Number Of Positive Antibodies Research Articles

A Role of Immunothrombosis in Acquired Antibody-Mediated Thrombophilia's: Classical Complement and Neutrophil Activation in APS and HIT

Introduction: Heparin induced thrombocytopenia (HIT) and APS (antiphospholipid syndrome) are acquired thrombophilia's characterized by both arterial and venous thromboembolic events (TE). As a common feature APS and HIT share the presence of pathological antibodies, which play a crucial role in disease pathogenesis. In HIT, antibodies are directed to platelet factor (PF) 4 and heparin, respectively, and in APS, antibodies target anti-phospholipids (anti-cardiolipin antibody, anti-Beta2- glycoprotein I and lupus anticoagulant). There is growing evidence that complement- and neutrophil activation in the form of neutrophil extracellular traps (NETs) play an important role in the pathogenesis of thrombosis, the so-called "immunothrombosis". Insufficient control with subsequent unrestrained complement activation results in thrombosis as seen in patients with paroxysmal nocturnal hemoglobinuria (PNH). Therapeutic inhibition of complement activation abrogates thrombosis in these patients, emphasizing the important role of complement activation in the pathogenesis of thrombosis. It has been demonstrated, that complement activation products efficiently induce neutrophil activation, and therapeutic complement inhibition in sepsis and PNH prevents neutrophil activation. Here, we investigate the role of complement activation and neutrophil activation in patients with APS and HIT, respectively. Methods To assess the association of complement activation and NET Formation, we measured complement activation products (C4bc, C3bc) and marks for neutrophil activation, such as nucleosomes and human neutrophil elastase-α1- antitrypsin complexes (HNE) by ELISA in plasma of 34 primary APS- and 26 HIT- patients and 31 healthy controls. Groups have been compared using the Mann- Whitney-Rank Test. Values are indicated as median and range, p<0.05 is considered statistically significant. Results Patients with APS and HIT showed sign. higher levels of complement activation products as compared to healthy controls. Complement activation mainly of the classical (C4b/c) and alternate pathway (C3b/c) in APS (11 nM and 263 nM) and in HIT (33 nM and 324 nM) were sign. higher (p<0.0001) as compared to the healthy controls (7 nM and 19 nM). C4bc levels were sign. higher in HIT as compared to APS patients (p<0001), but there was no difference in C3b/c levels between these two groups (see figure 1). Patients with HIT had a sig. (p<0.0001) higher nucleosomes (440 U/ml) and HNE (121 ng/ml) as compared to healthy controls (12 U/ml and 26 ng/ml). HNE levels were sign. (p<0.001) higher in APS patients (37 ng/ml) as compared to healthy controls (26 ng/ml), however no difference was observed in nucleosome levels (12 U/ml vs 14 U/ml) (see figure 2). Based on the number of positive antibodies, eleven APS patients have been stratified as high-risk and 24 APS patients as low- to intermediate risk. C4bc levels were sign. increased in the high-risk patients as compared to the low- to intermediate risk group, whereas no difference in C3b/c levels could be observed between the 2 groups. Similarly, HNE complexes sign. increased in the high-risk patients as compared to the low- to intermediate risk group, whereas no difference in nucleosome levels could be observed between the 2 groups. Six HIT patients (22%) had a diagnosed TE event whereas 17 patients (63%) had no diagnosed TE. In four patients (15%) no clinical data were available, and they have been excluded from subgroup analysis. Interestingly, the measured markers for complement - and neutrophil activation in HIT patient with and without thrombosis were not different. Conclusions We demonstrate complement activation in patients with HIT and APS. Based on the simultaneous raise in nucleosomes and HNE, there is evidence of NET formation in HIT, whereas only weak neutrophil activation can be found in APS. In APS classical complement activation and neutrophil activation correlates with APS risk stratification. Our results demonstrate classical complement pathway- and neutrophil activation in HIT and APS patients. These findings may form the base for new therapeutic strategies targeting complement and neutrophil activation. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Antiplatelet Antibody Testing in Immune Thrombocytopenia and Evans Syndrome: Longitudinal Serologic Evolution and Relation to Clinical Features

Introduction: Antiplatelet antibody (APA) testing is considered an adjunct laboratory test in the diagnosis of immune thrombocytopenia (ITP). While it is not routinely obtained owing to inconsistent sensitivity and specificity in prior studies (Neunert et al, 2011), the definition of ITP used in these studies was not standardized. Additionally, potential clinical utility of this testing beyond diagnosis has been suggested, such as correlations between certain serologic patterns and response to IVIG (Peng et al, 2014). In consideration of these prior findings, we undertook a retrospective analysis of APA testing [glycoprotein-specific testing done by the commercial PakAuto assay (Immucor, Brookfield, WI), a monoclonal antibody immobilization of platelet antigens (MAIPA) assay, with all testing performed by the same laboratory] utilizing standardized ITP and Evans syndrome (ES) diagnostic criteria for patient inclusion. We examined serologic evolution over time and relation of antibody positivity to disease severity and response to therapies.Methods: Data collected for analysis included dates and results of APA testing (including disease status and platelet count at time of testing), patient demographics, and disease characteristics. Satisfaction of the 2011 American Society of Hematology (ASH) ITP diagnostic criteria were required for ITP patient inclusion and standard definitions of disease severity and response to treatment from the ASH guidelines were used in analysis. Logistic regression was used to model the probability of disease severity (non-severe, severe, or refractory) and treatment response (to corticosteroids or IVIG) based on serologic findings. Longitudinal serologic evolution in patients with multiple APA assays were analyzed.Results: A total of 214 APA assays from 115 ITP patients and 12 ES patients were collected; results from eluate testing (direct assay) only were used in analysis. Baseline patient characteristics are listed in Table 1. Of 7 possible positive test serologic patterns, only 4 were seen (Figure 1); antibodies against both GPIIb/IIIa and GPIb/IX were required for the presence of antibodies against GPIa/IIa. A multinomial logistic regression model including disease severity, age, sex, duration of disease, and platelet count at time of APA assay found a statistically significant predictive relationship between an increasing number of positive antibodies and disease severity [relative to non-severe ITP, relative risk ratio for severe ITP and refractory ITP was 1.89 (P=0.001) and 2.38 (P=0.004), respectively, per one additional positive antibody, Figure 2]. Multiple logistic regression models including antibody positivity to each platelet glycoprotein (GPIIb/IIIa, GPIb/IX, or GPIa/IIa), age, sex, disease duration and splenectomy status found a significant predictive relation between presence of anti-GPIa/IIa and non-response to corticosteroid treatment (odds ratio, 0.082, P=0.008). No significant relation was found between an antibody and non-response to IVIG. Fifty patients had multiple (2 to 5) APA assays performed over months to years. In evaluation of serologic evolution over time, all 7 patients who entered clinical remission also converted from a positive to a negative serology; 22 patients had stable serologic findings over time; 5 patients had a reduction in number of positive antibodies; and 18 patients demonstrated evidence for epitope spreading, with an increase in the number of positive antibodies (Figure 3). The sensitivity of a positive APA assay for the presence of active ITP was 91%. The sensitivity and specificity of a negative APA assay for remission in a patient with previously confirmed ITP (N=40 assays in patients with clinical remission in the study) were 88% and 91%, respectively.Conclusions: The MAIPA-based direct APA assay is sensitive for the presence of active ITP in patients satisfying 2011 ASH diagnostic criteria. To our knowledge, this is the first study demonstrating that a higher number of positive glycoprotein-specific antibodies may predict for more severe disease as defined by 2011 ASH disease severity criteria. Anti-GPIa/IIa antibodies only occur in the setting of pre-existing positivity for both anti-GPIIb/IIIa and anti-GPIb/IX antibodies, possibly due to a distinctive sequence of epitope spreading. Serologic testing typically turns negative when a patient enters clinical remission. [Display omitted] DisclosuresAl-Samkari:Agios: Consultancy. Kuter:Novartis: Consultancy; Pfizer: Consultancy; Syntimmune: Consultancy; Argenx: Consultancy; Dova Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; ONO: Consultancy; Amgen Inc.: Consultancy; Bioverativ: Consultancy, Research Funding; Principia: Research Funding; Rigel: Consultancy, Research Funding; BMS: Research Funding; Protalex: Research Funding.

Autoantibodies against zinc transporter 8 are related to age and metabolic state in patients with newly diagnosed autoimmune diabetes

AimsTo assess the prevalence of ZnT8-ab and its correlation to other autoimmune markers and diabetic ketoacidosis occurrence in children and adults with T1DM onset.MethodsThe study included 367 patients (218 children; 149 adults) at the T1DM onset. Selected diabetes-related autoantibodies such as GAD-ab, IA2-ab, ZnT8-ab were tested before the initiation of insulin therapy. Diabetic ketoacidosis was defined as glucose concentration > 13.9 mmol/l, pH < 7.30, concentration of HCO3 < 15 mmol/l, presence of ketone bodies in the blood and urine.ResultsThe autoantibodies pattern differs in both study groups. Children were mostly positive for two (37.8%) and three (49.5%) autoantibodies, whereas adults for one (32.2%) and two (30.7%). The most frequently detected autoantibodies in youth were ZnT8-ab (81.1%) and IA2-ab (80.7%), while in adults GAD-ab (74.8%). ZnT8-ab (p < 0.0001) titers were significantly higher in children, but adults had higher titer of GAD-ab (p < 0.0001) and IA2-ab (p < 0.0001). Children developed more frequently diabetic ketoacidosis (28.4 vs. 10.7%, p = 0.0002). ZnT8-ab (p = 0.002) and IA2-ab (p = 0.008) were reported mostly in individuals with ketoacidosis. A correlation between the number of positive antibodies and the severity of ketoacidosis was observed (Rs − 0.129 p = 0.014). ZnT8-ab were associated with a greater risk of ketoacidosis independent of gender, age group and the autoantibodies number [OR = 2.44 (95% CI 1.0–5.94), p = 0.04].ConclusionsChildren are at greater risk of ketoacidosis at the diagnosis of diabetes. ZnT8-ab and IA2-ab are commonly detected in children, while adults have frequently higher titer of GAD-ab. ZnT8-ab are associated with more acute diabetes onset.

Serological evolution in women with positive antiphospholipid antibodies.

To explore the clinical and serological course of fertile women with positive antiphospholipid (aPL), and the factors and therapeutic implications associated with aPL negativization. Retrospective study including 105 women with a positive aPL serology between 1995 and 2013 attending the obstetric autoimmune pathology clinic of a tertiary facility. Patients were classified into the following 3 groups: patients with primary antiphospholipid syndrome (pAPS, 49), patients with a positive serology for aPL, not meeting clinical criteria (42), and patients with systemic lupus erythematosus and a positive aPL serology (14). They were also classified according to the serological aPL evolution: persistently negative aPL, transiently positive serology, and persistently positive serology according to established criteria. After a mean follow-up of 114.4 ± 37.2 months, 59% of patients had persistently negative antibodies, while 25.7% of patients presented persistently positive aPL serology. Multivariate analysis confirmed that smoking (OR = 4; 95% CI: 1.45-11.08; p = 0.008) was an independent risk factor for positive persistence. Persistent positivity as well as a higher antibody load was associated with higher risk for further pregnancy morbidity. In 29 patients, with persistently negative serology who were asymptomatic, treatment with low-dose aspirin was discontinued. No clinical events related to APS were reported after treatment withdrawal, during the 40.95 months of follow-up. A significant proportion of fertile women with aPL antibodies became negative during follow-up. Tobacco use and the number of positive antibodies are associated with persistently positive serology. Patients with persistently positive aPL serology suffer more obstetric complications. Treatment withdrawal might be safe in selected patients.

Distribution of C-Peptide and Its Determinants in North American Children at Risk for Type 1 Diabetes

OBJECTIVETo determine basal and stimulated C-peptide percentiles in North American children and adolescents at risk for type 1 diabetes (T1D) and to examine factors associated with this distribution in the Diabetes Prevention Trial–Type 1 (DPT-1).RESEARCH DESIGN AND METHODSWe included 582 subjects aged 4–18 years at randomization in the DPT-1 trials. A 2-h oral glucose tolerance test (OGTT) was performed at baseline and every 6 months during the 5-year follow-up period. The percentile values of C-peptide after baseline OGTT were estimated according to age, BMI Z score (BMIZ), and/or sex categories. Conditional quantile regression was used to examine the relationship between C-peptide percentiles and various independent variables.RESULTSThe basal and stimulated C-peptide levels increased significantly as age and BMIZ increased (P < 0.05). Both age and BMIZ had a stronger impact on the upper quartile of C-peptide distributions than the lower quartile. Sex was only significantly associated with stimulated C-peptide. Higher stimulated C-peptide levels were generally observed in girls compared with boys at the same age and BMIZ (P < 0.05). HLA type and number of positive antibodies and antibody titers (islet cell antibody [ICA], insulin autoantibody, GAD65A, and ICA512A) were not significantly associated with C-peptide distribution after adjustment for age, BMIZ, and sex.CONCLUSIONSAge-, sex-, and BMIZ-specific C-peptide percentiles can be estimated for North American children and adolescents at risk for T1D. They can be used as an assessment tool that could impact the recommendations in T1D prevention trials.

Pattern recognition receptor and autophagy gene variants are associated with development of antimicrobial antibodies in Crohnʼs disease

We sought to investigate whether variants in genes involved in bacterial sensing and autophagy (NOD2, TLR5, IRGM, ATG16L1) and the interleukin-23 signaling pathway (IL12B, IL23R, STAT3) were associated with development of antimicrobial antibodies in patients with Crohn's disease (CD). A cohort of 616 CD patients from a tertiary referral hospital (Mount Sinai Hospital, Toronto) was evaluated. DNA was tested for three CD-associated NOD2 variants (3020insC, G908R, R702W), variants in IRGM, ATG16L1, IL12B, IL23R, STAT3, and a TLR5-stop mutation. Serum was analyzed by enzyme-linked immunosorbent assay (ELISA) for anti-Saccharomyces cerevisiae (ASCA) IgG and IgA, anti-outer membrane porin C (anti-ompC), anti-Cbir1 flagellin, and anti-Pseudomonas fluorescens (anti-I2). NOD2 3020insC was associated with cumulative seroreactivity by quartile sum (P = 0.003) and number of positive antibodies (P = 0.02). NOD2 G908R was also associated with quartile sum (P = 0.05). Increased ASCA seropositivity was associated with NOD2 3020insC (odds ratio [OR] = 1.9, P = 0.02) and G908R (OR = 1.8, P = 0.05), and ATG16L1 T300A (OR = 1.4, P = 0.01) variants; ASCA-positive patients had an increased cumulative number of NOD2 3020insC and ATG16L1 T300A variants (P = 0.007). TLR5-stop mutation abrogated development of anti-flagellin in a dominant-negative fashion (OR = 0.5, P = 0.009). The IRGM CD risk variant was associated with increased anti-flagellin seropositivity (OR = 1.5, P = 0.03). IL12B, IL23R, and STAT3 variants did not contribute to development of antimicrobial antibodies. Variants in innate immune genes involved in pattern recognition and autophagy but not the interleukin-23 signaling pathway influence antimicrobial seroreactivity in CD. In particular, the additive effect of NOD2 3020insC and ATG16L1 T300A suggests a role for autophagy in development of ASCA.

Circulating auto-antibodies against nuclear and non-nuclear antigens in primary Sjögren's syndrome

The aim of this study was to analyze the prevalence and clinical significance of circulating auto-antibodies against nuclear and non-nuclear antigens in a large cohort of Spanish patients with primary Sjögren's syndrome (SS). We studied 335 patients diagnosed with primary SS seen consecutively in our department since 1994 and tested for anti-nuclear antibodies (ANA), anti-Ro/SS-A, anti-La/SS-B, anti-Sm, anti-ribonucleoprotein (anti-RNP), anti-smooth muscle antibodies (anti-SMA), anti-parietal cell antibodies (anti-PCA), anti-liver-kidney microsome type-1 (anti-LKM-1) antibodies and anti-mitochondrial antibodies (AMA). ANA were detected in 278 (83%) patients. The association of positive ANA with the presence of anti-Ro/SS-A and anti-La/SS-B antibodies reached statistical significance at a titre of ANA >1/80 (p<0.001), while the presence of anti-Sm and anti-RNP was associated with positive ANA at a titre > or =1/320 (p=0.037 for Sm and p=0.016 for RNP). ANA titres correlated with the number of positive antibodies against specific nuclear antigens (p<0.001) but not with the number of positive antibodies against non-nuclear antigens. We found positive anti-Ro/SS-A antibodies in 111 (33%) patients, anti-La/SS-B in 78 (23%), anti-RNP in 8 (2%) and anti-Sm in 4 (1%). Anti-SMA antibodies were detected in 208 (62%) patients, with no significant associations with clinical or analytical SS features, while anti-PCA antibodies were found in 90 (27%) patients and were associated with a higher prevalence of thyroiditis and liver involvement. AMA were detected in 28 (8%) patients, although only 14 presented clinical and/or analytical evidence of liver involvement. No patient presented anti-LKM antibodies. ANA play a central role in the immunological expression of primary SS, due to their frequency and close association with the underlying presence of one or more anti-ENA antibodies. Positivity for antibodies against non-nuclear antigens such as anti-PCA and AMA suggests an association with some organ-specific autoimmune diseases (thyroiditis and primary biliary cirrhosis), while the presence of anti-SMA, in spite of their high prevalence, has no clinical significance in primary SS.

Predisposition to atherosclerosis by infections: role of endothelial dysfunction.

Several microorganisms have been implicated in the pathogenesis of atherosclerosis. We hypothesized that infections may predispose to atherosclerosis by inflicting endothelial injury. Of 375 patients undergoing coronary angiography, 218 had assessment of endothelial function using intracoronary acetylcholine (ACH) and of endothelium-independent function with sodium nitroprusside and adenosine. Immunoglobulin-G antibody titers to cytomegalovirus, Chlamydia pneumoniae, Helicobacter pylori, hepatitis A virus, and herpes simplex virus-1 were measured. Pathogen burden was defined as the number of positive antibodies. Although positive serology to individual pathogens tended to be associated with increased incidence of coronary arteriosclerosis (CAD), the pathogen burden correlated with the presence of CAD, even after adjustment for risk factors (OR 1.3; 95% CI, 1.05 to 1.6, P=0.018). Moreover, the severity of CAD was independently associated with the pathogen burden (P=0.001). Pathogen burden was an independent predictor of endothelial dysfunction, determined as the percent change in coronary vascular resistance in response to ACH (P=0.009) but not the responses to sodium nitroprusside or adenosine. Pathogen burden was also an independent determinant of endothelial function in the subgroup with angiographically normal coronary arteries. The immunoglobulin-G antibody response to multiple pathogens (pathogen burden) is an independent risk factor for endothelial dysfunction and the presence and severity of CAD. Endothelial dysfunction provides the crucial link by which pathogens may contribute to atherogenesis.

Modern concepts for the prediction of type 1 diabetes.

Type 1 diabetes results from the destruction of the insulin-producing beta cells of the pancreas. The disease process is thought to be initiated by a complex interaction between genetic and environmental factors leading to a cellular and humoral autoimmune response against beta cell specific components. Over the past decade there has been important progress in identification of several diabetes specific autoantigens. The availability of recombinant antigens, most notably the enzyme glutamic acid decarboxylase (GAD) and the tyrosine phosphatase-like molecule IA-2, made it possible to develop simple, sensitive and specific radioimmunoassays for the detection of autoantibodies on a large scale. It has been shown that the combination of GAD antibodies (GADA) and IA-2 antibodies (IA-2-A) with insulin autoantibodies (IAA) can replace the conventional ICA test. This considerably facilitates screening programmes. Prospective studies in subjects with and without family history of type 1 diabetes conclusively demonstrate that the risk for type 1 diabetes is strongly correlated with the number of positive antibodies. The 5-10-years risk for type 1 diabetes varies from 0-1% in individuals with only one positive antibodies to 62-100% in subjects who were positive for three or more antibodies. Despite the identification of novel genetic markers associated with type 1 diabetes, the major histocompatibility complex, namely HLA-DQ alleles, still represents the strongest genetic risk factor. The analysis of HLA-DQ alleles may be important to discriminate between subjects at high or intermediate risk from antibody positive individuals carrying protective haplotypes. Assessment of the metabolic state using the first phase insulin response to intravenous glucose may provide additional information to predict rapid progression to diabetes. Screening for multiple antibodies is the most specific and sensitive strategy for identifying subject at risk for type 1 diabetes. Second-line genotyping or analysis of the first phase insulin response can serve as useful tool to improve the prediction of type 1 diabetes. The value of additional markers such as isotype specific autoantibodies or T-helper cells subsets measured by ELISPOT assays or FACS remains to be shown.