Normal inflammasome activation and low production of IL-23 by monocyte-derived macrophages from subjects with a history of reactive arthritis.

OBJECTIVES: The pathogenesis of reactive arthritis (ReA) is incompletely understood but may involve aberration(s) in the host's innate immune response towards infecting microbes. We therefore studied the production of interleukin (IL)-1ß, a marker of inflammasome activation, and of IL-6, IL-12, IL-23, and tumour necrosis factor (TNF)-α, promoters of T-cell differentiation, by peripheral blood mononuclear cells (PBMNs) and monocyte-derived macrophages from healthy subjects with a history of ReA. METHOD: The study included 10 human leucocyte antigen (HLA)-B27-positive healthy subjects with previous ReA triggered by Yersinia enterocolitica O:3 infection and 20 healthy reference subjects, of whom 10 were HLA-B27 positive. PBMNs and macrophages were cultured for 18 h with bacterial lipopolysaccharide (LPS), muramyl dipeptide (MDP), Yersinia, or their appropriate combinations. PBMNs were also stimulated with monosodium urate (MSU) crystals. Cytokine levels were measured using an enzyme-linked immunosorbent assay (ELISA) and the Luminex system. RESULTS: IL-1ß secretion was similar from cells of the ReA group and from the HLA-B27-positive and -negative reference groups. TNF-α production from macrophages upon co-stimulation of LPS and MDP increased in the order ReA group < HLA-B27-positive reference group < HLA-B27-negative reference group (p for a trend = 0.027). Similarly, Yersinia-induced TNF-α and IL-23 production increased in the same order (p for trend for TNF-α = 0.036; p for trend for IL-23 = 0.026). CONCLUSIONS: PBMNs and macrophages from healthy subjects with previous ReA show normal inflammasome activation and low TNF-α and IL-23 production. This low cytokine production may impair bacterial elimination and thereby contribute to the triggering of ReA.

Association study of FUT2 (rs601338) with celiac disease and inflammatory bowel disease in the Finnish population.

Homozygosity for a nonsense mutation in the fucosyltransferase 2 (FUT2) gene (rs601338G>A) leads to the absence of ABH blood groups (FUT2 non-secretor status) in body fluids. As the secretor status has been shown to be a major determinant for the gut microbial spectrum, assumed to be important in the gut immune homeostasis, we studied the association of rs601338-FUT2 with celiac disease (CelD) and inflammatory bowel disease (IBD) in the Finnish population. Rs601338 was genotyped in CelD (n = 909), dermatitis herpetiformis (DH) (n = 116), ulcerative colitis (UC) (n = 496) and Crohn's disease (CD) (n = 280) patients and healthy controls (n = 2738). CelD showed significant genotypic [P = 0.0074, odds ratio (OR): 1.28] and recessive (P = 0.015, OR: 1.28) association with the rs601338-AA genotype. This was also found in the combined CelD+DH dataset (genotype association: P = 0.0060, OR: 1.28; recessive association: P < 0.011, OR: 1.28). The A allele of rs601338 showed nominal association with dominant protection from UC (P = 0.044, OR: 0.82) and UC+CD (P = 0.035, OR: 0.84). The frequency of non-secretors (rs601338-GG) in controls, CelD, DH, UC and CD datasets was 14.7%, 18%, 18.1%, 14.3% and 16.1%, respectively. No association was evident in the DH or CD datasets alone. In conclusion, FUT2 non-secretor status is associated with CelD susceptibility and FUT2 secretor status may also play a role in IBD in the Finnish population.

Association of celiac disease genes with inflammatory bowel disease in Finnish and Swedish patients.

Some genetic loci may affect susceptibility to multiple immune system-related diseases. In the current study, we investigated whether the known susceptibility loci for celiac disease (CelD) also associate with Crohn's disease (CD) and/or ulcerative colitis (UC), the two main forms of inflammatory bowel disease (IBD), in Finnish patients. A total of 45 genetic markers were genotyped in a Finnish data set comprising 699 IBD patients and 2482 controls. Single-marker association with IBD and its subphenotypes was tested. A meta-analysis with a Swedish UC data set was also performed. A total of 12 single-nucleotide polymorphisms associated with CD and/or UC (P<0.05). In the subphenotype analysis, rs6974491-ELMO1 (P=0.0002, odds ratio (OR): 2.20) and rs2298428-UBE2L3 (P=5.44 × 10(-5), OR: 2.59) associated with pediatric UC and CD, respectively. In the meta-analysis, rs4819388-ICOSLG (P=0.00042, OR: 0.79) associated with UC. In the subphenotype meta-analysis, rs1738074-TAGAP (P=7.40 × 10(-5), OR: 0.61), rs6974491-ELMO1 (P=0.00052, OR: 1.73) and rs4819388-ICOSLG (P=0.00019, OR: 0.75) associated with familial UC, pediatric UC and sporadic UC, respectively. Multiple CelD risk loci also confer susceptibility for CD and/or UC in the Finnish and Swedish populations. Certain genetic risk variants may furthermore predispose an individual for developing a particular disease phenotype.

Correlation of faecal calprotectin and lactoferrin with an endoscopic score for Crohn's disease and histological findings.

BACKGROUND: Faecal calprotectin and lactoferrin increasingly serve as surrogate markers of disease activity in IBD. Data on the correlation of these markers with simple endoscopic score for Crohn's disease (SES-CD) and with histological findings are as yet limited. Aim To study the correlation of faecal calprotectin and lactoferrin with SES-CD and histology. METHODS: During 87 consecutive ileocolonoscopies, SES-CD was calculated and biopsy specimens were obtained from the ileum, colon and rectum. Faecal calprotectin and lactoferrin were measured. RESULTS: In ileocolonic or colonic disease, both faecal calprotectin and lactoferrin correlated significantly with colon SES-CD (P < 0.001) and colon histology (P < 0.001). In patients with normal calprotectin or lactoferrin levels, endoscopic and histology scores were significantly lower than in those with elevated concentrations (P < 0.001). In ileal CD, ileal SES-CD correlated with histology (P < 0.001), but not with faecal calprotectin (P = 0.161) or lactoferrin (P = 0.448). CONCLUSION: In ileocolonic and colonic disease, endoscopic score SES-CD and histological findings correlated significantly with faecal calprotectin and lactoferrin. A normal faecal-marker concentration was a reliable surrogate marker for endoscopically and histologically inactive CD. Ileal endoscopic score and histological findings failed, however, to correlate with faecal markers.

A novel modification of a flow cytometric assay of phosphorylated STAT1 in whole blood monocytes for immunomonitoring of patients on IFN alpha regimen.

We explored whether episodes stimulating leucocytes in vivo could be tracked from whole blood samples by monitoring activation of STAT1 by flow cytometry. The method was tested in hepatitis C patients (n = 9) that were on interferon (IFN)alpha regimen. CD14+ monocytes responded strongly to IFNalpha/gamma being sensitive indicators for recent immune activation. At 45 min after s.c. IFNalpha 91% of monocytes were phosphorylated STAT1+. The frequency of responding cells decreased to a base level within 6 h. Monocytes, however, had a long-term deficient phosphorylated STAT1 response to IFNalphain vitro that in patients on standard IFNalpha regimen lasted for 48 h. In patients on pegylated IFNalpha the phosphorylated STAT1 response was completely absent. We conclude that whole blood analysis of STAT1 activation by flow cytometry is applicable to monitor immune cells in patient material.

CARD15 frameshift mutation in patients with CROHN disease is associated with immune dysregulation.

BACKGROUND: Mutations in the caspase-activating recruitment domain 15 (CARD15) gene are associated with Crohn disease (CD). CARD15 is an intracellular receptor for bacterial lipopolysaccharides (LPS). LPS-induced activation of transfectants containing the frameshift mutation (1007fs) of CARD15 is impaired. The aim of this study was to investigate whether the presence of CARD15 1007fs affects activation of CD patients' own cells. Patients (4 homozygotes, 6 heterozygotes, and 6 wild-type) were matched according to clinical picture and medication. METHODS: Immune inflammatory status was evaluated by measuring monocyte HLA-DR and CD11b densities and the proportion of CD14dimCD16+ monocytes, and was found to be comparable in the three groups. Blood mononuclear cells were cultured overnight in serum-free medium alone, or the medium supplemented with LPS (0.1-10.0 ng/mL), a combination of IFN-gamma (100 IU/mL) and granulocyte-macrophage colony stimulating factor (GM-CSF) (5 ng/mL), or both. TNF and IL-10 levels in the culture supernatant were determined. RESULTS: LPS 0.1 or 1.0 ng/mL alone did not increase TNF levels. IFN-gamma/GM-CSF induced TNF release, and co-culture with LPS 1.0 or 10.0 ng/mL was strongly synergistic. CARD15 1007fs mutation was linked in a gene-dose-dependent manner to low TNF release induced by IFN-gamma/GM-CSF (P value for linear trend = 0.001). The degree of synergism in co-culture was normal or high, suggesting that 1007fs did not depress responses to LPS. IL-10 levels were not related to CARD15 1007fs. CONCLUSIONS: In CD patients, CARD15 1007fs is associated in a gene-dose-dependent manner to low mononuclear cell TNF release by IFN-gamma/GM-CSF but does not impair TNF release by LPS. This type of immune dysregulation may influence susceptibility to and/or phenotype of CD.

CARD15/NOD2 gene variants are associated with familially occurring and complicated forms of Crohn's disease.

BACKGROUND: Variants of the caspase activating recruitment domain 15/nucleotide oligomerisation domain 2 (CARD15/NOD2) gene have been associated with susceptibility to Crohn's disease (CD). AIM: Our aim was to evaluate the allele frequencies of the CARD15 variants R702W, G908R, and 1007fs in Finnish inflammatory bowel disease (IBD) patients and to search for possible associations between CARD15 variants and occurrence of familial forms of IBD or complicated forms of CD. PATIENTS AND METHODS: We investigated 198 sporadic CD patients, 46 probands with familial CD, 27 CD probands from mixed IBD families, 99 unrelated patients with ulcerative colitis (UC), and 300 control individuals for the occurrence of the CARD15 gene variants R702W, G908R, and 1007fs. RESULTS: In CD patients, the allele frequencies for the rare variants of these polymorphisms were 3.3%, 0.6%, and 4.8% (total 8.7%), and the corresponding frequencies in healthy controls were 1.8%, 0%, and 1.7% (total 3.5%) (8.7% v 3.5%; p<0.01). In UC patients allele frequencies were comparable with those in controls. The frequency of the 1007fs polymorphism variant allele was significantly higher among all CD patients than in controls (4.8% v 1.7%; p<0.01) but there was no significant difference in allele frequencies between the CD and UC groups. The 1007fs allele frequency was higher in familial CD than in non-familial cases with CD (10.9% v 3.5%; p<0.01). There were no significant differences in the allele frequencies of the R702W and G908R polymorphisms between CD patients, UC patients, and controls. We found that 15.5% of CD patients, 9.1% of UC patients, and 6.7% of controls carried at least one of the CARD15 variants. In CD patients carrying at least one of the three NOD2 variants, the ileum was affected more often than in non-carrier CD patients (90% v 73%; p<0.05), they had stricturing or penetrating disease more often than non-carriers (88% v 56%; p<0.01), and they had an increased need for bowel surgery. CONCLUSIONS: The frequency of NOD2 gene variants was lower in genetically homogenous Finns than in other populations. The 1007fs variant was associated with CD. The occurrence of CARD15 variants predicted ileal location as well as stricturing and penetrating forms of CD.

Familial and sporadic inflammatory bowel disease: comparison of clinical features and serological markers in a genetically homogeneous population.

BACKGROUND: The familial occurrence of inflammatory bowel disease (IBD) and the clinical features of familial and sporadic IBD in the genetically homogeneous Finnish population are evaluated. METHODS: 257 patients with Crohn disease (CD) and 436 with ulcerative colitis (UC) participated in the study. They were asked whether IBD was present (familial IBD) or absent (sporadic IBD) in their first-degree relatives. Data on the clinical course of the disease were collected from the patient records. Antibodies to Saccharomyces cerevisiae (ASCA) and anti-neutrophil cytoplasmic antibodies (ANCA) were determined from serum samples. RESULTS: Affected first-degree relatives were found in 15.6% of patients with CD and in 13.8% of patients with UC. In familial cases CD was more often located in the ileum (38% versus 21%) and less often in the ileocolon (35% versus 50%) (P< 0.05) than in sporadic cases. A greater percentage of CD patients than UC patients were smokers (47% versus 13%; P < 0.01). An elevated level of IgA and/or IgG antibodies for ASCA was found more often in CD patients than in UC patients (59% versus 14%; P < 0.01), while pANCA were found more often in UC than in CD patients (48% versus 12%; P < 0.01). The combination of pANCA-ASCA+ yielded a sensitivity, specificity and positive predictive value of 48%, 92% and 90%, respectively, for CD, and the combination of pANCA + ASCA- of 55%, 94% and 90%, respectively, for UC. CONCLUSIONS: The percentage of familial IBD cases in Finland is comparable to that reported elsewhere in Europe. No important clinical differences between patients with familial and sporadic forms of the disease were found. ASCA is associated with both familial and sporadic CD and pANCA with UC, but low sensitivity diminishes their value as a serological marker of IBD or as a differential diagnostic test between CD and UC.

Genetic analysis in Finnish families with inflammatory bowel disease supports linkage to chromosome 3p21.

In inflammatory bowel diseases (IBD), certain chromosomal candidate loci have been repeatedly identified by independent studies in different populations. To investigate the contribution of the loci on chromosomes 1, 3, 7, 12, 14, and 16 to the susceptibility of IBD in Finnish population, where the predominant feature is the excess of ulcerative colitis (UC) families compared to Crohn's disease (CD) families, we carried out linkage analyses using 93 Finnish, multiply-affected IBD families. We observed nominal evidence for linkage to chromosome 3p21, consistent with earlier reports. The lod scores peaked at D3S2432, with a maximum two-point lod score of 1.68 (P=0.0027). In addition, we studied whether risk of IBD is associated with functional variants of two positional candidate genes; the chemokine receptor CCR5 gene on chromosome 3p21 and the interleukin-4 receptor alpha-subunit gene (IL4RA) on chromosome 16. We did not find any significant correlation between a 32-bp deletion variant of CCR5 or a single nucleotide change A1902G (Gln576Arg) of IL4RA, and IBD phenotypes, with the exception that in the UC group homozygosity for the G1902 allele of IL4RA was less frequent (0.019 vs 0.049, P=0.038). In conclusion, our study, carried out in a genetically homogenous population, suggests that chromosome 3 may contain a susceptibility gene for IBD.

Comparison of iohexol and lactulose-mannitol tests as markers of disease activity in patients with inflammatory bowel disease.

Increased intestinal permeability has been proposed as one aetiological factor for inflammatory bowel diseases (IBD). We have previously found that intestinal permeability of a water-soluble contrast medium, iohexol, correlates with disease activity. The objective was to compare the iohexol test with the lactulose-mannitol ratio, which is a more extensively studied permeability marker, in patients with active IBD. Urinary excretion of iohexol was compared to the lactulose-mannitol ratio in 22 patients with an exacerbation of IBD and in 10 healthy controls. Median intestinal absorption of iohexol was 0.64% (range 0.13-3.8%) in the 22 patients and 0.3% (range 0.15-0.54%) in the controls (p = 0.016), whereas the median lactulose-mannitol ratio was 0.037 (range 0.01-0.260) in patients and 0.03 (range 0.004-0.063) in controls (N.S.). Correlation between urinary excretion of iohexol and lactulose-mannitol ratio was positive (R = +0.41, p = 0.018). The urinary excretion of iohexol correlated positively with endoscopic disease activity (R = +0.74, p < 0.001) and the modified Harvey-Bradshaw index (R = +0.44, p = 0.04). The lactulose-mannitol ratio correlated positively with endoscopic disease activity (R = +0.44, p = 0.05), but correlations with clinical index or c-reactive protein were poor. In conclusion, the iohexol test is a superior activity marker compared to the lactulose-mannitol ratio which probably reflects, instead, some pathogenic property of IBD.

Arg506Gln factor V mutation and Val34Leu factor XIII polymorphism in Finnish patients with inflammatory bowel disease.

BACKGROUND: Earlier studies have indicated that inflammatory bowel disease (IBD), including Crohn's disease (CD) and ulcerative colitis (UC), is associated with thromboembolic complications, whereas inherited disorders of coagulation occur less often than expected in IBD patients. The point mutation Arg506Gln of factor V (Factor V Leiden), resulting in resistance to activated protein C, is the commonest inherited form of thrombophilia. Alterations in circulating levels of factor XIII (FXIII) have been reported among IBD patients. We investigated whether Factor V Leiden or inherited Val34Leu polymorphism of FXIII would associate with IBD or its clinical outcome. METHODS: Factor V Leiden mutation and FXIII Val34Leu polymorphism were assayed in 328 unrelated Finnish patients with UC and 235 patients with CD by solid-phase minisequencing techniques. The control groups comprised 142 apparently healthy Finnish subjects and 120 Finnish blood donors. RESULTS: The frequency of Factor V Leiden mutation among IBD patients (4.5%) was not significantly different from that in subjects living in the same area (2.1%). No significant differences could be observed in the FXIII Val34Leu polymorphism allele frequencies between patients and controls. Clinical features of IBD, including the disease extent, requirement for immunosuppressive drugs, and occurrence of complications, seemed to be independent of these two clotting factor variants analyzed. CONCLUSIONS: Our data do not support an aetiologic or disease-modifying role for the factor V mutation or factor XIII Val34Leu polymorphism in IBD.

Long-term treatment of ulcerative colitis with ciprofloxacin: a prospective, double-blind, placebo-controlled study.

BACKGROUND & AIMS: Although bacterial bowel flora may be one of the contributing factors in the pathogenesis of chronic mucosal inflammation, antibiotic treatment has no established role in ulcerative colitis. The aim of the study was to evaluate the role of ciprofloxacin in the induction and maintenance of remission in ulcerative colitis in patients responding poorly to conventional therapy with steroids and mesalamine. METHODS: Ciprofloxacin (n = 38; 500-750 mg twice a day) or placebo (n = 45) was administered for 6 months in a double-blind, randomized study with a high but decreasing dose of prednisone and maintenance treatment with mesalamine including follow-up for the next 6 months. Clinical assessment and colonoscopic evaluation were performed at 0, 3, 6, and 12 months. Treatment failure, the primary end point, was defined as both symptomatic and endoscopic failure to respond. RESULTS: During the first 6 months, the treatment-failure rate was 21% in the ciprofloxacin-treated group and 44% in the placebo group (P = 0.02). Endoscopic and histological findings were used as secondary end points and showed better results in the ciprofloxacin group at 3 months but not at 6 months. CONCLUSIONS: Addition of a 6-month ciprofloxacin treatment for ulcerative colitis improved the results of conventional therapy with mesalamine and prednisone.

Association of gut inflammation with increased serum IgA class Klebsiella antibody concentrations in patients with axial ankylosing spondylitis (AS): implication for different aetiopathogenetic mechanisms for axial and peripheral AS?

OBJECTIVES: A role for Klebsiella pneumoniae in ankylosing spondylitis (AS) has been suggested because faecal carriage of Klebsiella and serum Klebsiella specific antibodies may be increased in this disease. This study has extended the earlier findings by comparing Klebsiella specific serum IgA class antibodies with inflammatory changes in the gut. METHODS: IgA antibodies to K pneumoniae, Escherichia coli, and Proteus mirabilis in serum samples of 25 patients with AS, of eight control patients, and of 100 healthy blood donors were measured by enzyme immunoassay. Gut inflammation of the patients was studied by ileocolonoscopy. RESULTS: Increased IgA antibody concentrations to K pneumoniae associated with gut inflammation in patients with axial form of AS. Such association was not seen in patients with peripheral form of AS. CONCLUSIONS: These findings may provide further evidence for the role of K pneumoniae in the pathogenesis of AS. However, at least some of the patients with axial AS without gut inflammation, as well as patients with peripheral AS did not have increased K pneumoniae antibody concentrations, which may be regarded as evidence against the direct role of K pneumoniae in the pathogenesis. The aetiopathogenetic mechanisms in the axial and peripheral form of AS may be different.

Chronic diarrhea due to a single strain of Aeromonas caviae.

Over a period of 17 months, Aeromonas caviae was cultured 15 times as the sole enteropathogen from the feces of a man who had developed chronic diarrhea after traveling to Turkey. Determination of rRNA gene restriction patterns confirmed that the seven isolates of Aeromonas caviae studied were identical (hybridization group [HG]4). After therapy with ciprofloxacin for four weeks, the patient was culture negative for the original isolate, and six months later a novel strain of Aeromonas media with a different ribopattern (HG 5A) was isolated from the feces of the patient. The patient responded again, both clinically and bacteriologically, to a four-week course of ciprofloxacin and has remained asymptomatic since then.

Budesonide enema in active haemorrhagic proctitis--a controlled trial against hydrocortisone foam enema.

BACKGROUND: The aim was to compare budesonide enema, 2 mg/100 mL (Entocort) and hydrocortisone acetate foam enema, 125 mg (Colifoam) in patients with active haemorrhagic proctitis. METHODS: The trial was a controlled, randomized, investigator-blind study with two parallel groups. Endoscopy, histology and diary cards were used to assess the response to therapy. Safety was assessed by laboratory tests and adverse event recording. RESULTS: Seventy-two patients were included. Investigations were made before treatment and after 2 and 4 weeks. Both treatment groups showed statistically significant improvement in endoscopic scores but significant differences between the groups were not found. In the hydrocortisone group, plasma cortisol was significantly lowered after 4 weeks compared with budesonide. Bowel habits and quality of life variables did not differ between the treatments. The recorded adverse events were mild or moderate and may have been due to the proctitis. CONCLUSIONS: These results suggest that budesonide enema is as effective as hydrocortisone foam enema, but without the potential for side-effects associated with suppression of plasma cortisol.