Determination of Reference Values of MDR-ABC Transporter Activities in CD3+ Lymphocytes of Healthy Volunteers Using a Flow Cytometry Based Method.

BACKGROUND: MDR transporters are important biomarkers of drug resistance in cancer and in autoimmune conditions. We determined the MDR1, MRP1 and BCRP activity in CD3+ lymphocytes using a flow cytometry based method from 120 healthy volunteers in order to describe normal reference values of the activity of these transporters. The effects of gender and age were also determined. METHODS: The Solvo MDQ Kit™ was used for measurements. In this assay, fluorescent reporter substrates (Calcein-AM for MDR1 and MRP1 and mitoxantrone for BCRP, respectively) are trapped in the cytoplasm and pumped out by MDR proteins depending on the presence or absence of specific inhibitors (verapamil for MDR1 and MRP1, indomethacin for MRP1 and KO134 for BCRP, respectively), allowing for quantitative, standardized assessment. Cell surface staining was applied to select CD3+ cells. RESULTS: MAF values of MRP1 and BCRP are independent from age. MAFC and MAF of MDR1 show negative correlation with the age of the studied subjects (P = 0.003, r = -0.27 and P = 0.0001, r = -0.34, respectively). No difference was detected in any of the four MAF values between men and women. Gender does not affect the presence or lack of correlation between MAF values and age. CONCLUSIONS: The determination of the functional activity of MDR-ABC transporters is achievable using a flow cytometry based standardized method. Having established the normal range of MAF values on CD3+ lymphocytes of a healthy population, our results allow for the development of novel flow cytometry based diagnostic tools. © 2018 International Clinical Cytometry Society.

Combination of IgG N-glycomics and corresponding transcriptomics data to identify anti-TNF-α treatment responders in inflammatory diseases.

Prediction of responsiveness in biological therapies is an important and challenging issue in different diseases. Analyzing glycosylation pattern changes of key serum glycoproteins is one of the possible avenues to follow disease remission. The aim of this study was to investigate the changes of serum IgG glycoforms in Crohn's disease (CD) and rheumatoid arthritis patients in response to antitumor necrosis factor alpha (anti-TNF-α) treatment. IgG was isolated from patient serum samples using Protein A affinity pull-down, followed by the release of N-glycans with peptide-N-glycosidase F. The released glycans were fluorescently tagged with 8-aminopyrene-1,3,6-trisulfonate and analyzed by CGE with laser-induced fluorescent detection. Significant alterations were detected between responders and nonresponders in both disease groups. In CD patients, disease-specific alteration was found in response to anti-TNF-α therapy, which was also confirmed by transcriptomics data analysis of the corresponding glycosyltransferases and glycosidases.

Peripheral blood derived gene panels predict response to infliximab in rheumatoid arthritis and Crohn's disease.

BACKGROUND: Biological therapies have been introduced for the treatment of chronic inflammatory diseases including rheumatoid arthritis (RA) and Crohn's disease (CD). The efficacy of biologics differs from patient to patient. Moreover these therapies are rather expensive, therefore treatment of primary non-responders should be avoided. METHOD: We addressed this issue by combining gene expression profiling and biostatistical approaches. We performed peripheral blood global gene expression profiling in order to filter the genome for target genes in cohorts of 20 CD and 19 RA patients. Then RT-quantitative PCR validation was performed, followed by multivariate analyses of genes in independent cohorts of 20 CD and 15 RA patients, in order to identify sets ofinterrelated genes that can separate responders from non-responders to the humanized chimeric anti-TNFalpha antibody infliximab at baseline. RESULTS: Gene panels separating responders from non-responders were identified using leave-one-out cross-validation test, and a pool of genes that should be tested on larger cohorts was created in both conditions. CONCLUSIONS: Our data show that peripheral blood gene expression profiles are suitable for determining gene panels with high discriminatory power to differentiate responders from non-responders in infliximab therapy at baseline in CD and RA, which could be cross-validated successfully. Biostatistical analysis of peripheral blood gene expression data leads to the identification of gene panels that can help predict responsiveness of therapy and support the clinical decision-making process.

Role of the N-terminal transmembrane region of the multidrug resistance protein MRP2 in routing to the apical membrane in MDCKII cells.

In polarized cells, the multidrug resistance protein MRP2 is localized in the apical plasma membrane, whereas MRP1, another multidrug resistance protein (MRP) family member, is localized in the basolateral membrane. MRP1 and MRP2 are thought to contain an N-terminal region of five transmembrane segments (TMD(0)) coupled to 2 times six transmembrane segments via an intracellular loop (L(0)). We previously demonstrated for MRP1 that a mutant lacking TMD(0) but still containing L(0), called L(0)DeltaMRP1, was functional and routed to the lateral plasma membrane. To investigate the role of the TMD(0)L(0) region of MRP2 in routing to the apical membrane, we generated mutants similar to those made for MRP1. In contrast to L(0)DeltaMRP1, L(0)DeltaMRP2 was associated with an intracellular compartment, most likely endosomes. Co-expression with TMD(0), however, resulted in apical localization of L(0)DeltaMRP2 and transport activity. Uptake experiments with vesicles containing L(0)DeltaMRP2 demonstrated that the molecule is able to transport LTC(4). An MRP2 mutant without TMD(0)L(0), DeltaMRP2, was only core-glycosylated and localized intracellularly. Co-expression of DeltaMRP2 with TMD(0)L(0) resulted in an increased protein level of DeltaMRP2, full glycosylation of the protein, routing to the apical membrane, and transport activity. Our results suggest that the TMD(0) region is required for routing to or stable association with the apical membrane.