Martin W. Laass

Identification of GP2, the major zymogen granule membrane glycoprotein, as the autoantigen of pancreatic antibodies in Crohn’s disease

Backround and aims: The aetiopathogenesis of Crohn’s disease, an inflammatory bowel disease (IBD), is not yet fully understood. Autoimmune mechanisms are thought to play a role in the development of Crohn’s disease, but the target antigens and the underlying pathways have not been sufficiently identified. Methods: Based on data from immunoblotting and matrix-assisted laser desorption ionisation time-of-flight (MALDI-TOF) mass spectrometry, the major antigenic target of pancreatic autoantibodies (PABs), which are specific for Crohn’s disease, was identified. Specificity of autoantibody reactivity was confirmed by enzyme-linked immunosorbent assay (ELISA) and indirect immunofluorescence (IIF) using purified rat and human recombinant GP2 synthesised in transiently transfected mammalian HEK 293 cells. Real-time polymerase chain reaction (rt-PCR) and IIF were used to detect mRNA and antigen localisation in human colon biopsies. Results: The major zymogen granule membrane glycoprotein 2 (GP2) was identified as the autoantigen of PABs in Crohn’s disease. PAB-positive sera from patients with Crohn’s disease (n = 42) displayed significantly higher IgG reactivity to rat GP2 in ELISA than either PAB-negative sera (n = 31), or sera from patients with ulcerative colitis (n = 49), or sera from blood donors (n = 69) (p<0.0001, respectively). Twenty-eight (66%) and 18 (43%) of 42 PAB-positive sera demonstrated IgG and IgA reactivity to human recombinant GP2 in IIF, respectively. Patients with PAB-negative Crohn’s disease (n = 31) were not reactive. GP2 mRNA transcription was significantly higher in colon biopsies from patients with Crohn’s disease (n = 4) compared to patients with ulcerative colitis (n = 4) (p = 0.0286). Immunochemical staining confirmed GP2 expression in human colon biopsies from patients with Crohn’s disease. Conclusion: Anti-GP2 autoantibodies constitute novel Crohn’s disease-specific markers, the quantification of which could significantly improve the serological diagnosis of IBD. The expression of GP2 in human enterocytes suggests an important role for anti-GP2 response in the pathogenesis of Crohn’s disease.

Diagnosis and classification of Crohn's disease

Crohns disease (CrD) is a chronic relapsing inflammatory bowel disease (IBD) potentially affecting any portion of the gastrointestinal tract from the mouth to the anus. CrD usually manifests between 15 and 30 years of age and presents typically with abdominal pain, fever, bloody or non-bloody diarrhoea, and weight loss. Paediatric patients may show failure to thrive, growth impairment, and delayed puberty additionally. Extraintestinal manifestations like arthritis, uveitis, and erythema nodosum are diagnosed in almost half of the patients. CrD is characterized by a discontinuous and ulcerous transmural inflammation often involving the ileocaecal region and leading to a stricturing or even fistulising phenotype in up to 50% of patients finally. Incidence and prevalence of CrD have been rising worldwide over the past decades. Although many details of the pathophysiology of CrD have been elucidated, no common aetiopathogenic model exists for all forms of CrD, presenting more an umbrella term for a phenotypically and genotypically heterogeneous clinical condition. In CrD, we see an inappropriate response of the innate and/or adaptive immune system to the intestinal microbiota in genetically predisposed individuals. The diagnosis of CrD is based mainly on patients history and clinical examination and supported by serologic, radiologic, endoscopic, and histologic findings. Antibodies to Saccharomyces cerevisiae and autoantigenic targets such as glycoprotein 2 may aid in differentiating CrD from UC. Their single use, however, is limited by low sensitivity requiring antibody profiling for an appropriate serologic diagnosis. This review focuses on diagnostic and classification criteria of CrD.

Diagnosis and classification of ulcerative colitis

Ulcerative colitis (UC) is a chronic relapsing inflammatory bowel disease (IBD) characterised by superficial mucosal ulceration, rectal bleeding, diarrhoea, and abdominal pain. In contrast to Crohns disease (CrD), UC is restricted to the colon and the inflammation is limited to the mucosal layer. Classic UC affects the colon in a retrograde and continuous fashion starting from the rectum and extending proximally. Dependent on the anatomic extent of involvement, UC can be classified as proctitis, left-sided colitis, or pancolitis. Inflammatory arthropathies and primary sclerosing cholangitis (PSC) are the most common and clinically most important extraintestinal manifestations of UC. The aetiopathogenesis of UC is incompletely understood, but immune-mediated mechanisms are responsible for dysregulated immune responses against intraluminal antigens in genetically predisposed individuals. The diagnosis is based on the history, as well as clinical, radiological, endoscopic and histological features. Autoantibodies, mainly antineutrophil cytoplasmic antibodies (ANCA) and anti-goblet cell antibodies (GAB) may be helpful in the early diagnosis of UC and in differentiating it from CrD.

XIAP variants in male Crohn's disease

Objective The genetic basis of inflammatory bowel disease (IBD) is incompletely understood. The aim of this study was to identify rare genetic variants involved in the pathogenesis of IBD. Design Exome sequencing and immunological profiling were performed in a patient with early onset Crohns disease (CD). The coding region of the gene encoding X-linked inhibitor of apoptosis protein (XIAP) was sequenced in samples of 275 paediatric IBD and 1047 adult-onset CD patients. XIAP genotyping was performed in samples of 2680 IBD patients and 2864 healthy controls. Functional effects of the variants identified were investigated in primary cells and cultured cell lines. Results Our results demonstrate the frequent occurrence of private variants in XIAP in about four percent of male patients with paediatric-onset CD. While XIAP mutations are known to be associated with the primary immunodeficiency (PID) X-linked lymphoproliferative disease type 2 (XLP2), CD patients described here exhibited intestinal inflammation in the absence of XLP2 and harboured a spectrum of mutations partially distinct from that observed in XLP2. The majority of XIAP variants identified was associated with a selective defect in NOD1/2 signalling, impaired NOD1/2-mediated activation of NF-κB, and altered NF-κB-dependent cytokine production. Conclusions This study reveals the unanticipated, frequent occurrence of XIAP variants in male paediatric-onset CD. The link between XIAP and NOD1/2, and the association of XIAP variants with XLP2, support the concept of PID in a subset of IBD patients. Moreover, these studies provide a rationale for the implementation of XIAP sequencing in clinical diagnostics in male patients with severe CD.

Probiotic Escherichia coli Nissle 1917 versus placebo for treating diarrhea of greater than 4 days duration in infants and toddlers.

Background: Administering probiotics can prevent or cure some forms of diarrhea. The efficacy of probiotic Escherichia coli Nissle 1917 (EcN) in infants and toddlers with diarrhea >4 days was tested by a double-blind trial. Methods: One hundred fifty-one children aged 1–47 months with nonspecific diarrhea were randomized to receive either EcN suspension (N = 75) or placebo (N = 76). Diarrhea had to meet the following definition: >3 watery or loose nonbloody stools in 24 hours of a diarrheal episode persisting for >4 consecutive days but ≤14 days. All children were well nourished or only moderately malnourished, mildly dehydrated, and received oral rehydration at study commencement. They were treated orally with 1–3 mL EcN suspension (1 mL contains 108 viable cells) or placebo daily for 21 days. Primary objective was to confirm a better response rate (reduction of daily stool frequency to ≤3 watery or loose stools over ≥4 days) with EcN. Results: The 7-day response was higher for the EcN group than placebo (EcN 78.7%, placebo 59.2%). Significant differences were observed on days 14 (EcN 93.3%, placebo 65.8%, P = 0.0017) and 21 (EcN 98.7%, placebo 71.1%, P < 0.001). Kaplan-Meier survival analysis resulted in a significant difference of 3.3 days between the groups (P < 0.0001); median time to response for EcN was 2.4 and 5.7 for placebo. EcN was safe and well tolerated. Conclusions: In the conditions of this trial EcN was a suitable remedy for diarrhea >4 days in young children.

IgG Antibodies against Deamidated Gliadin Peptides for Diagnosis of Celiac Disease in Patients with IgA Deficiency

BACKGROUND Assays for IgG antibodies against deamidated gliadin (IgG-anti-dGli) are comparable in performance with tests detecting IgA antibodies against tissue transglutaminase (IgA-anti-tTG) in diagnosing celiac disease (CD). IgA-anti-tTG are absent in IgA deficiency, a condition often associated with CD. In IgA deficiency, IgG-anti-tTG, which have a lower overall diagnostic accuracy, are routinely measured. We examined whether IgG-anti-dGli would be useful for diagnosing CD in patients with IgA deficiency. METHODS We studied 34 IgA-deficient CD patients, 185 IgA-competent newly diagnosed children with CD, 316 children without CD, 400 adult blood donors, and 6 control IgA-deficient individuals without CD. Anti-dGli and anti-tTG were measured by ELISA, and endomysium antibodies (EmA) were measured by immunofluorescence on monkey esophagus (IgA as well as IgG class for all antibodies). We calculated diagnostic sensitivity (percentage of patients above cutoff with 95% CIs) according to age-specific cutoffs for 95% diagnostic specificity and according to cutoffs proposed by the manufacturer of the assays. RESULTS No IgA-deficient CD patients were positive for any IgA-based antibody assay. Diagnostic sensitivity of IgG-anti-tTG was 91.2% (95% CI 76.3%-97.7%) according to age-specific cutoffs and 82.4% (66.1%-92.0%) according to manufacturer cutoffs. The diagnostic sensitivity of IgG-EmA was 75.8% (58.8%-87.4%) and the sensitivity of IgG-anti-dGli was 88.2% (72.8%-95.9%) according to both cutoffs. CONCLUSIONS IgG-anti-dGli and IgG-anti-tTG have comparable diagnostic sensitivities for IgA-deficient celiac patients. IgG-anti-dGli may be useful for diagnosing CD in IgA-deficient patients.

Autoantibodies to GP2, the major zymogen granule membrane glycoprotein, are new markers in Crohn's disease

BACKGROUND Crohns disease (CD) is an inflammatory bowel disease (IBD) characterized by reactivity against microbial and self antigens. Zymogen granule glycoprotein 2 (GP2) was identified as the major autoantigen of CD-specific pancreatic autoantibodies (PAB). METHODS Human GP2 was expressed in the Spodoptera frugiperda 9 (Sf9) cell line using the baculovirus system, purified by Ni-chelate chromatography, and used as antigen for anti-GP2 IgA and IgG assessment by enzyme-linked immunosorbent assays (ELISA). Antibodies to mannan of Saccharomyces cerevisiae (ASCA), PAB, and anti-GP2 were investigated in sera of 178 CD patients, 100 ulcerative colitis (UC) patients, and 162 blood donors (BD). RESULTS Anti-GP2 IgG and IgA were found in 48/72 (66.7%) and 23/72 (31.9%) PAB positive and 5/106 (4.7%) and 1/106 (0.9%) PAB negative CD patients (p<0.0001), respectively. CD patients displayed significantly higher reactivity to GP2 than UC patients and BD (p<0.0001), respectively. Occurrence of anti-GP2 antibodies correlated with PAB reactivity (Spearmens rho=0.493, p<0.00001). There was a significant relationship between the occurrence of ASCA IgG and anti-GP2 IgG (p=0.0307). CONCLUSIONS Anti-GP2 IgG and IgA constitute novel CD specific autoantibodies, the quantification of which could improve the serological diagnosis of IBD.

Diagnostic value, clinical utility and pathogenic significance of reactivity to the molecular targets of Crohn's disease specific-pancreatic autoantibodies

Pancreatic autoantibodies (PAB) giving characteristic staining patterns of the exocrine pancreas by indirect immunoflourescence appear to be specific markers of Crohns disease (CrD), being present in approximately 30% of patients with CrD and in less than 5% of patients with ulcerative colitis (UC). Some studies have suggested that PAB are associated with specific disease phenotypes and that their detection may be of clinical significance. Thorough investigation of the role of PAB in the immunopathogenesis of inflammatory bowel diseases (IBD) has been hampered due to the lack of identity of their antigenic targets. The recent identification of the pancreatic zymogen granule protein 2 (GP2) as the major target of PAB has led to the development of an enzyme immunoassay that helps determine the diagnostic and clinical relevance of antigen-specific immune responses. Recent studies have demonstrated that GP2 is expressed on the apical surface of intestinal membranous cells of the follicle-associated epithelium, and is essential for host-microbial interaction and the initiation of bacteria-specific mucosal immune responses. This review critically discusses recent reports investigating the diagnostic and clinical utility of GP2-specific autoantibody responses in patients with IBD. Hints towards a better understanding of the immunogenicity of GP2 are also provided.

Antibodies against deamidated gliadin as new and accurate biomarkers of childhood coeliac disease.

Objectives: Assays of tissue transglutaminase antibodies (anti-tTG) represent the cornerstone of serological coeliac disease (CD) diagnostics. Assays of antibodies against native gliadin (anti-nGli) lost importance due to low validity. We investigated the performance of new assays for antibodies against deamidated gliadin (anti-dGli) in childhood CD. Methods: We retrospectively compared children (142 with active CD and 160 without CD, diagnosis confirmed or excluded by intestinal biopsy) concerning (immunoglobulin [Ig] G and IgA) anti-nGli, anti-tTG, and 2 different anti-dGli assays. Results: IgG-anti-dGli1, IgG-anti-dGli2, and IgA-anti-tTG performed similarly. Area under the receiver-operating characteristic curve (AUC) was 98.6%, 98.9%, and 97.9%; accuracy was 94.7%, 95.7%, and 96.7%. Anti-dGli1 and anti-dGli2 (IgG and IgA) and IgA-anti-tTG performed significantly better than IgA-anti-nGli and IgG-anti-nGli. Both IgG-anti-dGli showed higher AUC and accuracy than IgA-anti-dGli and IgG-anti-tTG. Combined evaluation of IgA-anti-tTG with one of the IgG-anti-dGli tests reduced the rate of falsely classified patients. At enhanced cutoff (specificity >99%), sensitivity was above 67% for both IgG-anti-dGli and IgA-anti-tTG. If IgA-anti-tTG assay was combined with one of the IgG-anti-dGli tests, then the fraction of patients identified with more than 99% specificity as coeliacs increased significantly above 84.5%. Combined evaluation of the 2 IgG-anti-dGli tests did not improve the performance. Conclusions: The new IgA and IgG-anti-dGli tests outperform conventional anti-nGli assays. The validity of IgG-anti-dGli cannot be distinguished from IgA-anti-tTG. It should be studied prospectively whether antibody assays could replace biopsy in diagnosis of CD in a substantial segment of children.

Pancreatic-specific autoantibodies to glycoprotein 2 mirror disease location and behaviour in younger patients with Crohn's disease.

BackgroundGlycoprotein 2 (GP2) was discovered as the major autoantigen of Crohn’s disease (CD)-specific pancreatic autoantibodies (PAB). We investigated anti-GP2 IgA and IgG antibodies as novel serological parameters in CD and assessed their association with distinct disease phenotypes.MethodsAnti-GP2 and anti-Saccharomyces cerevisiae (ASCA) IgA and IgG were detected by ELISA employing recombinant human GP2 and phosphopeptidomannan, respectively and PAB by indirect immunofluorescence (IIF) in 271 sera, 169 with CD and 102 with ulcerative colitis (UC). As healthy controls 160 adult blood donors and 65 children were included.ResultsAnti-GP2 IgG and/or IgA were more prevalent in CD (51/169, 30.2%) than in UC (9/102, 8.9%) patients and in controls (9/225, 4%) (p < 0.001 respectively). ASCA IgG and/or IgA were present in 60/169 (35.5%) in CD and in 7/102 (6.9%) in UC patients (p < 0.001). CD patients with ileocolonic location (L3) showed a significantly higher prevalence of anti-GP2 and ASCA IgA and/or IgG (40/113 and 48/113, respectively; p < 0.05 for both comparisons), whereas CD patients with colonic location (L2) revealed a significantly diminished prevalence for these autoantibody specificities (2/32 and 5/32, respectively, p < 0.05 for both). Anti-GP2 IgG were significantly more prevalent in CD patients with stricturing behaviour (B2) and perianal disease (7/11, p < 0.02) and less prevalent in those with penetrating behaviour (B3) and perianal disease (4/31, p < 0.05). The occurrence of anti-GP2 IgA and/or IgG was significantly more prevalent in CD patients with age at diagnosis of ≤16 years (16/31, p < 0.009). Prevalence of one or more anti-GP2 or ASCA IgA and/or IgG was significantly higher in L3, B2, and A1 and lower in L2 (68/113, 27/41, 23/31, 6/32; p < 0.04, respectively).ConclusionsAnti-GP2 IgG and IgA, constituting novel CD specific autoantibodies, appear to be associated with distinct disease phenotypes identifying patients at a younger age, with ileocolonic location, and stricturing behaviour with perianal disease.