Gut inflammation and adaptive immunity amplify acetaminophen toxicity in bowel and liver.

BACKGROUND AND AIM: Prevention of liver failure arising from accidental or deliberate paracetamol (acetaminophen [APAP]) overdose remains a vexed health problem despite well-publicized guidelines for its early detection and treatment. It is recognized that the gut may aggravate liver pathology, via the gut-liver axis. The main aim of this study was to assess the role of the colon in APAP-induced liver toxicity. METHODS: Liver necrosis and colitis were studied following sublethal doses of APAP administered intraperitoneally to C57Bl/6 wild-type (WT) mice, as well as to C57Bl/6 Winnie mice, which develop a spontaneous colitis caused by a SNP in Muc2, and WT mice with acute DSS-induced colitis. Repeated APAP exposure was studied in WT and Rag1 ko mice that lack mature T and B lymphocytes. RESULTS: APAP overdose resulted in significant colonic injury in WT mice (P < 0.05), which resolved by 24 h. Underlying colitis was not associated with liver necrosis, but colitis exacerbated APAP-induced liver injury and extended APAP-colonic injury. Prior APAP exposure exacerbated both APAP-liver and APAP-colonic injury more so in WT than Rag1 ko mice. APAP impaired barrier function with increased intestinal permeability and associated bacterial translocation to the liver and spleen in mice with the Winnie phenotype. CONCLUSIONS: This study identifies novel roles for APAP in causing colitis, the amplification of APAP-liver toxicity where there is underlying colitis, and involvement of immune memory in APAP-toxicity. The latter could be key for decoding the poorly understood but important clinical entity of chronic APAP liver failure.

MUC13 overexpression in renal cell carcinoma plays a central role in tumor progression and drug resistance.

Metastatic renal cell carcinoma is a largely incurable disease, and existing treatments targeting angiogenesis and tyrosine kinase receptors are only partially effective. Here we reveal that MUC13, a cell surface mucin glycoprotein, is aberrantly expressed by most renal cell carcinomas, with increasing expression positively correlating with tumor grade. Importantly, we demonstrated that high MUC13 expression was a statistically significant independent predictor of poor survival in two independent cohorts, particularly in stage 1 cancers. In cultured renal cell carcinoma cells MUC13 promoted proliferation and induced the cell cycle regulator, cyclin D1, and inhibited apoptosis by inducing the anti-apoptotic proteins, BCL-xL and survivin. Silencing of MUC13 expression inhibited migration and invasion, and sensitized renal cancer cells to killing by the multi-kinase inhibitors used clinically, sorafenib and sunitinib, and reversed acquired resistance to these drugs. Furthermore, we demonstrated that MUC13 promotion of renal cancer cell growth and survival is mediated by activation of nuclear factor κB, a transcription factor known to regulate the expression of genes that play key roles in the development and progression of cancer. These results show that MUC13 has potential as a prognostic marker for aggressive early stage renal cell cancer and is a plausible target to sensitize these tumors to therapy.

A novel mouse model of veno-occlusive disease provides strategies to prevent thioguanine-induced hepatic toxicity.

OBJECTIVE: The anti-leukemic drugs, azathioprine and 6-mercaptopurine (6MP), are important in the treatment of inflammatory bowel disease but an alternative faster-acting, less-allergenic thiopurine, 6-thioguanine (6TG), can cause hepatic veno-occlusive disease/sinusoidal obstructive syndrome (SOS). Understanding of SOS has been hindered by inability to ethically perform serial liver biopsies on patients and the lack of an animal model. DESIGN: Normal and C57Bl/6 mice with specific genes altered to elucidate mechanisms responsible for 6TG-SOS, were gavaged daily for upto 28d with 6TG, 6MP or methylated metabolites. Animal survival was monitored and at sacrifice a histological score of SOS, haematology and liver biochemistry were measured. RESULTS: Only 6TG caused SOS, which was dose related. 6TG and to a lesser extent 6MP but not methylated metabolites were associated with dose-dependent haematopoietic toxicity. SOS was not detected with non-lethal doses of 6TG. SOS did not occur in hypoxanthine-phosphoribosyl transferase-deficient C57Bl/6 mice, demonstrating that 6TG-SOS requires thioguanine nucleotides. Hepatic inflammation was characteristic of SOS, and C57Bl/6 mice deficient in P- and E-selectins on the surface of vascular endothelial cells showed markedly reduced SOS, demonstrating a major role for leukocytes recruited from blood. Split dosing of 6TG markedly attenuated SOS but still effected immunosuppression and prevented spontaneous colitis in Winnie mice, which have a single nucleotide polymorphism mutation in Muc2. CONCLUSION: This novel model provides clinically relevant insights into how 6TG induces SOS, and how this dangerous adverse drug reaction may be avoided by either inhibition of endothelial activation or simple changes to dosing regimens of 6TG, while still being effective treatment for colitis.

A Nucleotide Analog Prevents Colitis-Associated Cancer via Beta-Catenin Independently of Inflammation and Autophagy.

BACKGROUND & AIMS: Chronic bowel inflammation increases the risk of colon cancer; colitis-associated cancer (CAC). Thiopurine treatments are associated with a reduction in dysplasia and CAC in inflammatory bowel disease (IBD). Abnormal Wnt/ß-catenin signalling is characteristic of >90% of colorectal cancers. Immunosuppression by thiopurines is via Rac1 GTPase, which also affects Wnt/ß-catenin signalling. Autophagy is implicated in colonic tumors, and topical delivery of the thiopurine thioguanine (TG) is known to alleviate colitis and augment autophagy. This study investigated the effects of TG in a murine model of CAC and potential mechanisms. METHODS: Colonic dysplasia was induced by exposure to azoxymethane (AOM) and dextran sodium sulfate (DSS) in wild-type (WT) mice and mice harboring intestinal epithelial cell-specific deletion of autophagy related 7 gene (Atg7ΔIEC). TG or vehicle was administered intrarectally, and the effect on tumor burden and ß-catenin activity was assessed. The mechanisms of action of TG were investigated in vitro and in vivo. RESULTS: TG ameliorated DSS colitis in wild-type but not Atg7ΔIEC mice, demonstrating that anti-inflammatory effects of locally delivered TG are autophagy-dependent. However, TG inhibited CAC in both wild-type and Atg7ΔIEC mice. This was associated with decreased ß-catenin activation/nuclear translocation demonstrating that TG's inhibition of tumorigenesis occurred independently of anti-inflammatory and pro-autophagic actions. These results were confirmed in cell lines, and the dependency on Rac1 GTPase was demonstrated by siRNA knockdown and overexpression of constitutively active Rac1. CONCLUSIONS: Our findings provide evidence for a new mechanism that could be exploited to improve CAC chemoprophylactic approaches.

The development and electronic delivery of case-based learning using a fast healthcare interoperability resource system.

HL7 International's Fast Healthcare Interoperability Resources (FHIR) standard provides a common format for sharing health data (eg, FHIR resources) and a RESTful Application Programming Interface (eg, FHIR API) for accessing those resources via a FHIR server connected to an electronic health record system or any other system storing clinical data. Substitutable Medical Applications and Reusable Technologies (SMART) leverages FHIR to create an electronic health record (EHR) agnostic app platform. It utilizes the OAuth standard to provide for authorization and authentication. This paper describes the development and informal evaluation of Case Based Learning on FHIR (CBL on FHIR), a prototype EHR-connected FHIR/SMART platform to provide interactive digital cases for use in medical education. The project goals were to provide a more interactive form of CBL than is possible on paper to more realistically simulate clinical decision making and to expose medical students to modern informatics systems and tools for use in patient care.

Colonic thioguanine pro-drug: Investigation of microbiome and novel host metabolism.

Thiopurines are analogues of endogenous purines. They are pro-drugs which require the purine salvage pathway to convert them to the active drug nucleotides (TGN). These drugs are used to maintain clinical remission in patients with inflammatory bowel diseases. In our recent Gut paper, we showed that thioguanine worked quickly to improve colitis in the absence in the host animal of the key guanine salvage enzyme, hypoxanthine-guanine-phosphoribosyltransferase (HPRT). Current evidence favours the proposition that active drug delivery to the host lacking HPRT requires translocation of TGN-loaded bacteria across the inflamed mucosal barrier, and most likely delivery by phagocytosis. Alternatively, the efficacy of thioguanine in treating colitis could be mediated by modulation of the community of the microbiota in the intestine, or there are novel host pathways for conversion of the thioguanine pro-drug to TGN.

The role of IL-22 in the resolution of sterile and nonsterile inflammation.

In a broad sense, inflammation can be conveniently characterised by two phases: the first phase, which is a pro-inflammatory, has evolved to clear infection and/or injured tissue; and the second phase concerns regeneration of normal tissue and restitution of normal physiology. Innate immune cell-derived pro-inflammatory cytokines and chemokines activate and recruit nonresident immune cells to the site of infection, thereby amplifying the inflammatory responses to clear infection or injury. This phase is followed by a cytokine milieu that promotes tissue regeneration. There is no absolute temporal distinction between these two phases, and cytokines may have dual pleiotropic effects depending on the timing of release, inflammatory microenvironment or concentrations. IL-22 is a cytokine with reported pro- and anti-inflammatory roles; in this review, we contend that this protein has primarily a function in restitution of normal tissue and physiology.

Critical assessment of thioguanine treatment for inflammatory bowel diseases: Is it time to rehabilitate this treatment?

OBJECTIVE: The potential therapeutic effect of thioguanine in the management of inflammatory bowel disease (IBD) is hindered due to association with vascular hepatotoxicity. The study aimed to assess the evidence for efficacy of thioguanine in IBD management and the association with nodular regenerative hyperplasia (NRH) and other thioguanine-related hepatotoxicities. METHODS: Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines were used for literature search. Due to the lack of randomized controlled trials (RCTs), the search was extended to observational studies. Quality of the included studies were graded A to C based on evaluation tools used to determine efficacy (subjective and objective grading tools) and nodular regenerative hyperplasia safety (liver biopsy and imaging tools). RESULTS: Two hundred and ninety studies were identified, but following Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines only 13 studies were evaluated for efficacy and safety of thioguanine. Outcome measures were consistent across the included studies. Thioguanine appeared efficacious and well-tolerated in patients who were intolerant/non-responsive to existing immunomodulators. There was a trend toward a positive association between dose of thioguanine and NRH but not with other adverse events such as liver biochemical abnormalities or with portal hypertension. CONCLUSIONS: The evidence to support thioguanine treatment is limited to observational studies. While encouraging, there is a need for prospective RCTs to determine the role of thioguanine in the management of IBD.

Microbial metabolism of thiopurines: A method to measure thioguanine nucleotides.

Thiopurines are anti-inflammatory prodrugs. We hypothesised that bacteria may contribute to conversion to active drug. Escherichia coli strain DH5α was evaluated to determine whether it could metabolise the thiopurine drugs, thioguanine or mercaptopurine, to thioguanine nucleotides. A rapid and reliable high performance liquid chromatography (ultraviolet detection) method was developed to quantify indirectly thioguanine nucleotides, by measuring thioguanine nucleoside.

Neutralizing IL-23 is superior to blocking IL-17 in suppressing intestinal inflammation in a spontaneous murine colitis model.

BACKGROUND: IL-23/T(H)17 inflammatory responses are regarded as central to the pathogenesis of inflammatory bowel disease, but clinically IL-17A antibodies have shown low efficacy and increased infections in Crohn's disease. Hence, we decided to closely examine the role of the IL-23/T(H)17 axis in 3 models of colitis. METHODS: IL-17A(-/-) and IL-17Ra(-/-) T cells were transferred into Rag1 and RaW mice to assess the role of IL-17A-IL-17Ra signaling in T cells during colitis. In Winnie mice with spontaneous colitis due to an epithelial defect, we studied the progression of colitis in the absence of IL-17A and the efficacy of neutralizing antibodies against the IL-17A or IL-23p19 cytokines. RESULTS: In transfer colitis models, IL-17A-deficient T cells failed to ameliorate disease, and IL-17Ra-deficient T cells were more colitogenic than wild-type T cells. In Winnie mice with an epithelial defect and spontaneous T(H)17-dominated inflammation, genetic deficiency of IL-17A did not suppress initiation of colitis but limited colitis progression. Furthermore, inhibition of IL-17A by monoclonal antibodies did not reduce colitis severity. In contrast, neutralizing IL-23 using an anti-p19 antibody significantly alleviated both emerging and established colitis, downregulating T(H)17 proinflammatory cytokine expression and diminishing neutrophil infiltration. CONCLUSIONS: Our results support clinical studies showing that IL-17 neutralization is not therapeutic but that targeting IL-23 suppresses intestinal inflammation. Effects of IL-23 distinct from its effects on maturation of IL-17A-producing lymphocytes may underlie the protection from inflammatory bowel disease conveyed by hypomorphic IL-23 receptor polymorphisms and contribute to the efficacy of IL-23 neutralizing antibodies in inflammatory bowel disease.

Splitting a therapeutic dose of thioguanine may avoid liver toxicity and be an efficacious treatment for severe inflammatory bowel disease: a 2-center observational cohort study.

BACKGROUND: Thioguanine (TG) is a treatment for inflammatory bowel disease, but association with nodular regenerative hyperplasia has restricted its use. We conjectured that splitting a therapeutic daily dose of TG would be efficacious and should avoid liver toxicity. METHODS: We report on 62 patients with severe inflammatory bowel disease not responding to prednisolone, conventional thiopurines, biologics, or calcineurin inhibitors. Patients were prescribed oral split-daily TG to avoid individual doses >0.3 mg/kg. Data on concomitant medication, clinical efficacy measured by Harvey-Bradshaw Index for Crohn's, or Simple Clinical Colitis Score for ulcerative/indeterminate colitis (UC), and some paired endoscopies were available. Safety was followed clinically and with bloods at 2 centers. All patients at the U.K. center had a liver biopsy or magnetic resonance imaging after 6 months. Twenty-one patients had serial ultrasounds at the Australian center. RESULTS: At 6 months, 19/21 of patients with Crohn's disease and 27/38 with ulcerative colitis had improved clinical activity. At study end, 53% of patients maintained improved clinical activity of steroids. Median duration of TG was 8 (0.3-45) months, median dose was 0.6 (0.3-1) mg/kg per day. Previous thiopurine-related adverse reactions were not encountered. Twenty-nine patients withdrew because of loss to follow-up, medical adverse events, or surgery. Possible early nodular regenerative hyperplasia was found on liver biopsy in 1 patient who was heterozygote deficient for thiopurine methyltransferase; the TG dose was lowered. TG was discontinued in a patient with nodular regenerative hyperplasia and concomitant antiphospholipid syndrome. There was 1 successful term pregnancy; cord blood and breast milk TG were low. CONCLUSIONS: Split-dose TG seemed well tolerated and efficacious in this retrospective study of patients with difficult inflammatory bowel disease.

Selenoprotein S is a marker but not a regulator of endoplasmic reticulum stress in intestinal epithelial cells.

Selenoproteins are candidate mediators of selenium-dependent protection against tumorigenesis and inflammation in the gut. Expression and roles of only a limited number of intestinal selenoproteins have been described so far. Selenoprotein S (SelS) has been linked to various inflammatory diseases and is suggested to be involved in endoplasmic reticulum (ER) homeostasis regulation and antioxidative protection in a cell-type-dependent manner, but its protein expression, regulation, and function in the gut are not known. We here analyzed the expression and localization of SelS in the healthy and inflamed gut and studied its regulation and function in intestinal epithelial cell lines. SelS was expressed in the intestinal epithelium of the small and large intestine and colocalized with markers of Paneth cells and macrophages. It was upregulated in inflamed ileal tissue from Crohn's disease patients and in two models of experimental colitis in mice. We detected SelS in colorectal cell lines, where it colocalized with the ER marker calnexin. SelS protein expression was unaffected by enterocytic differentiation but increased in response to selenium supplementation and after treatment with the ER stress inducer tunicamycin. On the other hand, depletion of SelS in LS174T, HT29, and Caco-2 cells by RNA interference did not cause or modulate ER stress and had no effect on hydrogen peroxide-induced cell death. In summary, we introduce SelS as a novel marker of Paneth cells and intestinal ER stress. Although it is upregulated in Crohn's disease, its role in disease etiology remains to be established.

Glucocorticoids alleviate intestinal ER stress by enhancing protein folding and degradation of misfolded proteins.

Endoplasmic reticulum (ER) stress in intestinal secretory cells has been linked with colitis in mice and inflammatory bowel disease (IBD). Endogenous intestinal glucocorticoids are important for homeostasis and glucocorticoid drugs are efficacious in IBD. In Winnie mice with intestinal ER stress caused by misfolding of the Muc2 mucin, the glucocorticoid dexamethasone (DEX) suppressed ER stress and activation of the unfolded protein response (UPR), substantially restoring goblet cell Muc2 production. In mice lacking inflammation, a glucocorticoid receptor antagonist increased ER stress, and DEX suppressed ER stress induced by the N-glycosylation inhibitor, tunicamycin (Tm). In cultured human intestinal secretory cells, in a glucocorticoid receptor-dependent manner, DEX suppressed ER stress and UPR activation induced by blocking N-glycosylation, reducing ER Ca(2+) or depleting glucose. DEX up-regulated genes encoding chaperones and elements of ER-associated degradation (ERAD), including EDEM1. Silencing EDEM1 partially inhibited DEX's suppression of misfolding-induced ER stress, showing that DEX enhances ERAD. DEX inhibited Tm-induced MUC2 precursor accumulation, promoted production of mature mucin, and restored ER exit and secretion of Winnie mutant recombinant Muc2 domains, consistent with enhanced protein folding. In IBD, glucocorticoids are likely to ameliorate ER stress by promoting correct folding of secreted proteins and enhancing removal of misfolded proteins from the ER.

Insights into N-calls of mitochondrial DNA sequencing using MitoChip v2.0.

BACKGROUND: Developments in DNA resequencing microarrays include mitochondrial DNA (mtDNA) sequencing and mutation detection. Failure by the microarray to identify a base, compared to the reference sequence, is designated an 'N-call.' This study re-examined the N-call distribution of mtDNA samples sequenced by the Affymetrix MitoChip v.2.0, based on the hypothesis that N-calls may represent insertions or deletions (indels) in mtDNA. FINDINGS: We analysed 16 patient mtDNA samples using MitoChip. N-calls by the proprietary GSEQ software were significantly reduced when either of the freeware on-line algorithms ResqMi or sPROFILER was utilized. With sPROFILER, this decrease in N-calls had no effect on the homoplasmic or heteroplasmic mutation levels compared to GSEQ software, but ResqMi produced a significant change in mutation load, as well as producing longer N-cell stretches. For these reasons, further analysis using ResqMi was not attempted. Conventional DNA sequencing of the longer N-calls stretches from sPROFILER revealed 7 insertions and 12 point mutations. Moreover, analysis of single-base N-calls of one mtDNA sample found 3 other point mutations. CONCLUSIONS: Our study is the first to analyse N-calls produced from GSEQ software for the MitoChipv2.0. By narrowing the focus to longer stretches of N-calls revealed by sPROFILER, conventional sequencing was able to identify unique insertions and point mutations. Shorter N-calls also harboured point mutations, but the absence of deletions among N-calls suggests that probe confirmation affects binding and thus N-calling. This study supports the contention that the GSEQ is more capable of assigning bases when used in conjunction with sPROFILER.