Thomas Orth

Noninvasive assessment of Crohn's disease activity: a comparison of 18F-fluorodeoxyglucose positron emission tomography, hydromagnetic resonance imaging, and granulocyte scintigraphy with labeled antibodies.

OBJECTIVES:Detection of disease activity in Crohns disease (CD) is of crucial importance for diagnosis and management of the disease. Noninvasive methods for monitoring are desirable and comprise hydromagnetic resonance imaging (hydro-MRI) and leukocyte scintigraphy. In addition, a recent case report indicated the potential of 18F-fluorodeoxyglucose positron emission tomography (FDG-PET) to assess CD activity. However, comparative prospective studies are lacking.METHODS:Between February, 1999 and August, 2000, 59 patients with CD were enrolled in a prospective study to assess disease activity by FDG-PET, hydro-MRI, and immunoscintigraphy with anti-nonspecific cross-reacting antigen 95 antigranulocyte antibodies. In 28 of these patients, colonoscopy could be performed. Twelve patients with irritable bowel syndrome and 20 tumor patients without gut inflammation served as controls. Results were compared by statistical analysis.RESULTS:FDG-PET detected 127 pathological findings (average maximum standardized uptake value = 4.4 ± 1.1) in the terminal/neoterminal ileum (37), small bowel (24), and colon (66) of 54 patients with CD, whereas no pathological findings were seen in five patients with CD, the control patients with irritable bowel syndrome, and the tumor patients without gut inflammation. In contrast, examination with hydro-MRI or granulocyte antibodies detected less pathological findings in CD patients. Forty-five of the detected foci were accessible to endoscopic verification. The correlation of the foci with endoscopic findings showed a high specificity (>89%) of all three methods to detect inflamed areas in the terminal ileum and colon of patients with CD, although analyses by hydro-MRI and granulocyte antibody scan had strikingly lower sensitivities (40.9% and 66.7%) than FDG-PET analysis (85.4%).CONCLUSIONS:FDG-PET appears to be a reliable noninvasive tool for simultaneous detection of inflamed areas in the small and large bowel of patients with CD. FDG-PET can be used to detect disease activity in the terminal ileum and colon of CD patients with high sensitivity and specificity.

Hydro-MRI in Crohn's disease: appraisal of disease activity.

RATIONALE AND OBJECTIVES To appraise the value of hydro-MRI in the assessment of activity in Crohns disease. METHODS After bowel opacification with 1000 mL of an orally administered 2.5% mannitol solution was achieved, axial and coronal breath-hold sequences (T2-weighted half-Fourier acquisition single-shot turbo spin-echo sequences with or without fat saturation, dynamic T1-weighted fast low-angle shot sequences, and contrast-enhanced T1-weighted fast low-angle shot with fat saturation sequences) were acquired in 82 patients with proved Crohns disease at 1.0 T. Enhancement of the bowel wall was correlated with other MRI findings, with the Crohns disease activity index (CDAI), and with levels of C-reactive protein (CRP). RESULTS In Crohns disease, contrast enhancement of the affected bowel wall was markedly increased in comparison with the normal bowel wall (+80% +/- 22% versus +43% +/- 12%; P = 3 x 10(-15)). Positive correlations could be established between the increase in bowel wall enhancement and many other MRI findings. Between the increase in bowel wall enhancement and the CDAI, only a poor correlation was found (r = 0.25, P = 0.02). There was no statistical correlation between the increase in bowel wall enhancement and CRP. CONCLUSIONS Hydro-MRI seems to be superior to the CDAI and CRP for the registration of Crohns disease activity. In particular, differentiation between an active and an inactive (scarred) stenosis, which is crucial for the choice of therapeutic procedures, seems to be more reliable by the interpretation of several morphological and functional parameters on hydro-MRI than by the use of CDAI and CRP.

Mycophenolate Mofetil Versus Azathioprine in Patients With Chronic Active Ulcerative Colitis: A 12-Month Pilot Study

OBJECTIVE:Ulcerative colitis (UC) is an inflammatory bowel disease (IBD) of unknown etiology frequently requiring long-term therapy for control of symptoms and prevention of relapse. Azathioprine (AZA) has been shown to be effective and safe in the treatment of chronic active UC. However, the alternatives to treatment with AZA are limited. Our aim was to compare the efficacy and safety of treatment with mycophenolate mofetil (MMF)/prednisolone versus standard immunosuppressive treatment with azathioprine (AZA)/prednisolone in patients with chronic active UC.METHODS:The study was designed as an open comparison of MMF versus AZA. Twenty-four patients with active UC (Rachmilewitz score ≥6 points) were randomly assigned to the MMF (20 mg/kg)/prednisolone or AZA (2 mg/kg)/prednisolone group. The initial prednisolone dosage was 50 mg and was tapered according to a standard protocol. Treatment was scheduled for 1 yr.RESULTS:The rates of remission were higher in the AZA/prednisolone group (n = 12) than in the MMF/prednisolone group (n = 12) throughout the study. Remission rates were 92% versus 67% after 4 wk, 92% versus 67% after 3 months, 92% versus 67% after 6 months, 83% versus 78% after 9 months, and 100% versus 88% after 1 yr. The number of patients not requiring steroids was higher in the AZA/prednisolone group than in the MMF/prednisolone group. Moreover, in the AZA/prednisolone group no severe adverse events were recorded, whereas in the MMF/prednisolone group two severe adverse events were observed: one patient discontinued MMF after 6 months because of recurrent upper airway infections, and one patient exhibited a bacterial meningitis after 9 months.CONCLUSIONS:Treatment with AZA/prednisolone appears to be more effective and safe compared to MMF/prednisolone in patients with chronic active UC. MMF might be an alternative treatment for patients with contraindications to AZA. To further evaluate the effects of MMF in active UC, a placebo-controlled double-blinded study appears warranted.

Antibodies to proteinase 3 mediate expression of vascular cell adhesion molecule-1 (VCAM-1).

VCAM‐1 was first identified as an adhesion molecule induced on human endothelial cells (HEC) by inflammatory cytokines such as IL‐1, tumour necrosis factor (TNF), and lipopolysaccharide (LPS). The molecule binds to a variety of leucocytes, including B cells, T cells, basophils, eosinophils and monocytes. Vascular expression of VCAM‐1 has been associated with a number of disease states, including rheumatoid arthritis and vasculitis. The detection of antineutrophil cytoplasmic antibodies (ANCA), especially to proteinase 3 (PR3), has become important in the diagnosis of Wegener’s granulomatosis (WG) and related vasculitides. Recently we were able to demonstrate a direct effect of anti‐PR3 antibodies on neutrophil–endothelial interactions (Blood 1993; 82:1221). Binding of anti‐PR3 antibodies to their antigen translocated into the membrane of HEC leads to an enhanced adhesion of neutrophils via induction of E‐selectin (Clin Exp Immunol 1993; 94:440). The aim of this study was to investigate the effect of anti‐PR3 antibodies on the expression of VCAM‐1. HEC was isolated from umbilical vein and cultured on microtitre plates. After preincubatiion with purified anti‐PR3 antibody, purified control antibodies (SS‐A, SS‐B, RNP) (IgG and F(ab′)2 fragments) or different cytokines (controls), VCAM‐1 was detected on the surface of unfixed HEC by cyto‐ELISA and polymerase chain reaction analysis. Incubation of HEC with anti‐PR3 antibodies led to a marked increase of endothelial VCAM‐1 expression with a peak after 8 h. Incubation with TNF‐α also led to maximal VCAM‐1 expression after 4–6 h (control). Increased adhesion of T lymphocytes to HEC after binding of anti‐PR3 antibodies to their antigen could be confirmed by performing adherence assays. This effect could be inhibited by antibodies to VLA‐4. In conclusion, we have been able to show that cytokine‐like effects of anti‐PR3 antibodies on HEC are not limited to induction of neutrophil adhesion. Anti‐PR3 antibodies may thus contribute to the regulation of T lymphocyte migration from the blood by HEC in ANCA‐related vasculitides.

Complete congenital heart block is associated with increased autoantibody titers against calreticulin

Complete congenital heart block (CCHB) is associated with anti‐Ro/SS‐A and anti‐La/SS‐B antibodies. Calreticulin, a calcium‐binding, multifunctional protein of the endoplasmic reticulum with C‐terminal KDEL‐sequence, is not part of the Ro/SS‐A ribonucleoprotein complex. In this study anti‐calreticulin autoantibody responses in serum samples from 18 infants with CCHB, their mothers and in a control group of 11 anti‐Ro/SS‐A or anti‐La/SS‐B positive infants without heart block and their mothers were analysed. Specific enzyme‐linked immunosorbent assays were performed. Nine out of 18 sera with CCHB contained IgG anti‐calreticulin antibodies. Four sera of those with IgG antibodies also had IgM antibodies. One serum contained anti‐calreticulin IgM antibodies only. In the non‐CCHB group two sera were positive for IgG and one serum was positive for IgM anti‐calreticulin antibodies. Sera of healthy infants were negative both for anti‐IgG and anti‐IgM calreticulin antibodies. Calreticulin is involved in calcium storage and therefore anti‐calreticulin antibodies might influence the development of CCHB. The new finding of IgM autoantibodies and the observed differences in antibody response in infants and mothers support the hypothesis of a fetally mediated and passively acquired autoimmune disease.

Actin is a target antigen of anti-neutrophil cytoplasmic antibodies (ANCA) in autoimmune hepatitis type-1

BACKGROUND/AIM Anti-neutrophil cytoplasmic antibodies (ANCA) are a group of autoantibodies first associated with Wegeners granulomatosis and microscopic polyangiitis. The significance of ANCA in autoimmune hepatitis remains uncertain; the nature of the antigen or antigens has not been defined yet. The purpose of this study was to identify the target antigen of ANCA in patients with autoimmune hepatitis. METHODS/RESULTS Sera from 32 type-1 autoimmune hepatitis patients were used in the present study. ANCA were detected in 24 of 32 sera (75%). A diffuse cytoplasmic staining pattern (C-ANCA) was detected in 14 patients; the P-ANCA pattern was observed in 10 patients. An extract of human neutrophils was prepared and subjected to SDS-PAGE and Western Blot analysis. A 43-kD dominant immunoreactive protein was found in 20 (63%) autoimmune hepatitis patients. Aminoacid sequence analysis of the 43 kD protein identified actin. Cytoplasmic or perinuclear staining pattern could be reduced after absorption of sera with actin and after removing anti-actin antibodies by affinity chromatography. This was observed for all C-ANCA and for 8 out of 10 P-ANCA. Moreover in double-staining indirect immunofluorescence, the same type of diffuse cytoplasmic staining was observed with autoimmune hepatitis-sera and anti-actin antibodies. In Western Blot analysis with actin, 17 (53%) patients gave a positive result, while 15 (47%) patients had a positive actin-ELISA. CONCLUSION This is the first report to identify the cytoskeletal protein actin as an ANCA antigen.

A novel rat model of chronic fibrosing cholangitis induced by local administration of a hapten reagent into the dilated bile duct is associated with increased TNF-α production and autoantibodies

BACKGROUND/AIM The cholangiopathies represent hepatobiliary diseases in which bile-duct epithelial cells are targets for destructive processes, including immune-mediated damage. We describe a novel rat model of chronic fibrosing cholangitis induced by administration of the hapten reagent 2,4,6-trinitrobenzenesulfonic acid (TNBS) into the dilated bile duct. METHODS The common bile duct was dilated due to a mild stenosis in 8-week-old female Lewis rats. TNBS (50 mg/kg) was injected during a second laparotomy. RESULTS TNBS-treatment reproducibly resulted in chronic fibrosing cholangitis. In retrograde cholangiography the bile ducts showed irregularities, beading and strictures. Alkaline phosphatase levels remained abnormal throughout the study period. Immunohistochemical staining showed an increased number of macrophages, CD3+ T-lympbocytes and MHC class II antigen upregulation. The spontaneous interferon-gamma, tumor necrosis factor-alpha and interleukin-10 production of liver-derived mononuclear cells was increased. Anti-neutrophil cytoplasmic antibodies with specificity against myeloperoxidase, catalase and actin were found between 1 and 12 weeks after TNBS injection. CONCLUSIONS We established a novel rat model of chronic fibrosing cholangitis with histologic, cholangiographic, serologic and immunologic similarities to human primary sclerosing cholangitis. This model may be used to study pathomechanisms of chronic cholangitis without concomitant inflammatory bowel disease.

Regulation of organic anion transporters in a new rat model of acute and chronic cholangitis resembling human primary sclerosing cholangitis

BACKGROUND/AIMS Primary sclerosing cholangitis (PSC) is a cholestatic liver disease of unknown etiology. Although the primary defect affects cholangiocytes, cholestatic injury of hepatocytes may promote further liver damage. Since down-regulation of hepatocellular organic anion transporters is implicated in the molecular pathogenesis of cholestasis, expression of these transporters was determined in a novel rat model, which closely resembles human PSC. METHODS Hepatic protein and mRNA expression of basolateral (Ntcp, Oatp1, 2 and 4) and canalicular (Mrp2, Bsep) organic anion transporters were analyzed 1, 4 and 12 weeks after induction of experimental PSC by 2,4,6-trinitrobenzenesulfonic acid (TNBS). RESULTS Specific down-regulation of basolateral and canalicular transport systems except Oatp4 and Bsep proteins occurred during the acute phase of inflammation. In chronic cholangitis 12 weeks after TNBS Mrp2 protein and mRNA remained down-regulated by 40-50% of controls (P<0.05). In addition Oatp1 protein was also reduced by 40+/-13% (P<0.05), whereas all other transporters returned to control values. CONCLUSIONS In chronic cholangitis only canalicular Mrp2 expression remained down-regulated. This might represent the first injury to hepatocytes in chronic cholangitis as an extension of liver injury from the level of cholangiocytes to hepatocytes in PSC.

Signal transduction pathways of membrane expression of proteinase 3 (PR-3) in human endothelial cells.

At present, the exact mechanism of the pathogenic effect of anti‐PR‐3 antibodies remains unknown. Interaction of anti‐neutrophil cytoplasmic antibodies (ANCAs) with human umbilical vein endothelial cells (HUVECs) may play a key role. Recently we were able to show that ANCAs recognize their target antigen, PR‐3, translocated into the membrane of HUVECs. The objective of this study was to investigate regulation, i.e. signal transduction pathways, of PR‐3 expression in endothelial cells. HUVECs were isolated according to the method of Jaffe et al. and cultured under standard conditions. A cyto‐enzyme‐linked immunosorbent assay (ELISA) with unfixed cells was performed. Membrane‐expressed PR‐3 was detected by affinity‐purified and monoclonal anti‐PR‐3 Ab. Tumour necrosis factor alpha (TNF‐α)‐induced membrane expression of PR‐3 could be blocked with the RNA synthesis inhibitor actinomycin D, the protein kinase C (PKC) and proteinase A (PKA) inhibitor staurosporine, the specific PKC inhibitor calphostin C, the c‐AMP‐dependent PKA inhibitor KT5720 and the tyrosine kinase inhibitor genistein in a dose‐dependent manner. The effect of calphostin C was the most significant. In addition, the effect of phorbol 12‐myristate 13‐acetate (PMA), a mediator of intracellular second messengers, was investigated. In our study, pretreatment of cells with PMA for 48 h led to a down‐regulation of PR‐3 expression. This effect, however, could be overridden by TNF‐α stimulation, i.e. TNF‐α‐induced membrane expression of PR‐3 was resistant to down‐regulation of PKC. In conclusion, our data suggest that translocation of PR‐3 in HUVECs is an active process depending on protein synthesis. PR‐3 expression by HUVECs may involve a PKC reactive to cytokines such as TNF‐α which induces PR‐3 expression at a transcriptional level.

Analysis of epitope spreading over an eleven-year period in a patient with systemic lupus erythematosus.

During a period of more than eleven years serum samples of a patient with Systemic Lupus Erythematosus were collected and analyzed for anti-nuclear autoantibodies. High titer of anti-La/SS-B were detectable in all serum samples. The La/SS-B epitopes remained constant. Besides anti-La/SS-B antibodies all serum samples contained traces of anti-Ro/SS-A including anti-Ro52 and anti-Ro60 antibodies. During disease flares anti-Ro/SS A antibodies were upregulated and anti-dsDNA antibodies appeared, thus supporting the concept of an antigen driven intermolecular epitope spreading to Ro/SS-A and dsDNA.