Andreas Koutsoumpas

Randomised, double-blind, placebo-controlled trial of fructo-oligosaccharides in active Crohn's disease

Introduction The commensal intestinal microbiota drive the inflammation associated with Crohns disease. However, bacteria such as bifidobacteria and Faecalibacterium prausnitzii appear to be immunoregulatory. In healthy subjects the intestinal microbiota are influenced by prebiotic carbohydrates such as fructo-oligosaccharides (FOS). Preliminary data suggest that FOS increase faecal bifidobacteria, induce immunoregulatory dendritic cell (DC) responses and reduce disease activity in patients with Crohns disease. Aims and methods To assess the impact of FOS in patients with active Crohns disease using an adequately powered randomised double-blind placebo-controlled trial with predefined clinical, microbiological and immunological end points. Patients with active Crohns disease were randomised to 15 g/day FOS or non-prebiotic placebo for 4 weeks. The primary end point was clinical response at week 4 (fall in Crohns Disease Activity Index of ≥70 points) in the intention-to-treat (ITT) population. Results 103 patients were randomised to receive FOS (n=54) or placebo (n=49). More patients receiving FOS (14 (26%) vs 4 (8%); p=0.018) withdrew before the 4-week end point. There was no significant difference in the number of patients achieving a clinical response between the FOS and placebo groups in the ITT analysis (12 (22%) vs 19 (39%), p=0.067). Patients receiving FOS had reduced proportions of interleukin (IL)-6-positive lamina propria DC and increased DC staining of IL-10 (p<0.05) but no change in IL-12p40 production. There were no significant differences in the faecal concentration of bifidobacteria and F prausnitzii between the groups at baseline or after the 4-week intervention. Conclusion An adequately powered placebo-controlled trial of FOS showed no clinical benefit in patients with active Crohns disease, despite impacting on DC function. ISRCTN50422530.

Smokers with active Crohn's disease have a clinically relevant dysbiosis of the gastrointestinal microbiota

Background: Patients with Crohns disease (CD) have an intestinal dysbiosis with components of the microbiota exerting differential immune effects. Smoking is associated with an increased incidence of CD, more frequent relapse, and greater burden of surgery. This study aimed to investigate the association between smoking and the intestinal microbiota in patients with active CD. Methods: Patients with active CD (n = 103) and healthy controls (n = 66) were recruited and demographic and clinical data recorded including current smoking behavior. Fecal samples were collected and analyzed by fluorescent in situ hybridization using probes targeting 16S rRNA of bacteria previously shown to be altered in active CD (bifidobacteria, bacteroides, Clostridium coccoides‐Eubacterium rectale, Escherichia coli, and Faecalibacterium prausnitzii). Results: In total, 29/101 (29%) patients with CD and 8/58 (14%) controls were current smokers (P = 0.032). Following multivariate analysis, smoking was found to have a significant and independent effect on the microbiota of patients with CD, with higher Bacteroides‐Prevotella in smokers (38.4%) compared with nonsmokers (28.1%) (F(1,93) = 12.6, P = 0.001). Healthy controls who smoked also had higher Bacteroides‐Prevotella (34.8%) than nonsmokers (24.1%) (F(1,55) = 4.5, P = 0.038). In the pooled multivariate analysis, patients with CD had higher bifidobacteria (F(1,156) = 30.5, P < 0.001), higher Bacteroides‐Prevotella (F(1,156) = 6.5, P = 0.012), and lower F. prausnitzii (F(1,156) = 3.8, P = 0.052) compared with healthy controls. Conclusions: Smokers have luminal microbiota that consist of significantly higher bacteroides. Investigation of whether this is one mechanism through which the negative effects of smoking on CD are mediated is warranted. (Inflamm Bowel Dis 2012;)

Relationship Between Human Intestinal Dendritic Cells, Gut Microbiota, and Disease Activity in Crohn's Disease

Background: Altered intestinal dendritic cell (DC) function underlies dysregulated T‐cell responses to bacteria in Crohns disease (CD) but it is unclear whether composition of the intestinal microbiota impacts local DC function. We assessed the relationship between DC function with disease activity and intestinal microbiota in patients with CD. Methods: Surface expression of Toll‐like receptor (TLR)‐2, TLR‐4, and spontaneous intracellular interleukin (IL)‐10, IL‐12p40, IL‐6 production by freshly isolated DC were analyzed by multicolor flow cytometry of cells extracted from rectal tissue of 10 controls and 28 CD patients. Myeloid DC were identified as CD11c+HLA‐DR+lin‐/dim cells (lin = anti‐CD3, CD14, CD16, CD19, CD34). Intestinal microbiota were analyzed by fluorescent in situ hybridization of fecal samples with oligonucleotide probes targeting 16S rRNA of bifidobacteria, bacteroides‐prevotella, C. coccoides‐E. rectale, and Faecalibacterium prausnitzii. Results: DC from CD produced higher amounts of IL‐12p40 and IL‐6 than control DC. IL‐6+ DC were associated with the CD Activity Index (r = 0.425; P = 0.024) and serum C‐reactive protein (CRP) (r = 0.643; P = 0.004). DC expression of TLR‐4 correlated with disease activity. IL‐12p40+ DC correlated with ratio of bacteroides: bifidobacteria (r = 0.535, P = 0.003). IL‐10+ DC correlated with bifidobacteria, and IL‐6+ DC correlated negatively with F. prausnitzii (r = −0.50; P = 0.008). The amount of TLR‐4 on DC correlated negatively with the concentration of F. prausnitzii. Conclusions: IL‐6 production by intestinal DC is increased in CD and correlates with disease activity and CRP. Bacterially driven local IL‐6 production by intestinal DC may overcome regulatory activity, resulting in unopposed effector function and tissue damage. Intestinal DC function may be influenced by the composition of the commensal microbiota. (Inflamm Bowel Dis 2011;)

Helicobacter pylori and autoimmune disease: Cause or bystander

Helicobacter pylori (H. pylori) is the main cause of chronic gastritis and a major risk factor for gastric cancer. This pathogen has also been considered a potential trigger of gastric autoimmunity, and in particular of autoimmune gastritis. However, a considerable number of reports have attempted to link H. pylori infection with the development of extra-gastrointestinal autoimmune disorders, affecting organs not immediately relevant to the stomach. This review discusses the current evidence in support or against the role of H. pylori as a potential trigger of autoimmune rheumatic and skin diseases, as well as organ specific autoimmune diseases. We discuss epidemiological, serological, immunological and experimental evidence associating this pathogen with autoimmune diseases. Although over one hundred autoimmune diseases have been investigated in relation to H. pylori, we discuss a select number of papers with a larger literature base, and include Sjögrens syndrome, rheumatoid arthritis, systemic lupus erythematosus, vasculitides, autoimmune skin conditions, idiopathic thrombocytopenic purpura, autoimmune thyroid disease, multiple sclerosis, neuromyelitis optica and autoimmune liver diseases. Specific mention is given to those studies reporting an association of anti-H. pylori antibodies with the presence of autoimmune disease-specific clinical parameters, as well as those failing to find such associations. We also provide helpful hints for future research.

Ileal Inflammation May Trigger the Development of GP2-Specific Pancreatic Autoantibodies in Patients with Crohn’s Disease

Why zymogen glycoprotein 2 (GP2), the Crohns disease (CD)-specific pancreatic autoantigen, is the major target of humoral autoimmunity in inflammatory bowel diseases (IBD) is uknown. Recent evidence demonstrates that GP2 is also present on the apical surface of microfold (M) intestinal cells. As the colon lacks GP2-rich M cells, we assumed that patients with colonic CD are seronegative for anti-GP2. Anti-GP2 antibodies were tested in 225 CDs, including 45 patients with colonic location (L2), 45 with terminal ileum (L1) and 135 with ileocolonic involvement; 225 patients with ulcerative colitis (UC) were also tested. Anti-GP2 reactivity was detected in 59 (26.2%) CDs and 15 (6.7%) UCs (P < 0.001). Only 5 CDs with L2 had anti-GP2 antibodies, compared to 54/180 (30.0%, P = 0.0128) of the CDs with L1 and L3. Anti-GP2 antibody positive CD patients had higher ASCA titres compared to seronegative cases. Amongst the 128 CD patients with previous surgical intervention, 45 (35.0%) were anti-GP2 antibody positive compared to 14/97 (14.0%) without surgical (P < 0.001). Our data support the assumption that ileal inflammation is required for the development of anti-GP2 antibodies in CD, and suggest that the intestine rather than the pancreatic juice is the antigenic source required for the initiation of anti-GP2 antibodies.

Diagnostic and clinical utility of antibodies against the nuclear body promyelocytic leukaemia and Sp100 antigens in patients with primary biliary cirrhosis.

BACKGROUND The lack of an immunoassay that detects antibodies to promyelocytic leukaemia (PML) protein, the primary biliary cirrhosis (PBC)-specific multiple nuclear dot (MND) antigen, has prompted us to develop a line immunoassay (LIA) for the simultaneous detection of PML and Sp100 MND-specific autoantibodies. METHODS PML and Sp100 were expressed in Escherichia coli, and analysed by SDS-PAGE and immunoblotting using a monoclonal antibody and MALDI-ToF fingerprinting. A quantitative PML and Sp100 LIA were developed and testing was performed in 150 anti-mitochondrial antibody (AMA) positive, 20 AMA-PBCs and 130 controls. RESULTS Thirty-five (23%) of 150 AMA+ PBCs (18 anti-MND+) were anti-PML+ (12%) or anti-Sp100+ (20%), 10 being anti-PML+/Sp100+, 5 single anti-PML+ and 20 single anti-Sp100+. Six (30%, 5 anti-MND+) AMA-PBCs were anti-PML+ or Sp100+. Only 2 (1.7%) pathological controls were anti-PML+ and/or anti-Sp100+. Levels of anti-PML correlated with those of anti-Sp100 (R=0.64, p<0.0001). The autoantibody profile largely remained unchanged over a 10year-follow up (52 patients, 352 samples). Anti-PML, Sp100 or MND-reactive PBCs were younger and had longer disease duration than the seronegative (p=0.06, for both). Anti-Sp100 levels correlated with the Mayo risk score (r=0.63, p=0.01). Anti-PML+/Sp100+ patients had more advanced disease compared to patients negative for anti-PML/Sp100 (p=0.04). CONCLUSION The new line immunoassay offers a robust and accurate method for the detection of clinically-relevant PBC-specific anti-MND antibodies.

Autoantibodies in Autoimmune Pancreatitis

Autoimmune pancreatitis (AIP) was first used to describe cases of pancreatitis with narrowing of the pancreatic duct, enlargement of the pancreas, hyper-γ-globulinaemia, and antinuclear antibody (ANA) positivity serologically. The main differential diagnosis, is pancreatic cancer, which can be ruled out through radiological, serological, and histological investigations. The targets of ANA in patients with autoimmune pancreatitis do not appear to be similar to those found in other rheumatological diseases, as dsDNA, SS-A, and SS-B are not frequently recognized by AIP-related ANA. Other disease-specific autoantibodies, such as, antimitochondrial, antineutrophil cytoplasmic antibodies or diabetes-specific autoantibodies are virtually absent. Further studies have focused on the identification of pancreas-specific autoantigens and reported significant reactivity to lactoferrin, carbonic anhydrase, pancreas secretory trypsin inhibitor, amylase-alpha, heat-shock protein, and plasminogen-binding protein. This paper discusses the findings of these investigations and their relevance to the diagnosis, management, and pathogenesis of autoimmune pancreatitis.

Anti-Helicobacter pylori antibody responses specific for VacA do not trigger primary biliary cirrhosis-specific antimitochondrial antibodies

Andreas Koutsoumpas, Maria Mytilinaiou, Dimitrios Polymeros, George N. Dalekos and Dimitrios Petrou Bogdanos, Liver Immunopathology, Institute of Liver Studies, King’s College London School of Medicine, King’s College Hospital, London, UK, 2nd Department of Medicine, Hepatogastroenterology Unit, Attikon University General Hospital, Athens, Department of Medicine, Academic Liver Unit and Research Laboratory of Internal Medicine, Medical School, University of Thessaly and Research Group of Investigational Medicine, Institute of Biomedical Research and Technology, Centre for Research and Technology – Thessaly (CE.RE.TE.TH), Larissa, Greece

Diagnostic and clinical significance of Crohn's disease-specific anti-MZGP2 pancreatic antibodies by a novel ELISA

BACKGROUND We developed a new IgA and IgG anti-MZGP2 antibody ELISAs based on recombinant isoform-4 of human zymogen granule protein-2 (GP2), which is the major autoantigen of Crohns disease (CrD)-specific pancreatic autoantibodies and assessed their clinical relevance in the largest inflammatory bowel disease (IBD) cohort tested to date. METHODS 832 sera were studied, including 617 consecutive IBD patients from 323 CrD and 294 ulcerative colitis (UC) follow-up in a tertiary centre, and 112 pathological and 103 normal controls. RESULTS Sensitivity of IgA anti-MZGP2 for CrD in the IBD population was 15% and specificity was 98% (95, 99), while the sensitivity and specificity of IgG anti-MZGP2 were 27% and 97%. IgA and IgG anti-MZGP2 combined testing led to a sensitivity of 31% and a specificity of 96%. Positivity for either ASCA (IgA or IgG) or anti-MZGP2 (IgA or IgG) showed a sensitivity of 75% (70, 80) and a specificity of 84% (79, 89). IgA anti-MZGP2 antibodies were more prevalent in CrD patients with early disease onset (p=0.011). Also, anti-MZGP2 positive patients more frequently had extensive disease with ileal involvement. Patients with longer disease duration were more likely to have IgG anti-MZGP2 antibodies. CONCLUSIONS Our novel ELISA confirms the high specificity of anti-MZGP2 antibodies for CrD and their association with disease severity phenotypes.

Peculiar antibody reactivity to human connexin 37 and its microbial mimics in patients with Crohn's disease

BACKGROUND/AIMS We found that pooled Crohns disease (CD) sera strongly react with a human gap-junction connexin 37 (Cx37) peptide and tested for anti-Cx37 antibody reactivity in sera from CD patients and controls. We also investigated whether peptide-recognition is due to Cx37/microbial molecular mimicry. METHODS The PSI-BLAST program was used for Cx37(121-135)/microbial alignment. Reactivity to biotinylated human Cx37(121-135) and its microbial mimics was determined by ELISA using sera from 44 CD, 30 ulcerative colitis and 28 healthy individuals. RESULTS Anti-Cx37(121-135) reactivity (1/200 dilution) was present in 30/44 (68%) CD cases and persisted at 1/1000 dilution. Database search shows that Cx37(121-135) contains the -ALTAV- motif which is cross-recognized by diabetes-specific phogrin and enteroviral immunity. Testing of 9 Cx37(121-135)-microbial mimics revealed 57-68% reactivity against human enterovirus C, Lactococcus lactis, coxsackie virus A24 and B4. Anti-Cx37(121-135) was inhibited by itself or the microbial mimics. No reactivity was found against the poliovirus, rubella, and Mycobacterium tuberculosis mimics, or the beta cell phogrin autoantigen. Microbial/Cx37 reactivity was not able to differentiate CD patients from UC or healthy controls, in terms of overall prevalence and antibody titres, but microbial mimics were unable to inhibit reactivity to human Cx37 in the majority of the controls. CONCLUSIONS Sera from CD patients react with connexin 37 and cross-react with specific Cx37-mimicking enteroviral peptides. Microbial/self reactivity can be seen in UC and healthy controls. The lack of responses to other Cx37(121-135) microbial mimics and the inability of the reactive microbes to inhibit reactivity to self is intriguing and warrants further investigation.