New serological markers in inflammatory bowel disease are associated with complicated disease behaviour.

BACKGROUND AND AIMS: Several antibodies have been associated with Crohn's disease and are associated with distinct clinical phenotypes. The aim of this study was to determine whether a panel of new antibodies against bacterial peptides and glycans could help in differentiating inflammatory bowel disease (IBD), and whether they were associated with particular clinical manifestations. METHODS: Antibodies against a mannan epitope of Saccharomyces cerevisiae (gASCA), laminaribioside (ALCA), chitobioside (ACCA), mannobioside (AMCA), outer membrane porins (Omp) and the atypical perinuclear antineutrophilic cytoplasmic antibody (pANCA) were tested in serum samples of 1225 IBD patients, 200 healthy controls and 113 patients with non-IBD gastrointestinal inflammation. Antibody responses were correlated with the type of disease and clinical characteristics. RESULTS: 76% of Crohn's disease patients had at least one of the tested antibodies. For differentiation between Crohn's disease and ulcerative colitis, the combination of gASCA and pANCA was most accurate. For differentiation between IBD, healthy controls and non-IBD gastrointestinal inflammation, the combination of gASCA, pANCA and ALCA had the best accuracy. Increasing amounts and levels of antibody responses against gASCA, ALCA, ACCA, AMCA and Omp were associated with more complicated disease behaviour (44.7% versus 53.6% versus 71.1% versus 82.0%, p < 0.001), and a higher frequency of Crohn's disease-related abdominal surgery (38.5% versus 48.8% versus 60.7% versus 75.4%, p < 0.001). CONCLUSIONS: Using this new panel of serological markers, the number and magnitude of immune responses to different microbial antigens were shown to be associated with the severity of the disease. With regard to the predictive role of serological markers, further prospective longitudinal studies are necessary.

Antibodies against laminaribioside and chitobioside are novel serologic markers in Crohn's disease.

BACKGROUND & AIMS: New serologic markers of inflammatory bowel disease may be useful for differentiating between Crohn's disease and ulcerative colitis and for disease stratification. We profiled sugar-binding antibodies to identify novel antiglycan antibodies that may be associated with inflammatory bowel disease. METHODS: Serum samples were obtained from patients with diagnosed Crohn's disease or ulcerative colitis and from control patients. The presence of antiglycan antibodies was evaluated using either a glycan array (GlycoChip; Glycominds, Ltd, Lod, Israel) in patients with Crohn's disease (n = 72) or ulcerative colitis (n = 56) and in healthy controls (n = 41) or using an enzyme-linked immunosorbent assay in patients with Crohn's disease (n = 124), ulcerative colitis (n = 106), and in control patients (n = 101). RESULTS: Inaddition to antibodies against mannan, antibodies to laminaribioside (Glc[beta1,3]Glc[beta]) and chitobioside (GlcNAc[beta1,4]GlcNAc[beta]) had the highest discriminative capability between Crohn's disease and ulcerative colitis (P < .001 and P < .05, respectively). Importantly, 44% (12/27) of anti-Saccharomyces cerevisiae antibody-negative Crohn's disease patients were positive for antilaminaribioside or antichitobioside. In patients with inflammatory bowel disease positive for antibodies against either laminaribioside, chitobioside, or mannan, the diagnosis of Crohn's disease was suggested with a sensitivity of 77.4% and specificity of 90.6%. Having at least 2 of these antibodies increased the specificity to 99.1%. In Crohn's disease, higher levels of antibodies against laminaribioside or mannan were significantly associated with small intestinal disease (P = .03 and P < .0001, respectively). CONCLUSIONS: Antilaminaribioside and antichitobioside carbohydrate antibodies are novel serologic markers associated with Crohn's disease. These antibodies may contribute to the diagnosis and improved stratification of Crohn's disease.

Intact cell adhesion to glycan microarrays.

A rapid and reproducible method was developed to detect and quantify carbohydrate-mediated cell adhesion to glycans arrayed on glass slides. Monosaccharides and oligosaccharides were covalently attached to glass slides in 1.7-mm-diameter spots (200 spots/slide) separated by a Teflon gasket. Primary chicken hepatocytes, which constitutively express a C-type lectin that binds to nonreducing terminal N-acetylglucosamine residues, were labeled with a fluorescent dye and incubated in 1.3-microL aliquots on the glycosylated spots. After incubating to allow cell adhesion, nonadherent cells were removed by immersing the slide in phosphate buffered saline, inverting, and centrifuging in a sealed custom acrylic chamber so that cells on the derivatized spots were subjected to a uniform and controlled centrifugal detachment force while avoiding an air-liquid interface. After centrifugation, adherent cells were fixed in place and detected by fluorescent imaging. Chicken hepatocytes bound to nonreducing terminal GlcNAc residues in different linkages and orientations but not to nonreducing terminal galactose or N-acetylgalactosamine residues. Addition of soluble GlcNAc (but not Gal) prior to incubation reduced cell adhesion to background levels. Extension of the method to CD4+ human T-cells on a 45-glycan diversity array revealed specific adhesion to the sialyl Lewis x structure. The described method is a robust approach to quantify selective cell adhesion using a wide variety of glycans and may contribute to the repertoire of tools for the study of glycomics.

In situ activation pattern of Met docking site following renal injury and hypertrophy.

BACKGROUND: Hepatocyte growth factor/scatter factor (HGF/SF) binds to its tyrosine kinase receptor, Met, thereby stimulating diverse cellular responses. The multifunctional docking site in the C-terminal domain mediates the signal of phosphorylated Met receptors to multiple transducers. The tyrosine at position 1356 of the Met docking site is crucial for cell motility and morphogenesis. METHODS: We examined the in situ distribution patterns of the Tyr1356-phosphorylated form of Met with a novel monoclonal antibody following renal injury and renal hypertrophy in rats. Sections of the kidney following either sham operation, transient ischaemia of one kidney or unilateral nephrectomy were analysed using indirect immunofluorescence staining and confocal laser scanning microscopy analysis of total Met protein levels and Tyr1356-phosphorylated Met (Met and pMet, respectively). RESULTS: At 6 h post-treatment, pMet increases in ischaemic kidneys compared with sham-operated kidneys, and these changes become substantial after 48 h in both medulla and cortex of ischaemic kidneys (P < 0.001). We also show significant up-regulation of Met predominantly in the medulla of ischaemic kidneys, 48 h following injury (P < 0.009). Inter-estingly, the stimulus for hypertrophy in the remnant kidney after uninephrectomy and the contra-lateral kidney during ischaemia is not accom-panied by significant up-regulation of Met or pMet staining compared with sham operation at both time points. CONCLUSIONS: We demonstrate in this work, for the first time, in situ detection of tyrosine kinase growth factor receptor docking site activation during pathological processes in the kidney. Using this methodology, we show a significant increase in Met docking site activity in both renal medulla and cortex solely following stimulation by ischaemia and repair.

A new kind of carbohydrate array, its use for profiling antiglycan antibodies, and the discovery of a novel human cellulose-binding antibody.

In this study, we use a novel glycan array to analyze the glycan-binding antibody repertoire in a pool of affinity-purified IgG collected from a healthy human population. The glycan array used is based on mono- and oligosaccharides covalently linked to the surface via a long linker at their reducing ends. They are thus presented to the medium with a well-defined orientation and are accessible for specific binding by glycan-binding proteins, such as antibodies and lectins. A novel anticellulose antibody was detected that binds specifically to beta4-linked saccharides with a preference for glucopyranose over galactopyranose residues. We also found previously known antiglycan antibodies against mono- and oligosaccharides that are constituents of commonly occurring bacterial polysaccharides. We propose that this array can facilitate high-throughput screening of glycan-binding proteins and the search for biomarkers for personalized medicine.