Thioredoxin-1 (Trx1) is a protein antioxidant involved in major cellular processes. Increased plasma levels of Trx1 have been associated with human diseases suggesting that Trx1 is a marker for oxidative stress with putative clinical use. However, the reported mean levels of Trx1 in the control cohorts vary a hundred-fold between studies (0.8-87 ng/ml), possibly due to methodological differences between the capture ELISA used in the different studies. The aim of this study was to investigate methodological aspects related to the ELISA measurement of Trx1. ELISAs utilizing different capture and detection combinations of antibodies to Trx1 and as well as recombinant human (rh) Trx1 standards from two sources were characterized. The different ELISAs were subsequently used to measure Trx1 in human plasma and cerebrospinal fluid samples (CSF) from healthy donors and from patients with various neurological diagnoses. The Trx1 standards differed in their content of monomeric and oligomeric Trx1, which affected the ELISAs composed of different antibody combinations. Thus, the levels of Trx1 determined in human plasma and CSF samples varied depending on the antibody used in the ELISAs and on the rhTrx1 standard. Furthermore, the relevance of preventing interference by heterophilic antibodies (HA) in human plasma and CSF was investigated. The addition of a HA blocking buffer to human samples drastically reduced the ELISA signals in many samples showing that HA are likely to cause false positive results unless they are blocked. In conclusion, the study shows that the design of a Trx1 ELISA in regards to antibodies and standards used has an impact on the measured Trx1 levels. Importantly, analyses of human plasma and CSF without preventing HA interference may obscure the obtained data. Overall, the results of this study are crucial for the improvement of future studies on the association of Trx1 levels with various diseases.
Aims: Th17 cells and their related cytokines play an important role in the pathogenesis of celiac disease (CD), and thioredoxin (Trx) is an extracellular TG2 activity regulator. This study evaluated Trx serum levels and the expression levels of IL17A, IL21, and Trx genes in biopsies of treated (gluten-free diet) and naïve (untreated) CD patients compared with healthy individuals.
Methods: Duodenal biopsies were collected from treated CD patients (n = 60), healthy controls (n = 60), and eight newly diagnosed celiac patients. IL17A, IL21, and Trx gene expression was assessed by quantitative polymerase chain reaction (qPCR) and compared with serum Trx levels assessed by enzyme-linked immunosorbent assay (ELISA).
Results: Expression levels of the IL21 and Trx genes were not significantly modulated in the CD group compared to the control group, whereas the IL17A gene in CD patients was transcribed at significantly higher levels among the CD group. Serum concentrations of Trx were significantly increased in treated CD patients compared to the control group.
Conclusions: We observed that IL17A gene is more highly expressed in duodenal biopsies of CD patients than controls, and that the serum levels of Trx are significantly higher in treated CD patients than controls. Therefore, the expression levels of these genes and gene products, respectively, could potentially be used as diagnostic biomarkers for CD patients, although more studies are needed to unravel the underlying molecular mechanisms.