Anti-tumour necrosis factor-α antibodies and B cell homeostasis in human inflammatory bowel diseases.

BACKGROUND: The expression of CD70 on T cells is greatly enhanced by antigen-presenting cell (APC)-associated signals, such as tumour necrosis factor(TNF)-α, which is constitutionally high in patients with inflammatory bowel disease (IBD). Experimentally, the chronic activation of CD27 as a result of the constitutive expression of CD70 leads to the demise of B cells in bone marrow (BM) and the secondary lymphoid organs. The aim of this study was to assess the number and phenotype of circulating B cell in untreated IBD patients and their counterparts treated with biological anti-TNF drugs. METHODS: The study involved 13 untreated IBD patients, 36 IBD patients treated with biological drugs, and 10 healthy controls. The B cell phenotypes were assessed by means of flow cytometry using monoclonal antibodies specific for CD20, CD19, CD3, CD27 and CD43. In order to evaluate B cell development in bone marrow and peripheral B cell activation, we identified four B cell subsets: hematogones (HBs: CD20+19+3-27-43+), memory B cells (MBs: CD20+19+3-27+43-), pre-plasmablasts (PPBs: CD20+19+3-27+43+), and plasmablasts (PBs: CD20-19+3-27+43+). RESULTS: The total number of B cells in the untreated patients was three times lower than that in the patients treated with biological drug (p<0.001), and half that in the healthy controls (p=0.03). The between-group differences (including the healthy donors) were statistically significant in the case of HBs and MBs, but not in the case of PPBs and PBs. Only one treated patient showed a transiently large increase in PPBs. There were statistically significant differences in all of the parameters between the untreated patients and those receiving biological therapy, and in some cases between the untreated patients and healthy controls, but never between the controls and the treated patients. Four non-responders to anti-TNF therapy had a smaller number of total circulating B cells than the untreated patients. CONCLUSIONS: Anti-TNF drugs disinhibit B cell production in IBD patients, but maintain the constant homeostasis of circulating B cells. The presence of individual variations may allow the activity of anti-TNF drugs to be monitored by studying B cell subgroups.

Significance of serum Il-9 levels in inflammatory bowel disease.

IL-9, which may be an inflammatory or regulatory cytokine, can be experimentally produced in a Th17 or modified Th2 context in the presence of T cell receptor (TCR) stimulation. The primary aim of this study was to measure serum IL-9 levels in patients with inflammatory bowel disease (IBD), and evaluate their relationships with the patients' clinical characteristics. The secondary aim was to determine the levels of interferon-γ (IFN (interferon)-γ), Th2 cytokines (IL-4, IL-5 and IL-13), and IL-6 in order to clarify the context of detectable peripheral cytokines in which IL-9 is produced.Venous blood samples of 43 IBD patients (20 with Crohn's disease [CD] and 23 with ulcerative colitis [UC]) were analysed by means of quantitative enzyme-linked immunosorbent assays using purified anti-human IL-4, IL-5, IL-13, IFN-γ, IL-9 and IL-6 antibodies, and the laboratory findings were statistically correlated with their clinical expression.None of the patients showed the peripheral presence of IL-4, IL-5 and IL-13. Forty (93%) were positive for IFN-γ, thus confirming the presence of Th1 in both UC and CD, and IFN-γ levels correlated with disease activity (P = 0.045). Eighteen patients (41%) were positive for IL-9, which was associated with a severe prognosis (P <0.001), and 72.2% of the IL-9-positive patients were also IL-6 positive. There was a significant correlation between disease severity and IL-9 in the CD patients (P <0.001), but not in the UC patients (P = 0.1).Our findings confirm the presence of common Th1 cytokines in UC and CD. However the IL-9 positivity indicates the presence of an alternative population of T cells that respond to antigen stimulation and condition the prognosis of IBD. The fact that the same serum IL-9 levels were differentially associated with clinical measures of CD and UC activity suggest that the same cytokine can be produced in different contexts.

Morphological distribution of µ chains and cd15 receptors in colorectal polyp and adenocarcinoma specimens.

BACKGROUND: We have recently investigated the localisation of immunoglobulin-producing cells (IPCs) in inflamed intestinal tissue samples from patients with inflammatory bowel disease (IBD), and identified two main patterns of B lymphocyte infiltration: one characterised by the moderate strong stromal localisation of small B1 cell-like IgM+/CD79+/CD20-/CD21-/CD23-/CD5 ± IPCs, and the other by the peri-glandular localisation of IPCs with irregular nuclei that had surface markers specific for a B cell subset (IgM and CD79), but quantitative differences in their λ and κ chains. The same patients were also tested for CD15+ receptors, which were localised on inflammatory cell surfaces or in the crypts of the intestinal epithelium. CD15+ receptor distribution in inflamed tissues was limited to the cell structures. The aim of the study was to analyse variations in IPCs and CD15+ cell morphology or distribution in bowel biopsy specimens taken from patients with pre-malignant polyps or adenocarcinomas. METHODS: IPCs were analysed by means of immunofluorescence using polyclonal goat anti-human µ chains. The pre-malignant polyp specimens were tested for B cell surface phenotype λ and κ chains, CD79, CD20, CD21 and CD23 using an immunoperoxidase method. CD15+ cells were evaluated using the immunoperoxidase method and monoclonal anti-CD15 IgM. RESULTS: The study involved 14 patients (four with pre-malignant polyps and 10 with colorectal adenocarcinomas). The distribution of µ chains and CD15 markers varied in all of the biopsies, but delineated normal cell structures in the pre-malignant polyp specimens. B cell surface phenotype analysis of µ chain-positive cells identified a subset of CD79+/CD20-/CD21-/CD23- IPCs. The IPCs in certain areas showed the sporadic disintegration of inflammatory cell membranes or the accumulation of fluorescence in individual cells. IPC membrane disintegration was particularly marked in all of the adenocarcinoma samples, in which the CD15 markers also showed epithelial cell involvement. Furthermore, six of the ten adenocarcinoma samples had atypical and reorganised membranes that expressed an excess of both receptors and isolated small portions of tissue within the tumour. CONCLUSION: The findings of this preliminary morphological study suggest the presence of membrane disintegration and remodelling mechanisms in the tumours. The newly-formed membranes expressed high concentrations of inflammatory cell receptors that can confer adhesive properties.

Unusual B cell morphology in inflammatory bowel disease.

B lymphocytes express various different types of surface immunoglobulins that are largely unrelated to other hematological lines, although some reports have described a relationship between malignant B cells and other cells such as macrophages. Multiple genes of hematopoietic lineage, including transcription factors, are co-expressed in hematopoietic stem cells and progenitors, a phenomenon referred to as "lineage priming". Changes in the expression levels and timing of transcription factors can induce the lineage conversion of committed cells, which indicates that the regulation of transcription factors might be particularly critical for maintaining hierarchical hematopoietic development. The aim of this study was to evaluate the surface markers of particular IgM-positive and irregularly nucleated cells detected in patients with inflammatory bowel disease (IBD), and to assess their association with diagnosis and inflammatory cell recruitment. Small intestine, colon and rectal biopsy specimens of 96 IBD patients were studied. Immunoglobulin-producing cells (IPCs) were analyzed by means of immunofluorescence using polyclonal rabbit anti-human Ig and goat anti-human IgM. The specimens positive for B cells with irregular nuclei were assessed using monoclonal antibodies specific for CD79, and λ and κ chains in order to confirm their B cell nature. CD15+ cells, an important marker of inflammatory cell recruitment, were also evaluated. Statistical correlations were sought between the histological findings and clinical expression. 34 (35.4%) of the 96 patients (64 with ulcerative colitis and 32 with Crohn's disease) presented a periglandial localization of IPCs with irregular nuclei, which showed surface markers specific for the B cell subset, such as IgM and CD79, but quantitative differences in λ and κ chains. These specimens also contained CD15-positive cells, which are usually absent in healthy controls. The quantitative aspects and localization of the CD15-positive cells correlated with the distribution of the IPCs with irregular nuclei. IPCs with irregular nuclei were significantly more frequent in those patients with Crohn's disease than in those with ulcerative colitis (p<0.001). The finding of a subpopulation of cells that simultaneously showed irregular nuclei and B cell markers, such as functional surface IgM, in patients with IBD suggests that an unusual subgroup of B cells that correlates with CD15 expression and a diagnosis of Crohn's disease may be observed in the inflammatory process.

The use of methotrexate for treatment of inflammatory bowel disease in clinical practice.

BACKGROUND: Methotrexate is considered a treatment for Crohn's disease, whilst few data in ulcerative colitis are available. AIM: To evaluate frequency, indications, efficacy and safety of methotrexate in inflammatory bowel disease patients. METHODS: 5420 case histories were reviewed. RESULTS: Methotrexate was prescribed to 112 patients (2.1%; 89 Crohn's disease, 23 ulcerative colitis). It was the first-line immunosuppressive option in 32 (28.6%), it was an alternative drug due to toxicity or failure of thiopurines in 80 (71.4%). Steroid-dependence represented the main indication both when it was used as first (13/32, 40.6%) and second option (41/80, 51.2%). Efficacy was considered optimal in 39/112 (34.8%), partial in 29/112 (25.9%), absent in 22/112 (19.6%), not assessable in 22/112 (19.6%). Side effects happened in 49 out of 112 patients (43.7%) (39 Crohn's disease, 10 ulcerative colitis), leading to drug discontinuation in 38 (33.9%). The occurrence of side effects was approximately fivefold higher in patients who did not receive folic acid (14/19, 73.7%) than in those who did (35/93, 37.6%): odds ratio 4.64, 95% confidence interval 1.54-14.00; p=0.005. CONCLUSIONS: The use of methotrexate appears to be negligible in clinical practice. However, our results suggest that, if appropriately used, methotrexate could be more widely administered to inflammatory bowel disease patients with complicated disease.

Clinical and laboratory aspects of Ro/SSA-52 autoantibodies.

Anti-Ro/SSA antibodies, which were described for the first time in systemic lupus erythematosus (SLE) and Sjögren's syndrome (SS), are the most prevalent extractable nuclear antigen (ENA) specificity identified in laboratories. Two types of anti-Ro/SSA antibodies have been described, anti-SSA-52 kDa (aSSA52) and anti-SSA-60 kDa (aSSA60), each specific to different antigens. Anti-Ro/SSA52 autoantibodies are more frequent than other autoantibodies possibly because of the antigen's accessible and ubiquitous nature. The sites involved and the symptoms associated with these autoantibodies depend on the antigen's structural variability. Isolated congenital complete atrioventricular block (CAVB) shows a close association with maternal anti-Ro/SSA and anti-La/SSB antibodies; the highest relative risks of CAVB are seen in offspring of mothers with antibodies against 52-kDa Ro and 48-kDa La proteins. Anti-Ro/SSA52 antibodies have little impact on adult rheumatic autoimmune diseases or adult cardiac arrhythmias, but the course of autoimmune liver diseases is greatly worsened by their presence, and solid tumours tend to relapse. Their diagnostic role in rheumatic diseases is controversial, although a significant association between isolated anti-Ro/SSA52-kDa positivity and myositis and to a lesser extent with systemic sclerosis (SSc) has been described. However, the majority of the specific diagnosis is mostly based on the simultaneous presence of other autoantibodies that seems diagnostically more relevant.

B lymphocyte intestinal homing in inflammatory bowel disease.

BACKGROUND: Inflammatory bowel disease (IBD) is thought to be due to an abnormal interaction between the host immune system and commensal microflora. Within the intestinal immune system, B cells produce physiologically natural antibodies but pathologically atypical anti-neutrophil antibodies (xANCAs) are frequently observed in patients with IBD. The objective is to investigate the localisation of immunoglobulin-producing cells (IPCs) in samples of inflamed intestinal tissue taken from patients with IBD, and their possible relationship with clinical features. METHODS: The IPCs in small intestinal, colonic and rectal biopsy specimens of patients with IBD were analysed by means of immunofluorescence using polyclonal rabbit anti-human Ig and goat anti-human IgM. The B cell phenotype of the IPC-positive samples was assessed using monoclonal antibodies specific for CD79, CD20, CD23, CD21, CD5, λ and κ chains. Statistical correlations were sought between the histological findings and clinical expression. RESULTS: The study involved 96 patients (64 with ulcerative colitis and 32 with Crohn's disease). Two different patterns of B lymphocyte infiltrates were found in the intestinal tissue: one was characterised by a strong to moderate stromal localisation of small IgM+/CD79+/CD20-/CD21-/CD23-/CD5± IPCs (42.7% of cases); in the other (57.3%) no such small IPCs were detected in stromal or epithelial tissues. IPCs were significantly less frequent in the patients with Crohn's disease than in those with ulcerative colitis (p = 0.004). CONCLUSION: Our findings suggest that different immunopathogenetic pathways underlie chronic intestinal inflammation with different clinical expressions. The presence of small B lymphocytes resembling B-1 cells also seemed to be negatively associated with Crohn's disease. It can therefore be inferred that the gut contains an alternative population of B cells that have a regulatory function.

[Frequency and clinical associations of antineutrophil cytoplasmic antibodies. A regional experience]. / Frequenza e associazioni cliniche degli anticorpi anti citoplasma dei neutrofili. Un'esperienza locale.

Serologic and clinical aspects of 50 positives patients for antineutrophil cytoplasmic antibodies (ANCA) have been evaluated (age range 7-94 years, mean age 43 years). 40 (80%) were females. Antineutrophil nuclear antibodies (pANNA), in which the antigenic specificity is unknown, were detected in seventeen patients (34%). About half of these cases (8 patients) had primary sclerosing cholangitis and other 7 patients had severe ulcerative colitis. Two pANNA patients, with increased susceptibility to infections, had undefined diagnosis. Both had thalassemic trait. Anti MPO were detected in 9 patients in which segmental lesions prevail and anti-PR3 were detected in 9 patients with granulomatous component. The patients with higher levels of these autoantibodies (40%) had the typical syndromes described in literature (vasculitis ANCA-related) although patients with lower autoantibodies levels (60%) mostly present variable clinical symptoms with unspecified diagnosis. Fourteen patients were positive for atypical ANCA detectable with commercial kits. They present variable clinical symptoms with unspecified diagnosis but show granulomatous or neoplastic lungs and bowel involvement. Both have mostly contact with environmental microorganisms. All cases are characterized by chronic inflammatory lesions in which the relapses correlate with infectious disorder.

[Anti-PR3 positive inflammatory bowel disease. Description of a case]. / Malattia infiammatoria cronica intestinale con positività anti-PR3. Descrizione di un caso.

We descrive the case of a patient with inflammatory bowel disease complicated by erythema nodosum and anti PR3 positivity. The patient was exposed to two different bacteria: salmonellosis was reported in patient history, while Staphylococcus aureus infection was diagnosed during the present hospital stay. Antibiotics and steroids are effective.

Lack of correlation between serum anti-HBcore detectability and hepatocellular carcinoma in patients with HCV-related cirrhosis.

BACKGROUND: While the likelihood of developing hepatocellular carcinoma (HCC) in patients coinfected with both HBV and HCV is increased, the role of previous exposure to HBV as a risk factor associated with tumor occurrence in subjects with HCV-related cirrhosis has not been fully investigated. AIM: To assess whether serum anti-HBc positivity, as a marker of previous HBV exposure, is associated with HCC development in HCV-related positive, hepatitis B surface antigen (HBsAg) negative patients with cirrhosis treated with alfa-interferon (IFN) monotherapy. PATIENTS AND: A database including 883 consecutive patients (557 men, mean age 54.7 yr) with histologically METHODS: proven cirrhosis treated with IFN between 1992 and 1997 was analyzed. All subjects have been surveilled every 6 months by ultrasound. Independent predictors of HCC were assessed by Cox multiple regression analysis. RESULTS: Mean follow-up was 96.1 months. Anti-HBc testing was available in 693 cases and, among them, 303 patients (43.7%) were anti-HBc seropositive. Anti-HBc positive patients were more often men (67.0%vs 58.7%, P= 0.03), had lower transaminase levels (3.3 +/- 2.0 vs 3.8 +/- 2.5 u.l.n., P= 0.004), and had higher rate of alcohol intake (38.3%vs 22.5%, P < 0.001) than anti-HBc negative patients. Overall, the incidence rates of HCC per 100 person-years were 1.84 (95% CI 1.34-2.47) in the anti-HBc positive patients and 1.86 (95% CI 1.41-2.42) in anti-HBc negative ones. By Cox multiple regression, there was no association of serum anti-HBc with HCC development (HR 1.03, 95% CI 0.69-1.52) or liver-related deaths incidence (HR 1.21; 95% CI 0.76-1.95). CONCLUSIONS: In comparison with anti-HBc negative subjects, serum anti-HBc positive patients with HCV-related/HBsAg negative cirrhosis treated with IFN monotherapy did not show a greater risk of HCC.

Sustained virological response to interferon-alpha is associated with improved outcome in HCV-related cirrhosis: a retrospective study.

UNLABELLED: The effect of achieving a sustained virological response (SVR) following interferon-alpha (IFNalpha) treatment on the clinical outcomes of patients with HCV-related cirrhosis is unknown. In an attempt to assess the risk of liver-related complications, HCC and liver-related mortality in patients with cirrhosis according to the response to IFNalpha treatment, a retrospective database was developed including all consecutive patients with HCV-related, histologically proven cirrhosis treated with IFNalpha monotherapy between January 1992 and December 1997. SVR was an undetectable serum HCV-RNA by PCR 24 weeks after IFNalpha discontinuation. HCC was assessed by ultrasound every 6 months. Independent predictors of all outcomes were assessed by Cox regression analysis. Of 920 patients, 124 (13.5%) were classified as achieving a SVR. During a mean follow-up of 96.1 months (range: 6-167) the incidence rates per 100 person-years of liver-related complications, HCC and liver-related death were 0, 0.66, and 0.19 among SVR and 1.88, 2.10, and 1.44 among non-SVR (P<0.001 by log-rank test). Multivariate analyses found that non-SVR was associated with a higher risk of liver-related complications (hazard ratio, HR, not applicable), HCC (HR 2.59; 95% CI 1.13-5.97) and liver-related mortality (HR 6.97; 95% CI 1.71-28.42) as compared to SVR. CONCLUSION: Thus, in patients with HCV-related, histologically proven cirrhosis, achievement of a SVR after IFNalpha therapy was associated with a reduction of liver-related mortality lowering both the risk of complications and HCC development. Irrespective of SVR achievement, all patients should continue surveillance because the risk of occurrence of HCC was not entirely avoided.

Bone mineral density in relation to efficacy and side effects of budesonide and prednisolone in Crohn's disease.

BACKGROUND & AIMS: Osteoporosis frequently occurs in Crohn's disease, often because of corticosteroids. Budesonide as controlled release capsules is a locally acting corticosteroid with low systemic bioavailability. We investigated its effects on bone compared with prednisolone. METHODS: In 34 international centers, 272 patients with Crohn's disease involving ileum and/or colon ascendens were randomized to once daily treatment with budesonide or prednisolone for 2 years at doses adapted to disease activity. One hundred eighty-one corticosteroid-free patients had active disease (98 had never received corticosteroids, corticosteroid naive; 83 had received corticosteroids previously, corticosteroid exposed), and 90 had quiescent disease, receiving long-term low doses of corticosteroids, corticosteroid-dependent; in 1 patient, no efficacy data were obtained. Bone mineral density and fractures were assessed in a double-blinded fashion; disease activity, side effects, and quality of life were monitored. RESULTS: Neither the corticosteroid-free nor the corticosteroid-dependent patients treated with budesonide differed significantly in bone mineral density from those receiving prednisolone. However, corticosteroid-naive patients receiving budesonide had smaller reductions in bone mineral density than those on prednisolone (mean, -1.04% vs -3.84%; P = .0084). Treatment-emergent corticosteroid side effects were less frequent with budesonide. Efficacy was similar in both groups. CONCLUSIONS: Treatment with budesonide is associated with better preserved bone mass compared with prednisolone in only the corticosteroid-naive patients with active ileocecal Crohn's disease. In both the corticosteroid-free and corticosteroid-dependent groups, budesonide and prednisolone were equally effective for up to 2 years, but budesonide caused fewer corticosteroid side effects.

Perianal fistulae following infliximab treatment: clinical and endosonographic outcome.

BACKGROUND: Management of perianal and rectovaginal fistulae complicating Crohn's disease (CD) is unsatisfactory. Infliximab is effective in the treatment of fistulating CD. However, reopening of fistulae is frequent, suggesting the persistence of deep fistula tracts despite superficial healing. In this study, the clinical and endosonographic behavior of perianal fistulae were evaluated following infliximab infusions, as well as the role of anal endosonography (AE) in predicting their outcome. METHODS: Thirty CD patients presenting with perianal and/or rectovaginal fistulae received an infusion of infliximab at a dose of 5 mg/kg at weeks 0 (entry into the study), 2, and 6. Laboratory and clinical assessments were repeated at same intervals and at week 10. AE was performed at entry and at week 10. Thereafter, the perianal region was re-examined every 6 months, and patients were investigated regarding draining of the fistula in the previous months. RESULTS: Fifteen patients (53.6%) showed closure of the fistulae at week 10, but only 5 patients had the fistula tracts disappeared at AE. Clinical and AE closure of rectovaginal fistulae was less prevalent than that of perianal fistulae [14.3% versus 63.6% at week 6 (p = 0.035); 28.6% versus 59.1% at week 10 (p = 0.21); 14.3% versus 22.7% at AE (p = 1.00)]. The behavior of fistulae was not affected by their number and AE classification, presence of rectal disease, or setons. Twenty patients with perianal fistulae were followed for a median of 15.5 months. Patients with closed perianal fistulae at week 10 and disappearance of fistulae tract at AE showed a lower relapse rate than those with endosonographic persistence of fistula tract. CONCLUSIONS: Infliximab can heal perianal and rectovaginal fistulae in approximately 60% and 30% of patients, respectively. Despite closure, most fistula tracts are still detectable at AE. Persistence of the internal tract is a condition at higher risk of fistula recurrence.