138 research outputs found
Automated evaluation of autoantibodies on human epithelial-2 cells as an approach to standardize cell-based immunofluorescence tests
INTRODUCTION: Analysis of autoantibodies (AAB) by indirect immunofluorescence (IIF) is a basic tool for the serological diagnosis of systemic rheumatic disorders. Automation of autoantibody IIF reading including pattern recognition may improve intra- and inter-laboratory variability and meet the demand for cost-effective assessment of large numbers of samples. Comparing automated and visual interpretation, the usefulness for routine laboratory diagnostics was investigated. METHODS: Autoantibody detection by IIF on human epithelial-2 (HEp-2) cells was conducted in a total of 1222 consecutive sera of patients with suspected systemic rheumatic diseases from a university routine laboratory (n = 924) and a private referral laboratory (n = 298). IIF results from routine diagnostics were compared with a novel automated interpretation system. RESULTS: Both diagnostic procedures showed a very good agreement in detecting AAB (kappa = 0.828) and differentiating respective immunofluorescence patterns. Only 98 (8.0%) of 1222 sera demonstrated discrepant results in the differentiation of positive from negative samples. The contingency coefficients of chi-square statistics were 0.646 for the university laboratory cohort with an agreement of 93.0% and 0.695 for the private laboratory cohort with an agreement of 90.6%, P < 0.0001, respectively. Comparing immunofluorescence patterns, 111 (15.3%) sera yielded differing results. CONCLUSIONS: Automated assessment of AAB by IIF on HEp-2 cells using an automated interpretation system is a reliable and robust method for positive/negative differentiation. Employing novel mathematical algorithms, automated interpretation provides reproducible detection of specific immunofluorescence patterns on HEp-2 cells. Automated interpretation can reduce drawbacks of IIF for AAB detection in routine diagnostics providing more reliable data for clinicians
Differences in Antiphospholipid Antibody Profile between Patients with Obstetric and Thrombotic Antiphospholipid Syndrome
Antiphospholipid syndrome (APS) is a systemic autoimmune condition characterised by the presence of antiphospholipid antibodies (aPL) associated with vascular thrombosis and/or pregnancy complications. In a cohort of 74 yet diagnosed APS individuals fulfilling Sydney laboratory criteria (twice positive for lupus anticoagulant, anticardiolipin, aCL, and/or anti-β2glycoprotein I, aβ2GPI), 33 out of 74 were obstetric APS (OAPS) and 41 thrombotic APS (TAPS) patients. 39% of TAPS patients were women. Although aPL detection was persistent, we observed an oscillatory aPL positivity in 56.7% and a transient seroconversion in 32.4% of APS patients at enrolment. Thus, we tested their sera in a line immunoassay that simultaneously detected IgG or IgM for criteria (aCL and aβ2GPI) and non-criteria (anti-phosphatidylserine, aPS; anti-phosphatidic acid, aPA; anti-phosphatidylinositol, aPI; anti-annexin 5, aA5; anti-prothrombin, aPT; anti-phosphatidylethanolamine; anti-phosphatidylglycerol, and anti-phosphatidylcholine) aPL. OAPS and TAPS patients displayed different but overlapping clusters based on their aPL reactivities. Specifically, while OAPS patients showed higher aPA, aPS, aA5, aβ2GPI and aPT IgM levels than TAPS patients, the latter displayed higher reactivity in aCL, aPI and aA5 IgG. Eventually, with a cut-off of the 99 th percentile established from a population of 79 healthy donors, TAPS patients significantly tested more positive for aCL and aA5 IgG than OAPS patients, who tested more positive for aPA, aPS and aβ2GPI IgM. Transiently seronegative APS patients showed non-criteria aPL positivity twice in sera obtained 3 months apart. Overall, our data show that APS patients presented clusters of aPL that define different profiles between OAPS and TAPS, and persistent non-criteria aPL positivity was observed in those who are transiently seronegativ
PR3-ANCAs Detected by Third-Generation ELISA Predicts Severe Disease and Poor Survival in Primary Sclerosing Cholangitis
A highly sensitive detection of anti-neutrophil cytoplasmic antibodies to serine
proteinase-3 (PR3-ANCAs) aids in the serological diagnosis of autoimmune liver disorders and the
prediction of severity in primary sclerosing cholangitis (PSC). Here, we evaluate a novel thirdgeneration ELISA for the detection of PR3-ANCAs. In total, 309 patients with PSC, 51 with primary
biliary cholangitis (PBC), and 120 healthy blood donors (BD) were analyzed. For the survival
analysis in PSC, the outcome was defined as liver-transplantation-free survival during the followup. Positive PR3-ANCA levels were found in 74/309 (24.0%) of patients with PSC. No BDs and one
patient with PBC demonstrated PR3-ANCA positivity. PR3-ANCAs were revealed as independent
predictors for a poor PSC outcome (study endpoint: liver transplantation/death, log-rank test, p =
0.02). PR3-ANCA positivity, lower albumin levels, and higher bilirubin concentrations were
independent risks of a poor survival (Cox proportional-hazards regression analysis, p < 0.05). The
Mayo risk score for PSC was associated with PR3-ANCA positivity (p = 0.01) and the disease
severity assessed with a model of end-stage liver disease (MELD) and extended MELD-Na (p < 0.05).
PR3-ANCAs detected by a third-generation ELISA are diagnostic and prognostic markers for PSC.
Their wider use could help to identify patients who are at-risk of a more severe disease
Over-Expression of LEDGF/p75 in HEp-2 Cells Enhances Autoimmune IgG Response in Patients with Benign Prostatic Hyperplasia : A Novel Diagnostic Approach with Therapeutic Consequence?
Lens epithelium-derived growth factor splice variant of 75 kDa (LEDGF/p75) is an autoantigen over-expressed in solid tumors and acts as a stress-related transcriptional co-activator.
Participation of autoimmune responses in the pathophysiology of benign prostatic hyperplasia
(PBH) and a corresponding immunosuppressive therapy by TNFalpha antagonists has been recently
suggested. Thus, autoAb testing could aid in the diagnosis of BPH patients profiting from such therapy. We generated CRISPR/Cas9 modified HEp-2 LEDGF knock-out (KO) and HEp-2 LEDGF/p75
over-expressing (OE) cells and examined IgG autoantibody reactivity to LEDGF/p75 in patients
with prostate cancer (PCa, n = 89), bladder cancer (BCa, n = 116), benign prostatic hyperplasia (BPH,
n = 103), and blood donors (BD, n = 60) by indirect immunofluorescence assay (IFA). Surprisingly,
we could not detect elevated binding of autoAbs against LEDGF/p75 in cancer patients, but autoAb
reactivity to LEDGF/p75 OE cells in about 50% of patients with BPH was unexpectedly significantly
increased. Furthermore, a line immunoassay enabling the detection of 18 different autoAbs revealed a
significantly increased occurrence of anti-dsDNA autoAbs in 34% of BPH patients in contrast to tumor
patients and BD. This finding was confirmed by anti-mitochondrial (mDNA) autoAb detection with
the Crithidia luciliae immunofluorescence test, which also showed a significantly higher prevalence
(34%) of anti-mDNA autoAbs in BPH. In summary, our study provided further evidence for the
occurrence of autoimmune responses in BPH. Furthermore, LEDGF/p75 over-expression renders
HEp-2 cells more autoantigenic and an ideal target for autoAb analysis in BPH with a potential
therapy consequence
Rediscovery of the Anti-Pancreatic Antibodies and Evaluation of their Prognostic Value in a Prospective Clinical Cohort of Crohn's Patients: The Importance of Specific Target Antigens [GP2 and CUZD1].
BACKGROUNDS
Glycoprotein 2[GP2] and CUB zona pellucida-like domain 1[CUZD1] belong to protein families involved in gut innate immunity processes and have recently been identified as specific targets of anti-pancreatic autoantibodies [PAbs] in Crohn's disease[CD]. We aimed to determine the prognostic potential of novel target-specific PAbs regarding long-term disease course of an adult CD patient cohort.
METHODS
Sera of 458 consecutive well-characterised IBD patients from a single referral IBD centre were tested by enzyme-linked immunosorbent assay [ELISA] with isoform 4 of recombinant GP2 [anti-MZGP2 and anti-GP2 IgA/IgG] and indirect immunofluorescence test [IIFT] system with GP2 and CUZD1 expressing transfected HEK 293 cells [anti-rPAg2 and rPAg1 IgA/IgG]. Clinical data were available on complicated disease or surgical interventions as well as disease activity and medical treatment during the prospective follow-up [median, 108 months].
RESULTS
Totals of 12.4% and 20.8% of CD patients were positive for IgA/IgG type of anti-GP2 and anti-CUZD1, respectively, with a significant difference compared with UC [p < 0.01]. Antibody status was stable over time. Agreement among three different anti-GP2 assays was good. Positivity for PAbs, mainly IgA subtypes, predicted a faster progression towards complicated disease course. In Kaplan-Meier analysis, time to surgery or development of perianal disease was associated with anti-GP2 IgA [pLogRank < 0.01] or anti-CUZD1 IgA [pLogRank < 0.001] positivity, respectively. Anti-CUZD1 IgA remained an independent predictor in the multivariate Cox-regression model (hazard ratio [HR]: 3.43, 95% confidence interval [CI]: 1.68-7.02, p < 0.001).
CONCLUSIONS
The present study has shown that specific PAbs [especially IgA subtype] predict complicated disease course including the development of perianal disease in CD
New Platform Technology for Comprehensive Serological Diagnostics of Autoimmune Diseases
Antibody assessment is an essential part in the serological diagnosis of autoimmune diseases. However, different diagnostic strategies have been proposed for the work up of sera in particular from patients with systemic autoimmune rheumatic disease (SARD). In general, screening for SARD-associated antibodies by indirect immunofluorescence (IIF) is followed by confirmatory testing covering different assay techniques. Due to lacking automation, standardization, modern data management, and human bias in IIF screening, this two-stage approach has recently been challenged by multiplex techniques particularly in laboratories with high workload. However, detection of antinuclear antibodies by IIF is still recommended to be the gold standard method for antibody screening in sera from patients with suspected SARD. To address the limitations of IIF and to meet the demand for cost-efficient autoantibody screening, automated IIF methods employing novel pattern recognition algorithms for image analysis have been introduced recently. In this respect, the AKLIDES technology has been the first commercially available platform for automated interpretation of cell-based IIF testing and provides multiplexing by addressable microbead immunoassays for confirmatory testing. This paper gives an overview of recently published studies demonstrating the advantages of this new technology for SARD serology
Virulence-associated genes, resistance genes and adhesion and probiotic activity tested by a new screening method
We established an automated screening method to characterize adhesion of
Escherichia coli to intestinal porcine epithelial cells (IPEC-J2) and their
probiotic activity against infection by enteropathogenic E. coli (EPEC). 104
intestinal E. coli isolates from domestic pigs were tested by PCR for the
occurrence of virulence-associated genes, genes coding for resistances to
antimicrobial agents and metals, and for phylogenetic origin by PCR. Adhesion
rates and probiotic activity were examined for correlation with the presence
of these genes. Finally, data were compared with those from 93 E. coli
isolates from wild boars. Isolates from domestic pigs carried a broad variety
of all tested genes and showed great diversity in gene patterns. Adhesions
varied with a maximum of 18.3 or 24.2 mean bacteria adherence per epithelial
cell after 2 or 6 hours respectively. Most isolates from domestic pigs and
wild boars showed low adherence, with no correlation between
adhesion/probiotic activity and E. coli genes or gene clusters. The gene
sfa/foc, encoding for a subunit of F1C fimbriae did show a positive
correlative association with adherence and probiotic activity; however E. coli
isolates from wild boars with the sfa/foc gene showed less adhesion and
probiotic activity than E. coli with the sfa/foc gene isolated from domestic
pigs after 6 hour incubation. In conclusion, screening porcine E. coli for
virulence associated genes genes, adhesion to intestinal epithelial cells, and
probiotic activity revealed a single important adhesion factor, several
probiotic candidates, and showed important differences between E. coli of
domestic pigs and wild boars
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