Anti-neutrophil cytoplasmic antibody (ANCA) in malaria is directed against cathepsin G

Autoantibodies of diverse specificities are detected in sera of patients with acute malaria. The clinical relevance of these autoantibodies is not clear, though there are reports associating some autoantibodies with specific disease manifestations. We have investigated the occurrence of ANCA in the sera of 93 patients during episodes of acute malaria. Sera were tested by indirect immunofluorescence (IIF) and by ELISA for antibodies to neutrophil cytoplasmic components proteinase 3 (PR3), myeloperoxidase (MPO), cathepsin G (CG), human leucocyte elastase (HLE), and lactoferrin (LF). Forty-seven sera samples (50.5%) were positive by IIF, all except one with the atypical ANCA pattern (a-ANCA). When screened by ELISA, anti-CG antibodies were detected in 52 samples (56%), while anti-PR3 and anti-MPO antibodies were detected in three and one samples, respectively. Antibody binding to HLE and LF was not significant. Anti-CG antibodies were detected in 93% of the IIF-positive sera. A combination of anti-CG and anti-PR3 antibodies was noted in three samples. Our study demonstrates the presence of ANCA in sera from patients with acute malaria, almost all with the a-ANCA pattern on IIF. The antibody specificity, noted for the first time in our study, appears to be predominantly directed against CG. The significance of CG and CG-ANCA in the pathogenesis and clinical manifestations of malaria has yet to be elucidated.     

Prevalence and characterization of perinuclear anti-neutrophil cytoplasmic antibodies (P-ANCA) directed against HMG1 and HMG2 in ulcerative colitis (UC)

In a previous study, we reported that the high mobility group (HMG) non-histone chromosomal proteins HMG1 and HMG2 were novel target antigens of P-ANCA. In this study, we determined the immunodiagnostic value of anti-HMG1/HMG2 antibodies in patients with UC. Sixty sera from patients with UC were tested for reactivity with HMG1 and HMG2 by means of ELISA. Anti-HMG1 antibody was detected in 32% of patients (40% of P-ANCA⁺ patients). Anti-HMG2 antibody was detected in 33% (40% of P-ANCA⁺ patients). Thirty-five percent of sera were positive for antibody to either HMG1 or HMG2 (43% of P-ANCA⁺ patients). P-ANCA⁺ patients expressed anti-HMG1/HMG2 antibodies with significantly greater frequency compared with P-ANCA^? patients. Furthermore, the anti-HMG1/HMG2 antibodies were significantly related to disease activity in UC. Sixteen of the 18 UC patients, who had high titres of anti-HMG1 or -HMG2 antibody during the active phase, showed lower titres in the inactive phase. Anti-HMG1/HMG2 antibodies appear to be useful as a marker for disease activity in UC.     

Are anti-neutrophil cytoplasmic antibodies (ANCA) clinically useful in inflammatory bowel disease (IBD)?

Since the first detection of ANCA in IBD, numerous studies have dealt with their prevalence, antigenic specificities, clinical significance, pathophysiological role, and their induction. This review summarizes the information obtained from those studies and shows that ANCA are not directly useful as diagnostic and prognostic factors in IBD. ANCA were detected in 50-85% of patients with ulcerative colitis (UC) and 10-20% of patients with Crohn's disease (CD). Multiple target antigens are recognized by these autoantibodies, including both cytoplasmic and nuclear proteins. A pathophysiological role for ANCA in IBD is far from clear. On the one hand, it is suggested that ANCA are genetic markers of susceptibility for IBD, and on the other hand, the induction of ANCA in those diseases may just be an epiphenomenon of chronic inflammation. We discuss recent evidence that ANCA may be induced by a break-through of tolerance towards bacterial antigens.     

Anti-neutrophil cytoplasmic antibodies (ANCA) directed against bactericidal/permeability increasing protein (BPI): a new seromarker for inflammatory bowel disease and associated disorders

It was our purpose to determine the immunodiagnostic value of ANCA directed against BPI in diseases known to be associated with ANCA, such as ANCA-associated vasculitides, inflammatory bowel disease (IBD) and the associated condition primary sclerosing cholangitis. The immunoreactivity of recombinant BPI (rBPI) was established in order to develop an ELISA specific for rBPI. By means of this assay, BPI-ANCA were assessed in sera of 178 patients with IBD or the associated disorder primary sclerosing cholangitis, 112 patients with ANCA-associated vasculitides, and in sera of 182 disease and 140 healthy controls. BPI-ANCA were found to be closely associated with IBD and primary sclerosing cholangitis (34% and 44% of ANCA-positive sera, respectively). By contrast, BPI-ANCA positivity was low (<10%) in the double-negative sera of patients with ANCA-associated vasculitides and in disease and healthy controls. BPI-ANCA appear to constitute an important marker for IBD and primary sclerosing cholangitis, but not for the ANCA-associated vasculitides.     

Anti-lactoferrin antibodies and other types of anti-neutrophil cytoplasmic antibodies (ANCA) in reactive arthritis and ankylosing spondylitis.

Fifty-five serum samples from patients with reactive arthritis (ReA), 40 from patients with ankylosing spondylitis (AS) and three from patients with chronic sacroiliac joint arthritis were analysed for the presence of ANCA of IgG class by means of enzyme immunosorbent assay using lactoferrin (Lf), myeloperoxidase (MPO) and antigen extracted from azurophil granules ('alpha-antigen') containing proteinase 3 (PR3) as substrate. IgG-ANCA were found in 31 (56%) patients with ReA. Twenty-three (42%) had anti-Lf antibodies, nine (16%) had anti-MPO and eight (15%) had anti-alpha-antigen antibodies, none of which reacted with PR3. Only six (14%) AS or sacroiliac joint arthritis patients had ANCA (P < 0.001). Three (7%) had anti-Lf, two (5%) anti-MPO and two (5%) anti-alpha-antigen antibodies. Yersinia and Salmonella bacteria were separated by SDS-PAGE and blots were incubated with serum from rabbits immunized with human Lf. The hyperimmune serum recognized a band of 78 kD from both bacteria which was not seen when preimmune serum was used. The reaction to the 78-kD antigen could be completely inhibited when anti-Lf antibodies were absorbed on Lf coupled to cyanogen bromide-activated Sepharose, possibly indicating cross-reacting epitopes in Lf and enterobacterial antigen.     

Anti-neutrophil cytoplasmic antibodies (ANCA) in inflammatory bowel disease: characterization and clinical correlates.

ANCA were detected by indirect immunofluorescence in 34 out of 67 patients with ulcerative colitis (UC, 51%) and in 14 out of 35 patients with Crohn's disease (CD, 40%). All but one ANCA-positive sera produced a perinuclear pattern of fluorescence (P-ANCA) on ethanol-fixed neutrophils. On paraformaldehyde-fixed neutrophils 76% of P-ANCA-positive sera in UC and 50% of P-ANCA-positive sera in CD produced cytoplasmic fluorescence, indicating that, indeed, cytoplasmic antigens are recognized by a considerable number of these sera. By Western blot analysis using whole neutrophil extract as a substrate 46% of sera from patients with UC and 32% of sera from patients with CD showed reactivity with either lactoferrin, polypeptides occurring as a doublet of 66/67 kD mol. wt, or polypeptides occurring as a doublet of 63/54 kD mol. wt, respectively. Identical patterns of reactivity have been observed among P-ANCA-positive sera from patients with autoimmune liver disease and rheumatoid arthritis. These data suggest that ANCA of restricted specificities are not specific for inflammatory bowel disease (IBD), but are present in diverse conditions characterized by chronic idiopathic inflammation.     

A proportion of proteinase 3 (PR3)-specific anti-neutrophil cytoplasmic antibodies (ANCA) only react with PR3 after cleavage of its N-terminal activation dipeptide

ANCA directed against PR3 are highly specific for Wegener's granulomatosis and microscopic polyangiitis, and have been implicated in the pathogenesis of small vessel vasculitis. Most PR3-ANCA are directed against conformational epitopes on PR3. This study was designed to determine whether the cleavage of the N-terminal activation dipeptide of PR3 is required for the binding of PR3-ANCA. Recombinant PR3 (rPR3) variants were expressed in the epithelial cell line, 293. As confirmed by radiosequencing, the rPR3 secreted into the 293 cell culture supernatant is N-terminally unprocessed. Two enzymatically inactive rPR3 mutants were expressed in 293 cells: rPR3-S176A and δ -rPR3-S176A. rPR3-S176A contains the N-propetide Ala-2-Glu-1, δ -rPR3-S176A does not. Culture supernatants of rPR3-S176A and δ -rPR3-S176A expressing 293 cells were used as sources of target antigen for PR3-ANCA testing by capture ELISA. Forty unselected consecutive PR3-ANCA⁺ sera were tested. With δ -rPR3-S176A as antigen all 40 were recognized, compared with only 34 of 40 when rPR3-S176A served as target antigen. The majority of the serum samples contained a mixture of antibodies reacting with epitopes accessible on the mature and on the proform of PR3. In conclusion, the cleavage of the N-terminal activation dipeptide of PR3 is not an absolute requirement for recognition by all PR3-ANCA. However, a substantial proportion of PR3-ANCA recognize (a) target antigen(s) exposed only after the conformational change of PR3 associated with the N-terminal processing. In 15% of sera this PR3-ANCA subset occurred exclusively. PR3-ANCA subtypes can be differentiated using specifically designed rPR3 variants as target antigens, and non-haematopoietic mammalian cells without regulated secretory pathway can be used for their expression.     

Anti-neutrophil cytoplasmic antibodies (ANCA) against bactericidal/permeability-increasing protein (BPI) and cystic fibrosis lung disease.

Persistent infection with Pseudomonas aeruginosa and inflammatory mechanisms play an important role in cystic fibrosis (CF) lung disease. ANCA against BPI, a potent host defence protein with anti-bacterial and anti-endotoxin properties, have been described in CF. We have assessed the relationship of anti-BPI antibodies to pulmonary disease severity in 148 CF subjects. IgA and IgG anti-BPI antibodies were found in 55.4% and 70.3% of CF patients, respectively, and higher levels were strongly associated with colonization with P. aeruginosa (P = 0.001 and 0.039 for IgA and IgG antibodies, respectively). IgA and IgG anti-BPI antibodies were independently associated with more severe lung disease as assessed by chest radiograph score (P = 0.023) and a significantly lower forced expiratory volume in 1 s (FEV1)% (P = 0.01). The pathophysiological relevance of the autoantibodies was investigated further by determining their epitope specificity and their effect on bacterial phagocytosis in vitro. Both isotypes of anti-BPI antibodies were specific for the C-terminus of BPI shown recently to be important for BPI-mediated opsonization, and in vitro affinity-purified anti-BPI antibodies significantly reduced BPI-induced phagocytosis of Escherichia coli compared with controls. These data indicate that anti-BPI autoantibodies are associated with colonization with P. aeruginosa and worse lung disease in CF. The inhibition of bacterial phagocytosis suggests that these autoantibodies may contribute to the persistence of P. aeruginosa in the CF lung and so play a role in perpetuating CF lung damage.     

Antineutrophil cytoplasmic antibodies (ANCA) directed against cathepsin G in ulcerative colitis, Crohn''s disease and primary sclerosing cholangitis.

Autoantibodies directed against polymorphonuclear neutrophils (PMN) have been observed in serum from patients with ulcerative colitis (UC), Crohn's disease (CD) and primary sclerosing cholangitis (PSC) using indirect immunofluorescence and fixed granulocyte ELISA. Our study demonstrates the presence in the serum of these patients of autoantibodies which bind to an azurophilic granule component distinct from proteinase 3, elastase and myeloperoxidase. These autoantibodies thus belong to the ANCA family, but their antigen specificity differs from the already characterized ANCA antigens. We have found that the same ANCA antigen target, named UC-antigen, was recognized by serum IgG from patients with UC, CD and PSC. It was purified by Matrex Gel Orange A dye affinity chromatography and subsequent immunoabsorption of contaminant proteinase 3 with immobilized anti-proteinase 3 MoAb. The identity between this UC antigen and cathepsin G was demonstrated by their coelution from Matrex Gel Orange A column and the parallel titration of cathepsin G-specific enzymatic activity and UC-ANCA binding, both in partially purified UC antigen and in highly pure cathepsin G. Furthermore, the use of cathepsin G ELISA confirmed that UC, CD and PSC patients' IgG did indeed bind to cathepsin G. Comparison of the results obtained with azurophilic granule- and cathepsin G-ELISA as well as inhibition of ANCA binding by anti-cathepsin G polyclonal antibodies, revealed that in some patients cathepsin G is the main azurophilic granule target of ANCA while others have other ANCA specificities. The fact that UC, CD and PSC are frequently associated with cathepsin G ANCA, while rarely occurring in other types of vasculitis, is intriguing but suggests that these diseases may have a common pathogenetic mechanism.     

Transforming growth factor-beta (TGF-β) expression and interaction with proteinase 3 (PR3) in anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis

TGF-β is a multifunctional cytokine modulating the onset and course of autoimmune diseases as shown in experimental models. The aim of this study was to investigate TGF-β expression in ANCA-associated vasculitis (AAV), and the possible interactions of this cytokine with lysosomal enzymes identified as ANCA autoantigens (e.g. PR3). This included TGF-β effects on the translocation of the lysosomal enzymes to the cell surface of polymorphonuclear neutrophils (PMN), and the presumed activation of non-bioactive, latent TGF-β by these enzymes. Patients with various types of systemic vasculitis (SV) were studied, including three different types of AAV (Wegener's granulomatosis (WG), Churg–Strauß syndrome (CSS) and microscopic polyangiitis (MPA)). Regardless of the type of assay applied, the TGF-β1 isoform was found to be overexpressed in SV, including AAV, and to correlate with disease activity as shown for WG. Mean TGF-β1 plasma levels in AAV patients ranged from 8.9 ng/ml (WG) to 13.3 ng/ml (CSS) (control 4.2 ng/ml; P< 0.01), while TGF-β2 levels were not elevated. Flow cytometry analysis showed TGF-β1 to be a potent translocation factor for PR3 comparable to other neutrophil-activating factors such as IL-8. PR3 membrane expression on primed PMN increased by up to 51% after incubation with TGF-β1. PR3 itself was revealed as a potent activator of latent TGF-β, thus mediating bioeffects of this cytokine. These findings, together with other features of TGF-β such as induction of angiogenesis and its strong chemotactic capacity, indicate that TGF-β might serve as a proinflammatory factor in SV, especially in AAV.     

Screening for anti-neutrophil cytoplasmic antibodies (ANCA): is indirect immunofluorescence the method of choice?

Detection of ANCA has become an important tool for the diagnosis and monitoring of disease activity in Wegener's granulomatosis (WG). Unfortunately, a group of sera positive by the standard method for ANCA detection, indirect immunofluorescence (IIF), are negative when more specific tests with purified proteins are used. In order to examine this discrepancy we examined groups of sera selected for being (i) C-ANCA-positive by IIF; (ii) positive in proteinase 3 (PR3)-ANCA ELISA; and (iii) from 24 patients with WG. The following assays were used: IIF, PR3-ANCA ELISA and capture PR3-ANCA ELISA using MoAbs against PR3. Furthermore, since granule enzymes are released during coagulation, we also measured ANCA in complex with PR3. To test if granule enzyme release had any influence on ANCA detection, both serum and EDTA-plasma were collected from a patient with active WG. No difference, however, was found. In the IIF-positive group (n = 60) 68% of the sera were positive in PR3-ANCA ELISA, 86% in capture PR3-ANCA ELISA and 80% were positive for the PR3/IgG-ANCA complex. In the PR3-ANCA ELISA group (n = 105) 88% of the sera were positive by IIF, 98% in capture PR3-ANCA ELISA and 53% in the PR3/IgG-ANCA assay. To evaluate the tests clinically sera from 24 patients with WG were examined. In the remission group (n = 10) two patients were positive by IIF, four in the PR3-ANCA ELISA, and five in the capture PR3-ANCA ELISA. Fourteen had active disease, and in this group 11/14 were positive by IIF, 10/14 in PR3-ANCA ELISA and 12/14 by capture-ELISA. The correlation between IIF and capture PR3-ANCA ELISA titre (r = 0.72, P = 0.0095) was better than between PR3-ANCA ELISA and IIF (r = 0.56, P = 0.043). It is concluded that the capture PR3-ANCA ELISA is more sensitive than PR3-ANCA ELISA, and that the capture ELISA can be used for screening of PR3-ANCA.     

Anti-neutrophil cytoplasmic antibodies (ANCA) from patients with systemic vasculitis recognize restricted epitopes of proteinase 3 involving the catalytic site

ANCA with specificity for proteinase 3 (PR3), a neutrophil primary granule enzyme, are of diagnostic value in Wegener's granulomatosis (WG) and certain other forms of systemic vasculitis. There is evidence to suggest that they play a pathogenic role in disease, and that the interaction of ANCA with PR3 is likely to be important. We showed, using a resonant mirror biosensor, that C-ANCA from different patients recognized the same or closely related epitopes on PR3. Studies using linear peptides in the SPOT system confirmed the highly restricted nature of this interaction and identified five linear epitopes. Fluid-phase inhibition studies, using a different set of peptides, validated the sequences involved. Using a computer-generated model of the structure of PR3, four of five epitopes were shown to be intimately linked with the catalytic site. The restricted number of epitopes, and their location at the catalytic site, has important implications for the role of C-ANCA in the pathogenesis of vasculitis.     

肾脏疾病患者血清中ANCA的荧光模式及其意义

目的：进一步探讨肾脏疾病患者血清中抗中性粒细胞细胞质抗体(ANCA)的荧光模式及其意义。方法：应用间接免疫荧光(IIF)和ELISA方法检测了251例肾脏疾病患者血清中ANCA及其靶抗原。结果：在251例肾脏疾病患者中，ANCA阳性51例占20．3％，其中10例为cANCA阳性(10／251)；30例为cANCA阳性(30／251);11例为aANCA阳性(11／251)。结论：同时用IIF法和ELISA法检测ANCA，可以提高ANCA检出的阳性率；不同ANCA荧光模式与疾病的种类病情及预后密切相关；提高对不典型ANCA和pANCA伴ANA荧光模式的鉴别，有助于临床对血管炎和其它自身免疫性疾病的诊断。     

Antineutrophil cytoplasmic antibodies, anti-Saccharomyces cerevisiae antibodies, and specific IgE to food allergens in children with inflammatory bowel diseases

Differential diagnosis between ulcerative colitis (UC) and Crohn's disease (CD) is difficult in the initial phases in pediatric patients with inflammatory bowel diseases (IBD). This study was performed to determine the significance of anti-neutrophil cytoplasmic antibodies (ANCA) and anti-Saccharomyces cerevisiae antibodies (ASCA) in IBD. ANCA were specified with regard to their antigenic specifity, significance to the diagnosis, and correlation of titer with the disease activity. The occurrence of food allergy was questioned, too. Serum samples from 44 children with UC (n = 23) or CD (n = 21) and from disease-control children (coeliac disease, n = 21) were analyzed for IgG ANCA, ANCA target antigens, IgA and IgG ASCA, and IgE to food allergens. Results show that ANCA occur more frequently in UC than in CD and disease-control (74, 24, and 10%, respectively). The presence of ANCA does not reflect disease activity. Antigenic specificity does not differ in any group. IgA-ASCA are found more often in patients with CD (76% versus 17% in UC). The testing for both ANCA and ASCA enabled clear-cut differential diagnosis between UC and CD based on the high specificity (ANCA+ ASCA- 92.5% for UC, ANCA- ASCA+ 93.2% for CD). Specific IgE to food allergens were found in 8.7, 14.3, and 23.8% of patients with UC, CD, and coeliac disease, respectively. We conclude that combined testing of ANCA and ASCA represents a valuable tool in the differential diagnosis between UC and CD in pediatric patients, minimizing invasive diagnostic procedures. Monitoring of ANCA, its specificity, and titer determination does not bring more information. Testing for specific IgE to food allergens may be considered in individual patients.     

Antibodies to selected minor target antigens in patients with anti-neutrophil cytoplasmic antibodies (ANCA)

In patients with anti-neutrophil cytoplasmic antibodies (ANCA)-associated vasculitis, indirect immunofluorescence (IF) distinguishes between cytoplasmic (C-ANCA) and perinuclear (P-ANCA) neutrophil staining patterns. In patients with primary systemic vasculitis such as Wegener's granulomatosis, microscopic polyangiitis and Churg-Strauss syndrome, these IF staining patterns correspond broadly with antibodies to the two major antigens: the C-ANCA pattern is associated generally with antibodies to serine protease 3 (PR3) and the P-ANCA pattern with antibodies to myeloperoxidase (MPO). However, some sera positive for ANCA by IF are negative for anti-PR3 and anti-MPO antibodies, suggesting the presence of antibodies to minor antigens of PMN granules. We tested sera from a previously well-defined clinical cohort of patients for antibodies to four possible minor antigens: bactericidal permeability increasing protein, elastase, cathepsin G and lactoferrin. IF-positive (+) sera had significantly higher antibody frequencies to the minor antigens than did the IF-negative (-) sera (P < 0.01). Patients with IF(+) PR3(-)MPO(-) sera showed the most varied reactivity to the minor antigens. Among the IF(+) groups, the IF(+) PR3(+)/MPO(-) sera showed the lowest reactivity to the minor antigens. Patients with well-defined ANCA specificities, e.g. the PR3-ANCA response associated with Wegener's granulomatosis, are less likely than are other patient subsets to have antibodies to minor antigen targets. Autoantibodies to these minor antigens contribute to the overall pattern of ANCA identified by IF and help to explain why the correlation between IF and enzyme immunoassays show discrepancies. While the pathophysiological significance of antibodies to minor target antigens needs further evaluation, they may be markers of inflammation associated with disease processes.     

Circulating antibodies to heat-shock protein 60 in Crohn''s disease and ulcerative colitis.

Heat-shock proteins (HSPs) are highly conserved immunogenic intracellular molecules that are induced by inflammatory mediators and may induce autoimmune phenomena in vivo. We have recently demonstrated the increased expression of HSP-60 in the colonocytes of patients with ulcerative colitis. To study further the role of HSP-60 in inflammatory bowel disease, we have now measured antibodies to recombinant mycobacterial HSP-65 (a member of the HSP-60 family) in patients with Crohn's disease, ulcerative colitis, healthy volunteers and, as disease controls, patients with confirmed bacterial diarrhoea. In comparison with healthy controls (n = 20; median level of 89 ELISA units; range 24-292), serum IgA HSP-60 antibodies were elevated in Crohn's disease (n = 21; 157; 57-364; P < 0.05) and active ulcerative colitis (n = 16; 188; 58-373; P < 0.01) but not bacterial diarrhoea (n = 10; 106; 51-285). Increased IgA HSP-60 antibody levels in patients with inflammatory bowel disease may occur as the result of HSP release from injured gut epithelium; alternatively, increased intestinal permeability could facilitate mucosal access of luminal antigens and the generation of cross-reactive anti-bacterial HSP antibodies.     

Genetic markers in clinically well defined patients with ulcerative colitis (UC)

Results of genetic association studies in UC are conflicting. We propose that the power of candidate gene studies will increase when disease heterogeneity is taken into account. Phenotype frequencies of molecularly defined HLA-DR alleles, polymorphisms in the tumour necrosis factor-alpha (TNF-α), lymphotoxin-alpha (LT-α), IL-1 receptor antagonist (IL-1Ra) and IL-1β genes were determined in 98 clinically well characterized UC patients with a mean period of follow up of 10 years, and ethnically matched healthy controls (HC). The alleles HLA-DRB1*0103 (phenotype frequency 6% versus 0.2%; P = 0.0002; odds ratio (OR) 27.6) and DRB1*15 (41% versus 26%; P = 0.001; OR = 2.0, compared with HC) were associated with overall disease susceptibility. Subgroup analysis revealed that DRB1*15 was only increased in females (53% versus 24%; P < 0.0001; OR = 3.5), but not in males. With regard to disease localization, all DRB1*0103⁺ patients had extensive disease (P < 0.002; OR = 33.5), and DRB1*15 was found in 59% of females with extensive colitis (P < 0.0001; OR = 4.4). DRB1*0103 was significantly increased in patients undergoing colectomy (P < 0.0002; OR = 84). No association between overall disease susceptibility and the cytokine gene polymorphisms were found. Subgroup analysis revealed several significant associations, but most did not retain significance when corrected for multiple comparisons. However, a noticeable finding was that haplotype TNF-C was significantly associated with progression in extent of disease (P = 0.003, OR = 20.4). This study provides additional evidence for the role of DRB1 alleles in the susceptibility to UC, and supports the hypothesis that these alleles may determine the severity of the disease. The cytokine gene polymorphisms evaluated in this study do not seem to be strong risk factors for the overall disease susceptibility in UC, but may be involved in determining the severity of the disease.     

Expression of a unique protein on colon cancer cells that reacts with a novel monoclonal antibody and ulcerative colitis serum.

We earlier developed a MoAb, 7E12H12 (IgM isotype), against a protein present in normal colonic epithelial cells. To examine if 7E12H12-reactive protein is expressed in colon cancer cells and is recognized by ulcerative colitis (UC)-associated autoantibody, we investigated several colon cancer cell lines. 7E12H12 reactivity against the cells was examined by indirect immunofluorescence assay and whole cell ELISA against six colon cancer cell lines HT-29, LoVo, COLO 205, DLD-1, LS 180 and SW 1116. A competitive ELISA was developed using 7E12H12 MoAb and patients' serum to examine the cross-reactive antibodies in the serum. Among the six colon cancer cell lines only LS 180, DLD-1 and SW 1116 reacted with 7E12H12 MoAb, while others did not. The mean (+/- s.e.m.) inhibition of the binding of 7E12H12 MoAb to LS 180 cells by UC serum (n = 51) was 42 +/- 2.1%, whereas in normal subjects (n = 17) it was 14 +/- 2.6%, in Crohn's disease (n = 19) it was 15.3 +/- 2.5%, in infectious diarrhoea (n = 10) it was 11% +/- 3%, and in systemic lupus erythematosus (n = 10) it was 2% +/- 0.6%. The inhibition by the UC group was significantly (P < 0.001 - < 0.0001) higher than any of the non-UC groups, and this inhibition was mainly by IgG1 antibody. The protein in the specific colon cancer cells recognized by the 7E12H12 MoAb cross-reacts with UC-IgG1 antibody and may provide an in vitro system to examine the autoimmune mechanisms in UC.     

Aberrant plasmacytoid dendritic cell distribution and function in patients with Crohn's disease and ulcerative colitis

Dendritic cell (DC) function is believed to be of critical importance for the pathogenesis of inflammatory bowel disease (IBD). To date, most research in animal models and the few human data available is restricted to myeloid DC, while plasmacytoid DC (pDC) capable of controlling both innate and adaptive immune responses have not yet been investigated systematically in human Crohn's disease (CD) or ulcerative colitis (UC). CD11c^‐, CD303⁺/CD304⁺ and CD123⁺ pDC from peripheral blood (n = 90), mucosal tissue (n = 28) or mesenteric lymph nodes (n = 40) (MLNs) of patients with UC and CD or controls were purified and cultured. Thereafter, pDC were enumerated, phenotyped and cytokine secretion measured by flow cytometry (FACS), immunohistochemistry and/or cytometric bead array, respectively. Interferon (IFN)‐α secretion following cytosine phosphatidyl guanine (CpG) A oligodeoxynucleotide (ODN) 2216 (5′‐GGGGGACGATCGTCGGGGGG‐3′) stimulation was assessed by enzyme‐linked immunosorbent assay (ELISA). We found a significantly higher frequency of pDC in the inflamed colonic mucosa and MLN of IBD patients. Moreover, the fraction of CD40 and CD86 expressing cultured peripheral blood pDC was significantly higher in flaring UC and CD patients and their secretion of tumour necrosis factor (TNF)‐α, interleukin (IL)‐6 and IL‐8 were increased significantly compared with controls. In contrast, the IFN‐α secretion of peripheral blood pDC isolated from flaring IBD, particularly in UC patients, was reduced significantly compared with controls. Our data suggest an aberrant distribution and function of pDC in IBD, contrary to their generally implicated role as inducers of tolerance. We speculate that the impaired IFN‐α secretion may relate to the hypothesized defect in innate immunity in IBD and could also impact upon the generation of regulatory T cells (T_reg).     

Autoimmunity to tropomyosin isoforms in ulcerative colitis (UC) patients and unaffected relatives

Autoimmunity to cytoskeletal protein tropomyosin (TM) has been demonstrated in UC. However, the TM isoforms involved in this IgG-mediated autoimmune response in UC and the possible presence of serum IgG antibodies against TM (hTMs IgG) in unaffected UC relatives are unknown. The aim of this study was to investigate the human TM (hTM) isoforms recognized by serum IgG from UC and to explore whether hTM IgG antibodies are present in healthy UC relatives. We studied 33 UC patients with 58 unaffected relatives, 31 Crohn's disease (CD) patients with 31 unaffected relatives and 20 controls (C). Serum IgG against four recombinant hTM isoforms (hTM1, 2, 3, 5) were tested by ELISA. p-ANCA were tested by ELISA and immunofluorescence. Serum hTM1 and hTM5 IgG were higher in UC patients than in CD and C (P < 0.005). Among UC patients 52% were seropositive for hTM1 and 64% for hTM5 (P < 0.001 versus CD and C). In UC, hTM5 IgG were higher in p-ANCA⁺ than in ANCA^? patients (P = 0.04). In UC relatives hTM1 IgG were higher than in CD relatives and C (P < 0.01). UC relatives were more frequently seropositive for hTM1 than hTM5 IgG (P = 0.001), while probands were more frequently seropositive for hTM5 IgG (P = 0.008). We conclude that autoimmunity to hTM1 and hTM5 is a feature of UC, while hTM1 IgG differentiate UC relatives from controls. A genetic susceptibility to immune recognition of hTM isoforms in UC is suggested.