The proximal cardia region of the stomach has a high incidence of inflammation, metaplasia and neoplasia. It demonstrates less acid buffering following meals than the more distal stomach. Novel high definition pHmetry was employed to investigate acidity at the cardia under fasting conditions and in response to a meal.
15 healthy subjects were studied. A custom-made 12-electrode pH catheter was clipped at the squamocolumnar junction with four electrodes recording proximal to and eight distal to the squamocolumnar junction. The most distal pH electrode was located at the catheter tip, and nine electrodes in the region of the squamocolumnar junction were 11 mm apart.
The electrode situated in the cardia 5.5 mm distal to the squamocolumnar junction differed from all other intragastric electrodes during fasting in recording minimal acidity (pH <4 = 2.2%) while all other intragastric electrodes recorded high intragastric acidity (pH <4 = >39%) (p<0.05). The cardia also differed from the rest of the stomach, showing a marked increase in acidity in response to the meal (from 2.2% fasting to 58.4% at 60–70 min after the meal; p<0.05) while the electrodes distal to the cardia all showed a marked decrease in acidity (p<0.05). These changes in acidity at the cardia following the meal caused the gastric acidity to extend 10 mm closer to the squamocolumnar junction.
Whereas the rest of the stomach shows a marked fall in acidity on ingesting a meal, the cardia paradoxically increases in acidity to become the most acidic region throughout the postprandial period.
Sulfate (SO42–) is an abundant component of intestinal mucins and its content is decreased in certain gastrointestinal diseases, including inflammatory bowel disease. In this study, the hyposulfataemic NaS1 sulfate transporter null (Nas1–/–) mice were used to investigate the physiological consequences of disturbed sulfate homeostasis on (1) intestinal sulfomucin content and mRNA expression; (2) intestinal permeability and proliferation; (3) dextran sulfate sodium (DSS)-induced colitis; and (4) intestinal barrier function against the bacterial pathogen, Campylobacter jejuni.
Intestinal sulfomucins and sialomucins were detected by high iron diamine staining, permeability was assessed by fluorescein isothiocyanate (FITC)–dextran uptake, and proliferation was assessed by 5-bromodeoxyuridine (BrdU) incorporation. Nas1–/– and wild-type (Nas1+/+) mice received DSS in drinking water, and intestinal damage was assessed by histological, clinical and haematological measurements. Mice were orally inoculated with C jejuni, and intestinal and systemic infection was assessed. Ileal mRNA expression profiles of Nas1–/– and Nas1+/+ mice were determined by cDNA microarrays and validated by quantitative real-time PCR.
Nas1–/– mice exhibited reduced intestinal sulfomucin content, enhanced intestinal permeability and DSS-induced colitis, and developed systemic infections when challenged orally with C jejuni. The transcriptional profile of 41 genes was altered in Nas1–/– mice, with the most upregulated gene being pancreatic lipase-related protein 2 and the most downregulated gene being carbonic anhydrase 1 (Car1).
Sulfate homeostasis is essential for maintaining a normal intestinal metabolic state, and hyposulfataemia leads to reduced intestinal sulfomucin content, enhanced susceptibility to toxin-induced colitis and impaired intestinal barrier to bacterial infection.
Patients with Crohn’s disease have an increased risk for systemic thromboembolism. Their platelets are hyperactive and possess an elevated endogenous content of CD40 ligand (CD40L), a tumour necrosis factor family protein member. Under basal conditions and after stimulation, these platelets express more CD40L on their surface and release higher amounts of soluble (s)CD40L than control platelets, through a mechanism that might be mediated by matrix metalloproteinases (MMPs).
The aim of this work is to study whether enhanced sCD40L release secondary to changes in the platelet content of MMPs contributes to the higher state of activation of platelets from patients with Crohn’s disease.
State of activation, CD40L and metalloproteinases content of platelets isolated from patients with Crohn’s disease and age- and sex-matched control individuals were analysed, respectively, by flow cytometry, western blot and gelatin zymography.
The hyperactive state of platelets from patients with Crohn’s disease might rely on their enhanced release of sCD40L, since its inhibition by a broad-range inhibitor of MMPs (GM6001) reduced fibrinogen binding induced by platelet stimulation. Analysis of the content of MMPs in platelets from patients with Crohn’s disease showed an exclusive increase in MMP-9 activity. Moreover, MMP-9 inhibition diminished sCD40L release and fibrinogen binding to activated platelets.
The results suggest that platelets from patients with Crohn’s disease release more sCD40L than controls as a consequence of their higher endogenous content of CD40L and of MMP-9, which is involved in CD40L shedding. The increased levels of released sCD40L might be responsible, at least in part, for the high state of activation of platelets from patients with Crohn’s disease.
Inflammatory bowel diseases (IBDs) are associated with uncontrolled innate and adaptive immunity against normal constituents, including commensal bacteria and microbial products. Mesenchymal stem cells (MSCs) suppress effector T cell responses and have beneficial effects in various immune disorders. This work investigates the therapeutic effects of human adipose-derived MSCs (hASCs) in various models of IBD and sepsis.
Acute and chronic colitis was induced in mice with dextran sulfate sodium. Sepsis was induced by caecal ligation and puncture or by endotoxin injection. Colitic and septic mice were treated intraperitoneally with hASCs or murine ASCs, and diverse disease clinical signs and mortality were determined. The levels of various inflammatory cytokines and chemokines, T helper 1(Th1)-type response and generation of regulatory T cells (Treg) were determined in affected organs.
Systemic infusion of ASCs significantly ameliorated the clinical and histopathological severity of colitis, abrogating weight loss, diarrhoea and inflammation, and increasing survival. The therapeutic effect was associated with downregulation of the Th1-driven inflammatory responses. ASCs decreased a wide panel of inflammatory cytokines and chemokines and increased interleuklin 10 (IL10), acting on macrophages. hASCs also impaired Th1 cell activation in both colonic mucosa and draining lymph nodes. The induction of IL10-secreting Treg was partially involved in the therapeutic effect of hASCs. Moreover, ASCs protected from severe sepsis by reducing the infiltration of inflammatory cells in various target organs and by downregulating the production of various inflammatory mediators.
hASCs emerge as key regulators of immune/inflammatory responses in vivo and as attractive candidates for cell-based treatments for IBD and sepsis.
To evaluate the efficacy of adalimumab in the healing of draining fistulas in patients with active Crohn’s disease (CD).
A phase III, multicentre, randomised, double-blind, placebo controlled study with an open-label extension was conducted in 92 sites.
A subgroup of adults with moderate to severely active CD (CD activity index 220–450) for >=4 months who had draining fistulas at baseline.
All patients received initial open-label adalimumab induction therapy (80 mg/40 mg at weeks 0/2). At week 4, all patients were randomly assigned to receive double-blind placebo or adalimumab 40 mg every other week or weekly to week 56 (irrespective of fistula status). Patients completing week 56 of therapy were then eligible to enroll in an open-label extension.
Complete fistula healing/closure (assessed at every visit) was defined as no drainage, either spontaneous or with gentle compression.
Of 854 patients enrolled, 117 had draining fistulas at both screening and baseline (70 randomly assigned to adalimumab and 47 to placebo). The mean number of draining fistulas per day was significantly decreased in adalimumab-treated patients compared with placebo-treated patients during the double-blind treatment period. Of all patients with healed fistulas at week 56 (both adalimumab and placebo groups), 90% (28/31) maintained healing following 1 year of open-label adalimumab therapy (observed analysis).
In patients with active CD, adalimumab therapy was more effective than placebo for inducing fistula healing. Complete fistula healing was sustained for up to 2 years by most patients in an open-label extension trial.
ClinicalTrials.gov Identifier: NCT00077779 and NCT00195715.
Emerging evidence suggests that highly treatment-resistant tumour-initiating cells (TICs) play a central role in the pathogenesis of pancreatic cancer. Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) is considered to be a novel anticancer agent; however, recent studies have shown that many pancreatic cancer cells are resistant to apoptosis induction by TRAIL due to TRAIL-activated nuclear factor-B (NF-B) signalling. Several chemopreventive agents are able to inhibit NF-B, and favourable results have been obtained—for example, for the broccoli compound sulforaphane—in preventing metastasis in clinical studies. The aim of the study was to identify TICs in pancreatic carcinoma for analysis of resistance mechanisms and for definition of sensitising agents.
TICs were defined by expression patterns of a CD44+/CD24–, CD44+/CD24+ or CD44+/CD133+ phenotype and correlation to growth in immunodeficient mice, differentiation grade, clonogenic growth, sphere formation, aldehyde dehydrogenase (ALDH) activity and therapy resistance.
Mechanistically, specific binding of transcriptionally active cRel-containing NF-B complexes in TICs was observed. Sulforaphane prevented NF-B binding, downregulated apoptosis inhibitors and induced apoptosis, together with prevention of clonogenicity. Gemcitabine, the chemopreventive agents resveratrol and wogonin, and the death ligand TRAIL were less effective. In a xenograft model, sulforaphane strongly blocked tumour growth and angiogenesis, while combination with TRAIL had an additive effect without obvious cytotoxicity in normal cells. Freshly isolated patient tumour cells expressing markers for TICs could be sensitised by sulforaphane for TRAIL-induced cytotoxity.
The data provide new insights into resistance mechanisms of TICs and suggest the combination of sulforaphane with TRAIL as a promising strategy for targeting of pancreatic TICs.
Dendritic cell (DC) defects may contribute to chronicity in hepatitis C virus (HCV) infection and determine response to PEG–interferon and ribavirin therapy via poor T cell stimulation. Studies to date have produced inconsistent results regarding DC maturation and function: no large study has examined DCs before and after therapy.
We examined if DC defects in maturation and chemotaxis are present by comparing therapeutic responders to non-responders.
We analysed peripheral DCs of 64 HCV genotype 1-infected patients from the Virahep-C study 2 weeks before and 24 weeks after therapy. We used flow cytometry to enumerate plasmacytoid DC (pDC) and myeloid DCs (mDC) and quantify expression of chemokine receptors and maturation markers. Chemotaxis was measured with an in vitro assay.
Pre-treatment frequencies of pDCs and mDCs were significantly lower in HCV patients than controls and successful therapy normalised pDCs. Levels of CXCR3 and CXCR4 on pDCs were higher at baseline compared to normal controls and decreased with therapy. Pre-therapy levels of co-stimulatory marker CD40 and the maturation marker CD83 were higher in pDCs of patients chronically infected with HCV compared to normal patients, and levels of both markers dropped significantly with therapy in the SVR+ group only. Other maturation markers (CD86 and CCR7) were not elevated suggesting a partially activated phenotype. Baseline chemotaxis of pDCs to CXCL12 and CXCL10 predicted failure of antiviral response and correlated with the histological activity index inflammation score.
Plasmacytoid DC defects exist in chronic HCV and successful antiviral therapy normalises many phenotypic and functional abnormalities.
Innate immunity appears to be silent in acutely heptitis B virus (HBV)-infected chimpanzees, as shown by microarray analysis of intrahepatic gene expression. Whether this observation also applies to HBV pathogenesis in man remains undefined. The aim of this study was thus to characterise natural killer (NK) and CD56+ natural T (NT) cell responses early after human HBV infection and their relationship to the induction of adaptive immunity.
Two HBV-seronegative blood donors who became hepatitis B surface antigen (HBsAg) and HBV DNA positive but had persistently normal alanine aminotransferase (ALT) were followed from a very early stage of HBV infection. The phenotype (CD69 and NKG2D) and function (cytotoxicity and interferon (IFN) production) of NK and NT cells were analysed. CD4- and CD8-mediated responses were studied in parallel with overlapping peptides covering the entire HBV sequence by ex vivo intracellular cytokine staining (ICS) for IFN, interleukin 2 (IL2), IL4 and IL10, and by ex vivo Elispot for IFN. Healthy subjects, and patients with chronic and acute HBV infection were studied for comparison.
An early induction of both innate and adaptive responses was observed. NK and NT cells showed faster kinetics than HBV-specific T cells with an earlier peak of activity, while CD4+ and CD8+ cell responses were mounted with a similar profile, with higher frequencies of IFN-producing CD8+ cells at the peak of the response.
The innate immune system is able to sense HBV infection, as shown by the early development of NK and NT cell responses, which probably contribute to contain the HBV infection and to allow timely induction of adaptive responses.
In 95% of patients with primary biliary cirrhosis (PBC) antimitochondrial antibodies (AMAs) can be detected reacting with at least one of the five components of the M2 antigen identified as the 2-oxoacid dehydrogenase complex (OADC). However, among our PBC sera 15–20% are anti-M2 negative by ELISA and western blotting but in the immunofluorescence test (IFT) they show the typical AMA staining. The aim of the present study was to characterise the target antigen(s) of these non-M2-related AMAs.
We analysed sera from 27 patients with clinically and histologically proven PBC being AMA positive by the IFT but anti-M2 negative by ELISA and western blotting. They were tested by western blotting against various 100 000 g supernatants obtained after sonication of mitochondria from rat liver, bovine heart and pig kidney. These were further separated by isopycnic sucrose density centrifugation using different sucrose density fractions.
Fourteen of the 27 AMA positive/anti-M2 negative sera (52%) reacted in the western blotting with a 60 kDa protein and eight (29%) with an 80 kDa protein, both present in the 100 000 g supernatant from bovine heart mitochondria accumulating at sucrose densities of 1.14–1.16. An identity of these determinants with any of the M2-related antigens could be excluded. In the 60 kDa band components of the mitochondrial enzymes F1F0-ATPase, ubiquinone cytochrome c reductase and acyl CoA dehydrogenase were detected by MALDI–TOF analysis; the 80 kDa protein could not be further characterised.
AMA positive/anti-M2 negative PBC sera contain antibodies to further mitochondrial antigens at 60 and 80 kDa which do not correspond to any of the M2 determinants. Those antibodies can be detected to a lesser extent in sera from patients with classical anti-M2 positive PBC but not in patients with other hepatic and non-hepatic disorders and may, therefore, represent additional marker antibodies for the serological diagnosis of PBC.
Progression of chronic cholestatic disorders towards ductopenia results from the dysregulation of cholangiocyte survival, with cell death by apoptosis prevailing over compensatory proliferation. Currently, no therapy is available to sustain cholangiocyte survival in the course of those disorders. It was recently shown that cholangiocytes express the glucagon-like peptide-1 receptor (GLP-1R); its activation results in enhanced proliferative reaction to cholestasis. The GLP-1R selective agonist exendin-4 sustains pancreatic β cell proliferation and prevents cell death by apoptosis. Exendin-4 is now employed in humans as a novel therapy for diabetes. The aim of the present study was to verify whether exendin-4 is effective in preventing cholangiocyte apoptosis.
In vitro, tests were carried out to determine if exendin-4 is able to prevent apoptosis of cholangiocytes isolated from normal rats induced by glycochenodeoxycholic acid (GCDCA); in vivo, animals subjected to 1 week of bile duct ligation and to a single intraperitoneal injection of CCl4 were treated with exendin-4 for 3 days.
Exendin-4 prevented GCDCA-induced Bax mitochondrial translocation, cytochrome c release and an increase in caspase 3 activity. Phosphatidylinositol 3-kinase, but not cAMP/protein kinase A or Ca2+/calmodulin-dependent protein kinase inhibitors, neutralised the effects of exendin-4. In vivo, exendin-4 administration prevented the increase in TUNEL (terminal deoxynucleotidyl transferase-mediated triphosphate end-labelling)-positive cholangiocytes and the loss of bile ducts observed in bile duct-ligated rats treated with CCl4.
Exendin-4 prevents cholangiocyte apoptosis both in vitro and in vivo; such an effect is due to the ability of exendin-4 to counteract the activation of the mitochondrial pathway of apoptosis. These findings support the hypothesis that exendin-4 may be effective in slowing down the progression of cholangiopathies to ductopenia.