoted genotoxin-induced NF-κB activation by increasing phosphorylation of ATM and SUMOylation of NF-κB essential modulator. Moreover, elevated expression of cytoplasmic MUC13 and NF-κB correlated with colorectal cancer progression and metastases. Our demonstration that MUC13 enhances NF-κB signaling in response to both TNF and DNA-damaging agents provides a new molecular target for specific inhibition of NF-κB activation. As proof of principle, silencing MUC13 sensitized colorectal cancer cells to killing by cytotoxic drugs and inflammatory signals and abolished chemotherapy-induced enrichment of CD133+ CD44+ cancer stem cells, slowed xenograft growth in mice, and synergized with 5-fluourouracil to induce tumor regression. Therefore, these data indicate that combining chemotherapy and MUC13 antagonism could improve the treatment of metastatic cancers.

Comparison of the efficacy and safety of Eudragit-L-coated mesalazine tablets with ethylcellulose-coated mesalazine tablets in patients with mild to moderately active ulcerative colitis.

BACKGROUND: There are no comparative studies of coated mesalazine. AIM: To compare the efficacy and tolerability of Eudragit-L- and ethylcellulose-coated mesalazine tablets in patients with mild to moderately active ulcerative colitis. METHODS: A double-blind, double-dummy, randomized parallel group trial was performed across 18 centres in Australia, and 20 in Eastern Europe. Patients were treated with 3 g mesalazine for 8 weeks with the primary efficacy end point being clinical remission. RESULTS: Of 215 patients, 69% achieved clinical remission in both treatment groups (P < 0.001; chi-square test) with no differences in frequency of adverse events. In the Australian cohort (n = 63), the Eudragit-L group had a higher remission rate (73% vs. 36%) and responded 13 days faster, compared with those in the European group (67% vs. 84%, and 2 days respectively). No clear reasons for differences in treatment responses were identified. CONCLUSIONS: Eudragit-L and ethylcellulose-coated mesalazine tablets are well tolerated and equally effective in achieving remission in mild-moderately active ulcerative colitis over 8 weeks.

Efficacy of thiopurines and adalimumab in preventing Crohn's disease recurrence in high-risk patients - a POCER study analysis.

BACKGROUND: Crohn's disease recurs in the majority of patients after intestinal resection. AIM: To compare the relative efficacy of thiopurines and anti-TNF therapy in patients at high risk of disease recurrence. METHODS: As part of a larger study comparing post-operative management strategies, patients at high risk of recurrence (smoker, perforating disease, ≥2nd operation) were treated after resection of all macroscopic disease with 3 months metronidazole together with either azathioprine 2 mg/kg/day or mercaptopurine 1.5 mg/kg/day. Thiopurine-intolerant patients received adalimumab induction then 40 mg fortnightly. Patients underwent colonoscopy at 6 months with endoscopic recurrence assessed blind to treatment. RESULTS: A total of 101 patients [50% male; median (IQR) age 36 (25-46) years] were included. There were no differences in disease history between thiopurine- and adalimumab-treated patients. Fifteen patients withdrew prior to 6 months, five due to symptom recurrence (of whom four were colonoscoped). Endoscopic recurrence (Rutgeerts score i2-i4) occurred in 33 of 73 (45%) thiopurine vs. 6 of 28 (21%) adalimumab-treated patients [intention-to-treat (ITT); P = 0.028] or 24 of 62 (39%) vs. 3 of 24 (13%) respectively [per-protocol analysis (PPA); P = 0.020]. Complete mucosal endoscopic normality (Rutgeerts i0) occurred in 17/73 (23%) vs. 15/28 (54%) (ITT; P = 0.003) and in 27% vs. 63% (PPA; P = 0.002). The most advanced disease (Rutgeerts i3 and i4) occurred in 8% vs. 4% (thiopurine vs. adalimumab). CONCLUSIONS: In Crohn's disease patients at high risk of post-operative recurrence adalimumab is superior to thiopurines in preventing early disease recurrence.

Pressor effect of ethanol in the isolated perfused human placental lobule.

Pharmacologically relevant concentrations of ethanol (0.01-0.3 g/%) were perfused via the fetal circulation in isolated human placental lobules. This resulted in a dose-related rise in fetal arterial pressure, which at 0.3 g/% ethanol, was 10.1 +/- 1.1 mmHg above the pre-drug baseline. The pressor responses to ethanol were (i) rapid in onset, reaching a stable plateau within 5-10 min following administration, (ii) readily reversible by perfusion with drug-free media, (iii) non-tachyphylactic and (iv) largely inhibited by the cyclo-oxygenase inhibitor, inhibitor, indomethacin (5 microM). This pressor action of ethanol in the fetal circulation may contribute to the pathogenesis of the fetal alcohol syndrome as well as represent an underlying mechanism of ethanol-induced hypertension.

Hydrogen sulphide and total acid-volatile sulphide in faeces, determined with a direct spectrophotometric method.

Hydrogen sulphide may be important in human bowel pathophysiology. Available methods for determination of hydrogen sulphide and total sulphide have not been validated for faeces. This paper reports on a method for direct determination of sulphide in faecal homogenates, and techniques for collecting faeces to permit determination of both hydrogen sulphide and total sulphide. Mean total sulphide and mean hydrogen sulphide concentrations were 0.66 and 0.17 mumol/g wet faeces, respectively. These concentrations are much higher than previous estimates. There was no significant difference between methanogenic and non-methanogenic faeces. Significant sulphidogenesis occurred in both methanogenic and non-methanogenic in vitro faecal incubations, with both sulphate and cysteine sources. These results do not support previous claims that sulphidogenesis occurs only in non-methanogenic faeces.

The effect of C5a and U46619 on the isolated, perfused human placental lobule: development of a method for the online estimation of tissue fluid accumulation.

A method for the automatic and simultaneous determination of perfusion pressure and fluid accumulation in the isolated, perfused human placental lobule is described. We demonstrated that the inflammatory mediator, C5a, a C5a agonist analogue peptide, and the thromboxane mimetic U46619 caused increased fetal perfusion pressure and increased tissue weight when administered via the fetal arterial circulation. Occlusion of the fetal venous effluent tubing caused significantly greater increases in tissue weight than the pharmacological agents. Detectable increases in tissue weight occurred within 47 +/- 3 sec (n = 21) following pressure increases caused by the pharmacological agents. In each case, the increase in tissue weight was accompanied by an increased permeability of the materno-fetal barrier, shown by the transfer of Evans blue albumin from the fetal circulation to the maternal compartment.

Reversibility of tachyphylaxis to C5A in guinea pig tissues, perfused human placental lobule, and umbilical artery.

The spasmogenic effect of C5a is mediated by histamine and/or eicosanoids. Tachyphylaxis to this effect of C5a occurs rapidly, but the spasmogenic effects of C5a on a guinea pig lung parenchymal strips, field-stimulated ventricular papillary muscle, and human umbilical artery were completely restored by a 1-h period of drug-free rest, whereas that of guinea pig ileum was not. Perfusion of the isolated human placental lobule with C5a caused a transient pressor response that was largely abolished by indomethacin (5 microM), indicating mediation by cyclooxygenase metabolites. This pressor response to C5a was also completely restored following a 1-h rest period. The results show that tissue rest reverses tachyphylaxis to the spasmogenic effects of C5a in tissues where the response is mediated by cyclooxygenase metabolites. Where the response is mediated by histamine released by mast cells, restoration does not occur, presumably because of the catastrophic nature of mast cell degranulation. Histamine released in guinea pig papillary muscle by C5a may be from non-mast-cell sources.

MUC1 and MUC13 differentially regulate epithelial inflammation in response to inflammatory and infectious stimuli.

The MUC1 cell-surface mucin is highly expressed on the gastric mucosal surface, while MUC13 is highly expressed on the intestinal mucosal surface. Polymorphisms in both MUC1 and MUC13 have been linked to inflammatory bowel diseases. MUC1 can act as a decoy molecule on the apical cell surface of epithelial cells and thereby limit bacterial adherence, infection, and inflammation. In this study, we examined whether and how MUC1 and MUC13 modulate infectious and inflammatory signaling. Using gastrointestinal tissue from Muc1- or Muc13-deficient mice in ex vivo culture, MUC1 small interfering RNA (siRNA) silencing in MKN7 gastric epithelial cells, and MUC13 siRNA silencing in LS513 intestinal epithelial cells, we showed that loss of MUC1 increased chemokine secretion, whereas loss of MUC13 decreased chemokine secretion in response to tumor necrosis factor-α. Anti-inflammatory activity of MUC1 and pro-inflammatory activity of MUC13 were also seen after exposure to pathogens, NOD1 (nucleotide-binding oligomerisation domain-containing protein-1), and Toll-like receptor ligands. MUC1 and MUC13 both regulate chemokine secretion in gastrointestinal epithelial cells through a nuclear factor-κB-dependent pathway, although MUC13 modulation could also involve other pathways. Our studies demonstrate that MUC1 and MUC13 are important components of gastrointestinal homeostasis and that disruption or inappropriate expression of these mucins could predispose to infectious and inflammatory disease and inflammation-induced cancer.

An intestinal epithelial defect conferring ER stress results in inflammation involving both innate and adaptive immunity.

We recently characterized Winnie mice carrying a missense mutation in Muc2, leading to severe endoplasmic reticulum stress in intestinal goblet cells and spontaneous colitis. In this study, we characterized the immune responses due to this intestinal epithelial dysfunction. In Winnie, there was a fourfold increase in activated dendritic cells (DCs; CD11c(+) major histocompatibility complex (MHC) class II(hi)) in the colonic lamina propria accompanied by decreased colonic secretion of an inhibitor of DC activation, thymic stromal lymphopoietin (TSLP). Winnie also displayed a significant increase in mRNA expression of the mucosal T(H)17 signature genes Il17a, IL17f, Tgfb, and Ccr6, particularly in the distal colon. Winnie mesenteric lymph node leukocytes secreted multiple T(H)1, T(H)2, and T(H)17 cytokines on activation, with a large increase in interleukin-17A (IL-17A) progressively with age. A major source of mucosal IL-17A in Winnie was CD4(+) T lymphocytes. Loss of T and B lymphocytes in Rag1(-/-) × Winnie (RaW) crosses did not prevent spontaneous inflammation but did prevent progression with age in the colon but not the cecum. Adoptive transfer of naive T cells into RaW mice caused more rapid and severe colitis than in Rag1(-/-), indicating that the epithelial defect results in an intestinal microenvironment conducive to T-cell activation. Thus, the Winnie primary epithelial defect results in complex multicytokine-mediated colitis involving both innate and adaptive immune components with a prominent IL-23/T(H)17 response, similar to that of human ulcerative colitis.

Low-dose azathioprine or mercaptopurine in combination with allopurinol can bypass many adverse drug reactions in patients with inflammatory bowel disease.

BACKGROUND: The thiopurine drugs, azathioprine and mercaptopurine (MP), are established treatments for IBD. However, therapeutic failure caused by adverse drug reactions occurs frequently. AIM: To study combination of allopurinol with reduced-dose thiopurine in an attempt to avoid adverse drug reactions in the treatment of IBD. METHODS: Patients with drug reactions to full-dose thiopurines were recruited for combination therapy in two IBD centres in this retrospective study. Dosing was guided by measuring thiopurine methyltransferase (for UK patients) or thioguanine nucleotides and methyl-6MP (Australian patients). Response was monitored by clinical activity indices. RESULTS: Of 41 patients, 25 had non-hepatic and 16 had hepatitic reactions. Clinical remission was achieved in 32 patients (78%) with a median follow-up of 41 weeks (range 0.5-400). Patients who did not respond to combination therapy tended to fail early with the same adverse reaction. The relative risk of having an adverse reaction with methyl-6MP in the top interquartile range was 2.7 (1.3-28) times that with methyl-6MP in the lower three quartiles (95% confidence interval). CONCLUSION: The combined experience from our centres is the largest reported experience of this combination therapy strategy in IBD, and the first to provide evidence for benefit in thiopurine and allopurinol co-therapy to avoid non-hepatitic adverse drug reactions.

Alkyl halides, super hydrogen production and the pathogenesis of pneumatosis cystoides coli.

BACKGROUND AND AIMS: The colons of patients with pneumatosis cystoides coli produce excessive H2. Exposure to alkyl halides could explain this. Six consecutive patients who had pneumatosis cystoides coli while taking chloral hydrate (1-5+ g/day) are reported. Patients 2 and 3 were investigated after they had ceased chloral hydrate treatment. One produced methane, the other did not. (Pneumatosis cystoides coli patients are non-methanogenic according to the literature.) Both had overnight fasting breath H2 of less than 10 ppm. A literature review disclosed just one patient who was using chloral at the time of diagnosed pneumatosis cystoides coli, but an epidemic of the disease in workers exposed to trichloroethylene. METHODS: (i) In vitro experiments with human faeces: chloral or closely related alkyl halides were added to anaerobic faecal cultures derived from four methane-producing and three non-methanogenic human subjects. H2 and CH4 gases were measured. (ii) In vivo animal experiment: chloral hydrate was added to drinking water of four Wistar rats, and faecal H2 compared with control rats. RESULTS: Alkyl halides increased H2 up to 900 times in methanogenic and 10 times in non-methanogenic faecal cultures. The Ki of chloral was 0.2 mM. Methanogenesis was inhibited in concert with the increase in net H2. In the rat experiment, chloral hydrate increased H2 10 times, but did not cause pneumatosis. CONCLUSIONS: Chloral and trichloroethylene are alkyl halides chemically similar to chloroform, a potent inhibitor of H2 consumption by methanogens and acetogens. These bacteria are the most important H2-consuming species in the colon. It is postulated that exposure to these alkyl halides increases net H2 production, which sets the scene for "counterperfusion supersaturation" and the formation of gas cysts. In recent times, very low prescribing rates for chloral have caused primary pneumatosis cystoides to become extremely rare. As with primary pneumatosis, secondary pneumatosis cystoides, which occurs if there is small bowel bacterial overgrowth distal to a proximally located gut obstruction, is predicted by counterperfusion supersaturation. "Inherent unsaturation" due to metabolism of O2 is a safety factor, which could explain why gas bubbles do not form more often in tissue with high H2 tension.

Inhibition of methanogenesis by human bile.

The factors that regulate methanogenesis in humans have not been established. The presence of bile acid, which is lost into the colon from the small intestine, may be an important regulatory factor of methanogenesis. To examine this possibility, the effect of human bile on methane production by faecal cultures, and the in vivo effect of biliary diversion on breath methane excretion in a methanogenic choledochostomy patient, were investigated. Faecal suspensions (0.1%) from five methanogenic humans were incubated anaerobically with bile (0.3-30%) from three choledochostomy patients, and headspace methane measured by gas chromatography. All biles inhibited headspace methane. Inhibition of methanogenesis was dose dependent, plateaued at 10-30% bile concentration, and was abolished by 0.6% cholestyramine. The maximum inhibition by bile, median (range), was 38 (0.9-56)% of control methane values. Reversal of the bile fistula in the fourth choledochostomy patient converted that subject from methanogenic to 'non-methanogenic' status, It is concluded that inhibition of methanogens in the caecum by bile acid could significantly reduce the number of methanogens in the colon. This and the effect of transit time could explain much of the known epidemiology of 'non-methanogenesis', which has been related to obesity, (comparatively) fast colonic transit in healthy persons, and to small intestinal Crohn's disease.

Does counterperfusion supersaturation cause gas cysts in pneumatosis cystoides coli, and can breathing heliox reduce them?

The pathogenesis of pneumatosis cystoides coli remains obscure in the absence of an explanation for why pockets of gas should form in the first place and why they should be maintained in the wall and mesentery of the colon. Counterperfusion supersaturation could explain the formation and location of the gas cysts, which occur mostly near blood vessels on the mesenteric border of the colon, and the absence of methane gas in them. The hypothesis can be tested by treating patients with pneumatosis cystoides coli with heliox.

A possible role for bile acid in the control of methanogenesis and the accumulation of hydrogen gas in the human colon.

This study investigated a possible role for primary bile acid in the control of methanogenesis in the human colon. Production of hydrogen and methane was measured in anaerobic faecal cultures derived from faeces of six 'non-methanogenic' and three methanogenic healthy humans. Using a sensitive technique for gas measurement, methane was detected in all faecal cultures, including those from 'non-methanogenic' humans. Bile acid inhibited methanogenesis in a dose-response fashion in the in vitro 'non-methanogenic' and methanogenic faecal cultures. Inhibition was significant at bile acid concentrations > 0.05%. Methanogenesis correlated with methanogen (methanogenic bacteria) numbers. If this inhibition occurs in vivo, then it would explain much of the epidemiology of non-methanogenesis in humans. From an analysis of net hydrogen production by the faecal cultures, it is inferred that bile acid inhibits other hydrogen-consuming bacteria in addition to methanogens. These in vitro data suggest a major role for bile acid in the accumulation of hydrogen gas in the colon. Possible links between bile acid induced accumulation of gas and irritable bowel syndrome are discussed.

The effect of dietary nitrate on nitrate and nitrite excretion in man.

Dietary nitrate and nitrite may affect colonic pathophysiology. These anions influence fermentation, and nitrite has been shown to augment sodium absorption by the colon and participate in the formation of N-nitroso compounds. There is, however, no general agreement as to how much dietary nitrate and nitrite reaches the colon. To help resolve this question, balance studies were performed on six healthy ileostomy subjects who were given diets that varied in nitrate content from 0.83 to 5.20 mmol/d. Nitrate and nitrite excretion in ileal effluent and urine were measured by anion-exchange chromatography with conductivity detection. There was no significant nitrite in the diets, urine or ileal effluent. Dietary nitrate was largely excreted in urine (1.31-4.25 mmol/d). The urinary excretion findings indicated net synthesis of nitrate at low dietary intakes and net catabolism of nitrate at high intakes. Nitrate losses in ileal effluent were very low (0.03-0.05 mmol/d, 0.03-0.06 mmol/kg) and unrelated to intake for all the diets. It is concluded that dietary nitrate and nitrite do not enter the colon from the small intestine in amounts that would affect fermentation and mucosal metabolism in man. The possibility of significant amounts of nitrate reaching the colon via blood in normal subjects has not been excluded.

Active chronic peptic ulcer disease complicated by gastric outlet obstruction in pregnancy.

A case of active peptic ulcer disease in a primigravida at 29 weeks' gestation is presented, with a literature review. This particular case was complicated by gastric outlet obstruction, a complication which has not been previously reported in the obstetric literature. Although an uncommon diagnosis in pregnancy, the investigations for peptic ulcer disease should be actively pursued. Treatment can prevent serious complications.