Creation of a gene expression classifier for predicting Parkinson's disease rate of progression.

Parkinson's disease (PD) etiology is heterogeneous, genetic, and multi-factorial, resulting in a varied disease from a mild slow progression to a more severe rapid progression. Prognostic information on the nature of the patient's disease at diagnosis aids the physician in counseling patients on treatment options and life planning. In a cohort of PD patients from the PPMI study, the relative gene expression levels of SKP1A, UBE2K, ALDH1A1, PSMC4, HSPA8 and LAMB2 were measured in baseline blood samples by real-time quantitative PCR. At baseline PD patients were up to 2 years from diagnosis, H&Y scale ≤ 2 and PD treatment naïve. PD-Prediction algorithm comprised of ALDH1A1, LAMB2, UBE2K, SKP1A and age was created by logistic regression for predicting progression to ≤ 70% Modified Schwab and England Activities of Daily Living (S&E-ADL). In relation to patients negative for PD-Prediction (n = 180), patients positive (n = 30) for Cutoff-1 (at 82% specificity, 80.0% sensitivity) had positive hazard ratio (HR+) of 10.6 (95% CI, 2.2-50.1), and positive (n = 23) for Cutoff-2 (at 93% specificity, 47% sensitivity) had HR+ of 17.1 (95% CI, 3.2-89.9) to progress to ≤ 70% S&E-ADL within 3 years (P value < 0.0001). Likewise, patients positive for PD-Prediction Cutoff-1 (n = 49) had HR+ 4.3 (95% CI, 1.6-11.6) for faster time to H&Y 3 in relation to patients negative (n = 170) for PD-Prediction (P value = 0.0002). Our findings show an algorithm that seems to predict fast PD progression and may potentially be used as a tool to assist the physician in choosing an optimal treatment plan, improving the patient's quality of life and overall health outcome.

Serum biomarker gMS-Classifier2: predicting conversion to clinically definite multiple sclerosis.

BACKGROUND: Anti-glycan antibodies can be found in autoimmune diseases. IgM against glycan P63 was identified in clinically isolated syndromes (CIS) and included in gMS-Classifier2, an algorithm designed with the aim of identifying patients at risk of a second demyelinating attack. OBJECTIVE: To determine the value of gMS-Classifier2 as an early and independent predictor of conversion to clinically definite multiple sclerosis (CDMS). METHODS: Data were prospectively acquired from a CIS cohort. gMS-Classifier2 was determined in patients first seen between 1995 and 2007 with ≥ two 200 µL serum aliquots (N = 249). The primary endpoint was time to conversion to CDMS at two years, the factor tested was gMS-Classifier2 status (positive/negative) or units; other exploratory time points were 5 years and total time of follow-up. RESULTS: Seventy-five patients (30.1%) were gMS-Classifier2 positive. Conversion to CDMS occurred in 31/75 (41.3%) of positive and 45/174 (25.9%) of negative patients (p = 0.017) at two years. Median time to CDMS was 37.8 months (95% CI 10.4-65.3) for positive and 83.9 months (95% CI 57.5-110.5) for negative patients. gMS-Classifier2 status predicted conversion to CDMS within two years of follow-up (HR = 1.8, 95% CI 1.1-2.8; p = 0.014). gMS-Classifier2 units were also independent predictors when tested with either Barkhof criteria and OCB (HR = 1.2, CI 1.0-1.5, p = 0.020) or with T2 lesions and OCB (HR = 1.3, CI 1.1-1.5, p = 0.008). Similar results were obtained at 5 years of follow-up. Discrimination measures showed a significant change in the area under the curve (ΔAUC) when adding gMS-Classifier2 to a model with either Barkhof criteria (ΔAUC 0.0415, p = 0.012) or number of T2 lesions (ΔAUC 0.0467, p = 0.009), but not when OCB were added to these models. CONCLUSIONS: gMS-Classifier2 is an independent predictor of early conversion to CDMS and could be of clinical relevance, particularly in cases in which OCB are not available.

Anti-GalNAcß: a novel anti-glycan autoantibody associated with pregnancy loss in women with antiphospholipid syndrome and in a mouse experimental model.

OBJECTIVES: We evaluated the presence of anti-glycan antibodies (aGA) in patients with antiphospholipid syndrome (APS), and associations between aGA and clinical features of the disease. METHODS: Sera from APS patients and healthy controls were analyzed for aGA levels by ELISA. Analysis of the association of specific aGA with clinical manifestations of APS was performed. Selected aGA were affinity-purified and injected intravenously into naive mice which were tested for fetal loss. Matrigel invasion assay was performed for detection of choriocarcinoma cells (JAR) invasion and proliferation in the presence of selected aGA. Culture fluid of JAR invasion assays was analyzed for the presence of MMP2 and MMP9. RESULTS: High levels of several aGA were found in APS sera, of which anti-GalNAc-ß was significantly associated with recurrent pregnancy loss. Naive mice infused intravenously with anti-GalNAc-ß developed increased fetal loss. Anti-GalNAc-ß significantly inhibited the in-vitro percentage of JAR invasiveness and the secretion of MMP2 and MMP9 by human JAR cells. CONCLUSIONS: APS sera contain significant levels of aGA directed against several glycans. Anti-GalNAc-ß Ab is specifically associated with recurrent pregnancy loss both in human patients and experimental mouse model. The pathogenic effects of anti-GalNAc-ß include inhibition of JAR cells invasiveness accompanied by decreased MMP2 and MMP9 secretion.

Characterization of changes in serum anti-glycan antibodies in Crohn's disease--a longitudinal analysis.

INTRODUCTION: Anti-glycan antibodies are a promising tool for differential diagnosis and disease stratification of patients with Crohn's disease (CD). We longitudinally assessed level and status changes of anti-glycan antibodies over time in individual CD patients as well as determinants of this phenomenon. METHODS: 859 serum samples derived from a cohort of 253 inflammatory bowel disease (IBD) patients (207 CD, 46 ulcerative colitis (UC)) were tested for the presence of anti-laminarin (Anti-L), anti-chitin (Anti-C), anti-chitobioside (ACCA), anti-laminaribioside (ALCA), anti-mannobioside (AMCA) and anti-Saccharomyces cerevisiae (gASCA) antibodies by ELISA. All patients had at least two and up to eleven serum samples taken during the disease course. RESULTS: Median follow-up time for CD was 17.4 months (Interquartile range (IQR) 8.0, 31.6 months) and for UC 10.9 months (IQR 4.9, 21.0 months). In a subgroup of CD subjects marked changes in the overall immune response (quartile sum score) and levels of individual markers were observed over time. The marker status (positive versus negative) remained widely stable. Neither clinical phenotype nor NOD2 genotype was associated with the observed fluctuations. In a longitudinal analysis neither changes in disease activity nor CD behavior led to alterations in the levels of the glycan markers. The ability of the panel to discriminate CD from UC or its association with CD phenotypes remained stable during follow-up. In the serum of UC patients neither significant level nor status changes were observed. CONCLUSIONS: While the levels of anti-glycan antibodies fluctuate in a subgroup of CD patients the antibody status is widely stable over time.

Association of the novel serologic anti-glycan antibodies anti-laminarin and anti-chitin with complicated Crohn's disease behavior.

BACKGROUND: We tested a panel of novel serological anti-glycan antibodies including the previously unpublished anti-laminarin IgA (Anti-L) and anti-chitin IgA (Anti-C) carbohydrate antibodies for the presence in Crohn's disease (CD) patients, diagnosis and differentiation of CD, association with complicated disease behavior, and marker stability over time. METHODS: The presence of Anti-L, Anti-C, anti-chitobioside IgA (ACCA), anti-laminaribioside IgG (ALCA), anti-mannobioside IgG (AMCA), and anti-Saccaromyces cervisiae IgG (gASCA) carbohydrate antibodies were tested in serum samples from 824 participants (363 CD, 130 ulcerative colitis [UC], 74 other gastrointestinal diseases, and 257 noninflammatory bowel/gastrointestinal disease controls) of the German IBD-network by enzyme-linked immunosorbent assay (ELISA; Glycominds, Lod, Israel) and for perinuclear antineutrophil cytoplasmic antibody (pANCA) by immunofluorescence. RESULTS: In all, 77.4% of the CD patients were positive for at least 1 of the anti-glycan antibodies. gASCA or the combination of gASCA/pANCA remained most accurate for the diagnosis of CD, but the combined use of the antibodies improved differentiation of CD from UC. Several single markers as well as an increasing antibody response were independently linked to a severe disease phenotype, as shown for the occurrence of complications, CD-related surgery, early disease onset, and ileal disease location. This was observed for both quantitative and qualitative antibody responses. The antibody status remained stable over time in most IBD patients. CONCLUSIONS: A panel of anti-glycan antibodies including the novel Anti-L and Anti-C may aid in differentiation of CD from UC, is associated with complicated CD behavior and IBD-related surgery, and is stable over time in a large patient cohort.

Serum anti-glycan antibodies predict complicated Crohn's disease behavior: a cohort study.

BACKGROUND: A high proportion of patients with Crohn's disease (CD) over time develop complications like fistulae and strictures, requiring surgery. We tested a panel of antiglycan antibodies for predicting the occurrence of complications and CD-related surgery in an adult patient cohort. METHODS: Serum samples of 149 CD patients of the German inflammatory bowel disease (IBD) network were tested for the presence of anti-laminarin IgA (Anti-L), anti-chitin IgA (Anti-C), anti-chitobioside IgA (ACCA), anti-laminaribioside IgG (ALCA), anti-mannobioside IgG (AMCA), and anti-Saccaromyces cerevisiae IgG (gASCA) carbohydrate antibodies by enzyme-linked immunosorbent assay (ELISA) (IBDX(R) panel, Glycominds, Lod, Israel) in a blinded fashion. Clinical data were available on occurrence of complicated disease or CD-related surgery as well as disease activity, onset, and location. RESULTS: The median follow-up of the patients without any previous complication or surgery at time of sample procurement was 53.7 months. Overall, 26.3% developed a complication and 17.1% underwent CD-related surgery, respectively. Positivity for gASCA, AMCA, ACCA, and Anti-L alone or an increasing frequency of positive serum antibodies independently predicted a faster progression toward a more severe disease course. Once a complication or surgery had occurred only positivity for Anti-L or more than 3 markers out of the whole panel indicated progression to an additional surgery or complication. The antibody status of most patients remained stable over time. CONCLUSIONS: This is the first study showing the clinical value of serum antiglycan antibodies for prediction of a more complicated disease course in adult patients with CD.

Serum anti-GAGA4 IgM antibodies differentiate relapsing remitting and secondary progressive multiple sclerosis from primary progressive multiple sclerosis and other neurological diseases.

The serum level of IgM antibodies against Glc(alpha1,4)Glc(alpha) (GAGA4) is higher in relapsing remitting multiple sclerosis (RRMS) compared to other neurological disease (OND) patients and healthy controls (HC). Detecting the level of anti-GAGA4 antibody by enzyme immunoassay and total IgM, we confirmed that anti-GAGA4 IgM can differentiate RRMS from OND patients and HC. Moreover, secondary progressive MS (SPMS) and RRMS patients have similar levels of anti-GAGA4 demonstrating the biomarker's presence throughout the disease. Interestingly, the anti-GAGA4 assay may also differentiate between primary progressive MS (PPMS) and RRMS/SPMS patients, since nearly all PPMS patients were negative for the assay.

Novel anti-glycan antibodies related to inflammatory bowel disease diagnosis and phenotype.

OBJECTIVES: We sought to evaluate whether two novel immunoglobulin A (IgA) cell wall polysaccharide antibodies, anti-laminarin (anti-L) and anti-chitin (anti-C), aid in the diagnosis and phenotype differentiation of Crohn's disease (CD) and ulcerative colitis (UC). METHODS: A cohort of 818 individuals with inflammatory bowel disease (IBD; 517 CD and 301 UC) from two IBD tertiary referral centers, with median ages of 33 and 39 years, respectively, and disease duration of 8.9 years, were phenotyped using the Montreal classification, and analyzed for seven anti-glycan antibodies (gASCA (anti-Saccharomyces cerevisiae) IgG, gASCA IgA, anti-chitobioside (GlcNAc(beta1,4)GlcNAc(beta)), anti-laminaribioside (Glc(beta1,3)Glb(beta)), anti-mannobioside (Man(alpha1,3)Man(alpha)), anti-L, and anti-C) and perinuclear atypical neutrophil cytoplasmic antibodies (pANCA). RESULTS: In the CD patient population, 73% were positive for >/=1 anti-glycan antibody. All glycan markers were specific for CD (85.4-97.7%) and more prevalent in CD vs. UC (P<0.0015). gASCA IgG and IgA best differentiated CD from UC followed by anti-L (area under the curve 0.818, 0.815, and 0.702, respectively). The addition of anti-L and anti-C to gASCA IgG and pANCA improved discrimination between CD and UC (P<0.001). Adding anti-L to gASCA and pANCA differentiated colonic CD and UC (P=0.02). An increasing number of positive antibodies was associated with early CD onset, penetrating phenotype, perianal disease, and the need for surgery (P<0.001). Anti-L was associated with ileocolonic CD (odds ratio (OR) 2.28, 95% confidence interval (CI) 1.40-3.69; P=0.001), and anti-C with penetrating (OR 2.75, 95% CI 1.50-5.04; P=0.001) and perianal disease (OR 1.95, 95% CI 1.06-3.59; P=0.03). CONCLUSIONS: Anti-L and anti-C improve differentiation between CD and UC. Anti-L may also differentiate between isolated colonic CD and UC. Both anti-L and anti-C are independently associated with a more aggressive CD phenotype.

New serological markers for inflammatory bowel disease are associated with earlier age at onset, complicated disease behavior, risk for surgery, and NOD2/CARD15 genotype in a Hungarian IBD cohort.

BACKGROUND: Antibodies to Saccharomyces cerevisiae (S. cerevisiae) (ASCA) and porin protein-C of Escherichia coli (anti-OmpC) are associated with disease phenotype and may be of diagnostic importance in inflammatory bowel diseases (IBD). Our aim was to determine whether a panel of new antibodies against bacterial proteins and carbohydrates could help differentiate among the various forms of IBD, and whether they were associated with particular clinical manifestations in a Hungarian cohort of IBD patients. METHODS: Six hundred fifty-two well-characterized, unrelated, consecutive IBD patients (CD [Crohn's disease] 557, men/women 262/295, duration 8.1 +/- 11.3 yr; ulcerative colitis [UC] 95, men/women 44/51, duration 8.9 +/- 9.8 yr) and 100 healthy and 48 non-IBD gastrointestinal (GI) controls were investigated. Sera were assayed for anti-OmpC and antibodies against a mannan epitope of S. cerevisiae (gASCA), laminaribioside (ALCA), chitobioside (ACCA), and mannobioside (AMCA). TLR4 and NOD2/CARD15 variants were tested by polymerase chain reaction/restriction fragment length polymorphism (PCR-RFLP). Detailed clinical phenotypes were determined by reviewing the patients' medical charts. RESULTS: Sixty-six percent of the CD patients had at least one of the investigated antibodies. Among glycan antibodies, gASCA or the combination of gASCA and atypical perinuclear antineutrophil cytoplasmic antibodies (pANCA) was most accurate for differentiating between CD and UC. ASCA and gASCA assays performed similarly. Increasing amount and level of antibody responses toward gASCA, ALCA, ACCA, AMCA, and OmpC were associated with more complicated disease behavior (P < 0.0001) and need for surgery in CD (P= 0.023). A serological dosage effect was also observed. gASCA and AMCA antibodies were associated with NOD2/CARD15, in addition to a gene-dosage effect. No serotype-phenotype associations were found in UC. CONCLUSIONS: Antibody response to this new panel of serological markers was associated with complicated disease phenotype, NOD2/CARD15 genotype, and a need for surgery in this eastern European IBD cohort.

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.

Serum anti-Glc(alpha1,4)Glc(alpha) antibodies as a biomarker for relapsing-remitting multiple sclerosis.

There is an unmet need to develop specific biomarkers for multiple sclerosis (MS) to aid in the diagnosis, improve the management of patients and the monitoring of the effectiveness of treatment. We have screened serum from patients with relapsing-remitting MS (RRMS, n = 107) against a library of glycans on a glycan chip, and have found significantly higher levels of IgM anti-Glc(alpha1,4)Glc(alpha) antibodies (anti-Galpha4Galpha antibodies) than in patients suffering from other neurological diseases (OND, n = 50, p < 0.0001), and other autoimmune diseases (OAD, n = 27, p = 0.02). No significant differences were found relative to patients having primary progressive MS (n = 16). No significant differences were detected between the levels of IgM anti-Galpha4Galpha antibodies in sera from patients with RRMS in relapsing versus remitting state, and in patients treated with immunotherapy versus untreated patients. To test whether the highly significant difference in the levels of IgM anti-Galpha4Galpha between RRMS and OND group is due to general increase in IgM levels, we have measured total serum IgM in a subgroup of 62 MS and 48 OND patients. Although the total IgM was significantly lower in the OND than the RRMS group (p = 0.0007), analysis of covariance (ANCOVA) reveled no statistically significant relationship to the covariate (total IgM). Furthermore, following normalizing the values to total IgM the difference in the levels of IgM anti-Galpha4Galpha between the MS and OND groups was found highly significant (p < < 0.0001). The present findings support further assessment of serum anti-Galpha4Galpha antibodies as a potential biomarker for MS, which may confirm disease diagnosis and aid in its management.

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.

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.