Impact des polymorphismes génétiques de la P-glycoprotéine sur le transport des immunosuppresseurs et des inhibiteurs de tyrosine kinases

ABCB1 encodes P-glycoprotein, which transports many drugs, including calcineurin inhibitors used in solid organ transplantation and tyrosine kinase inhibitors used in the treatment of chronic myeloid leukaemia. ABCB1 genetic polymorphisms, especially the coding ABCB1 1199G>A (Ser400Asn) (Rs2229109), 1236C>T(Gly412Gly) (Rs1128503), 2677G>T (Ala893Ser) (Rs2032582) and 3435C>T (Ile1145Ile) (Rs1045642), were reported to influence the clinical and pharmacological response towards calcineurin inhibitors and tyrosine kinase inhibitors. In this context, the main goal of our work was to assess the impact of these polymorphisms on intracellular accumulation and/or pharmacological effect of tacrolimus, cyclosporine A, imatinib, nilotinib, dasatinib and ponatinib in cell lines expressing several variants of the ABCB1 protein. Our results allowed confirming, for the first time, that Asn400 variant protein increases the intracellular accumulation of tacrolimus compared to the wild-type protein while its effect was unchanged for cyclosporine A. We also demonstrated that the Asn400 variant protein decreases the antiproliferative effect of imatinib, nilotinib and dasatinib compared to the wild-type protein (results confirmed for imatinib and nilotinib by a decreased intracellular accumulation). In relation to the three other polymorphisms investigated (1236C>T, 2677G>T and 3435C>T), the protein encoded by the CGC wild-type haplotype is associated to a lower in the antiproliferative effect compared to other haplotypes but only towards imatinib (results confirmed by a decreased intracellular accumulation). The importance of our results should be further clinically confirmed in patients treated by calcineurin inhibitors or by tyrosine kinase inhibitors.Les polymorphismes génétiques d’ABCB1 (gène codant pour la P-glycoprotéine) et plus particulièrement, les polymorphismes codants ABCB1 1199G>A (Ser400Asn (Rs2229109), 1236C>T (Gly412Gly) (Rs1128503), 2677G>T (Ala893Ser) (Rs2032582)et 3435C>T (Ile1145Ile) (Rs1045642), semblent influencer la réponse clinique vis-à vis des inhibiteurs de la calcineurine utilisés en transplantation d’organe et des inhibiteurs de tyrosine kinases utilisés dans le traitement de la leucémie myéloïde chronique. Dans ce contexte, le principal objectif de nos travaux consistait à évaluer l’impact de ces polymorphismes sur l’accumulation intracellulaire ainsi que sur la réponse pharmacologique du tacrolimus, de la ciclosporine A, de l’imatinib, du nilotinib, du dasatinib et du ponatinib dans des modèles cellulaires exprimant différents variants de la protéine ABCB1. Nos résultats ont permis de confirmer, pour la première fois in vitro, que la protéine variante Asn400 augmente l’accumulation intracellulaire du tacrolimus par rapport à la protéine sauvage alors que celle-ci semble, au contraire, inchangée lors d’une exposition à la ciclosporineA. Nous avons également constaté que cette protéine variante Asn400 diminue l’effet antiprolifératif de l’imatinib, du nilotinib et du dasatinib par rapport à la protéine sauvage (observation confirmée par une diminution de l’accumulation intracellulaire de l’imatinib et du nilotinib). En ce qui concerne les trois autres polymorphismes étudiés (1236C>T, 2677G>T et 3435C>T), la protéine codée par l’haplotype sauvage CGC est associée à une diminution de l’effet antiprolifératif par rapport aux autres haplotypes mais uniquement vis-à-vis de l’imatinib (observation confirmée par une diminution de l’accumulation intracellulaire). La relevance de ces résultats doit être ultérieurement confirmée en clinique chez des patients traités par ces inhibiteurs de la calcineurine ou par ces inhibiteurs de tyrosine kinases.(BIFA - Sciences biomédicales et pharmaceutiques) -- UCL, 201

Impact of ABCB1 1236C > T-2677G > T-3435C > T polymorphisms on the anti-proliferative activity of imatinib, nilotinib, dasatinib and ponatinib.

Overexpression of ABCB1 (also called P-glycoprotein) confers resistance to multiple anticancer drugs, including tyrosine kinase inhibitors (TKIs). Several ABCB1 single nucleotide polymorphisms affect the transporter activity. The most common ABCB1 variants are 1236C > T, 2677G > T, 3435C > T and have been associated with clinical response to imatinib in chronic myelogenous leukaemia (CML) in some studies. We evaluated the impact of these polymorphisms on the anti-proliferative effect and the intracellular accumulation of TKIs (imatinib, nilotinib, dasatinib and ponatinib) in transfected HEK293 and K562 cells. ABCB1 overexpression increased the resistance of cells to doxorubicin, vinblastine and TKIs. Imatinib anti-proliferative effect and accumulation were decreased to a larger extent in cells expressing the ABCB1 wild-type protein compared with the 1236T-2677T-3435T variant relatively to control cells. By contrast, ABCB1 polymorphisms influenced the activity of nilotinib, dasatinib and ponatinib to a much lesser extent. In conclusion, our data suggest that wild-type ABCB1 exports imatinib more efficiently than the 1236T-2677T-3435T variant protein, providing a molecular basis for the reported association between ABCB1 polymorphisms and the response to imatinib in CML. Our results also point to a weaker impact of ABCB1 polymorphisms on the activity of nilotinib, dasatinib and ponatinib

ABCB1 1199G>A polymorphism (rs2229109) affects the transport of imatinib, nilotinib and dasatinib.

AIM: ABCB1 (or P-glycoprotein) is implicated in the multidrug-resistance phenotype, including the resistance toward anticancer drugs such as tyrosine kinase inhibitors (TKIs). The purpose of this study was to evaluate in vitro the influence of the ABCB1 1199G>A SNP on ABCB1 transport activity toward selected TKIs (imatinib, nilotinib and dasatinib) that are currently used in chronic myelogenous leukemia. MATERIAL & METHODS: Two different cell lines, HEK293 and K562, were stably transfected with ABCB1 1199G wild-type or ABCB1 1199A variant allele. The impact of this polymorphism on accumulation and antiproliferative effects of imatinib, nilotinib and dasatinib was evaluated. RESULTS: In K562 models, the expression of Asn400 variant protein was associated with lower antiproliferative effects of imatinib, nilotinib and dasatinib compared with Ser400 wild-type protein. Moreover, in HEK293 cells, imatinib and nilotinib intracellular accumulation were lower in variant compared with wild-type models. CONCLUSION: Imatinib, nilotinib and dasatinib are transported more efficiently by the ABCB1 variant (Asn400) compared with the wild-type (Ser400) protein. The impact of ABCB1 1199G>A SNP on TKI response should be further investigated in chronic myelogenous leukemia patients

Short Communication: An Insertion of Seven Amino Acids in the Envelope Cytoplasmic Tail of HIV-2 Selected During Disease Progression Enhances Viral Replication.

The cytoplasmic tail (CT) of the HIV-2 envelope glycoprotein (Env) includes amino acid (aa) sequences that are similar to lentiviral lytic peptides (LLP) described in other lentiviruses. Within the putative LLP-2 region, we previously observed insertions of 3 or 7 aa in sequences deduced from plasma viral RNA of symptomatic HIV-2-infected individuals. Based on these observations, we reproduced the insertions in a molecular clone to assess their impact on replicative fitness and cell death in vitro. Using a molecular clone of the HIV-2 reference strain, site-directed mutagenesis experiments allowed the generation of plasmids with the insertion LTAI or LQRALTAI in the Env protein. The clone with 7 aa insertion enhanced viral release 8 to 11 times in infected T cells and cell viability was impaired by more than 20%, compared with the wild-type HIV-2 virus. The effect of the 3 aa insertion was milder, with a nonsignificant trend to enhance viral replication and cell death compared with the wild-type virus. Interestingly, the insertions in the Env proteins did not induce a significant increase of viral infectivity, as revealed by the infectivity assay using TZM-bl cells. The insertions in the Env CT observed in vivo from disease progressors may, therefore, be involved in the higher viral load observed in these individuals. This study may open the way to the development of a prognostic marker related to the HIV-2 infection progression

Large-scale, molecular and serological SARS-CoV-2 screening of healthcare workers in a 4-site public hospital in Belgium after COVID-19 outbreak.

We read with great interest the study of Chen Y et al., who analyzed, during the Chinese epidemic peak, the seroprevalence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) among 105 healthcare workers (HCWs) exposed to COVID-19 patients.1 They found 17.14% of seropositive asymptomatic or paucisymptomatic HCWs although their nasopharyngeal swab samples were SARS-CoV-2 RNA negative. Our purpose was to document at the end of the Belgium epidemic the seroprevalence of SARS-CoV-2 in HCWs exposed to COVID-19 at varying degrees and to compare these rates with those observed by other teams worldwide. Another objective was to highlight SARS-CoV-2 carriage in a priori healthy staff members to sensitize them to the need to respect individual protection measures and distancing to avoid patient contamination. [...

ABCB1 1199G>A genetic polymorphism (Rs2229109) influences the intracellular accumulation of tacrolimus in HEK293 and K562 recombinant cell lines

OBJECTIVE: ATP-binding cassette, subfamily B, member 1 (ABCB1) transporter, or P-glycoprotein, is an efflux protein implicated in the absorption and the distribution of various compounds, including tacrolimus and cyclosporine A. In vivo studies suggest an association between the ABCB1 1199G>A single nucleotide polymorphism (SNP) and tacrolimus intracellular accumulation. The aim of the present experimental study was to clarify in vitro the impact of the coding ABCB1 1199G>A SNP on ABCB1 transport activity towards both immunosuppressive drugs. METHOD: Two recombinant cell lines, i.e. Human Embryonic Kidney (HEK293) and Human Myelogenous Leukemia (K562) cells, overexpressing ABCB1 carrying either the wild-type allele (1199G) or its mutated counterpart (1199A), were generated. The impact of the 1199G>A SNP on ABCB1 activity towards rhodamine (Rh123), doxorubicin, vinblastine, tacrolimus and cyclosporine A was assessed by accumulation, cytotoxicity and/or kinetic experiments. RESULTS: Tacrolimus accumulation was strongly decreased in cells overexpressing the wild-type protein (1199G) compared to control cells, confirming the ability of ABCB1 to transport tacrolimus. By contrast, overexpression of the variant protein (1199A) had nearly no effect on tacrolimus intracellular accumulation whatever the model used and the concentration tested. Unlike tacrolimus, our results also indicate that cyclosporine A, Rh123 and doxorubicin are transported in a similar extent by the wild-type and variant ABCB1 proteins while the variant protein seems to be more efficient for the transport of vinblastine. CONCLUSION: ABCB1 encoded by the 1199G wild-type allele transports more efficiently tacrolimus in comparison to the 1199A variant protein. This observation indicates that the amino-acid substitution (Ser400Asn) encoded by the 1199A allele drastically decreases the ability of ABCB1 to drive the efflux of tacrolimus in a substrate-specific manner, in agreement with our previously published clinical data. Our study emphasizes the importance of the ABCB1 1199G>A polymorphism for ABCB1 activity and its potential to explain differences in drug response

First evaluation of the Next-Generation Sequencing platform for the detection of HIV-1 drug resistance mutations in Belgium.

INTRODUCTION: The WHO urges action against the threat posed by HIV drug resistance. It is well known that the sensitivity of Next-Generation Sequencing (NGS) is greater than that of Sanger Sequencing (SS). The objective of this study was to evaluate the performance of the novel NGS HIV-1 drug resistance monitoring system. MATERIALS & METHODS: NGS analyses were performed on 67 plasma samples from HIV-1 infected patients using the Sentosa SQ HIV Genotyping Assay from Vela-Dx. This kit was used on a semi-automated Ion Torrent-based platform. Sequences were compared to those obtained by SS. Samples were analysed in the same and in separate runs. Quality controls (QC) were added to control sequencing processes of protease (PRO), reverse transcriptase (RT) and integrase (INT) regions. RESULTS: Of the 41 analysed samples, 33 (80.5%) had identical drug resistance interpretation reports. Discrepant results were observed for eight samples. Five of them were only detected by NGS and had drug resistance mutations (DRMs) with an allelic frequency below the limit of detection of the SS method (between 6.3 to 20.5%). Two DRMs were only identified using the SS method. The sequences were similar in 98.2% of cases (counting variants as mismatches) and homologous in 99.9% if missed variants. Duplicated samples in a single run were similar in 95.7% (99.9%) of cases. Duplicated samples in two different runs were 98% (100%) homologous. QC results were manually assessed with a score of 340/340 for detection of DRMs in PRO and RT and 100% for INT sequencing. CONCLUSIONS: This is the first preliminary evaluation in Belgium employing the Sentosa SQ HIV Genotyping Assay. The NGS appears to be a promising tool for the detection of DRMs in HIV-1 patients and showed a higher sensitivity compared to SS. Large studies assessing the clinical relevance of low frequency DRMs are needed

Evaluation of the analytical performance of six rapid diagnostic tests for the detection of viral hepatitis B and C in Lubumbashi, Democratic Republic of Congo.

Rapid diagnostic tests (RDTs) are widely used in Lubumbashi for the diagnosis of viral hepatitis B and C. To date, there are no works that have been carried out in Lubumbashi to independently assess the performance of such tests. This study aimed at assessing the effectiveness of RDTs for the detection of HBsAg and anti-HCV antibodies in order to identify infected blood donors in Lubumbashi. A total of 300 serum samples (100 HBsAg positive samples; 100 anti-HCV positive samples and 100 HBsAg and anti-HCV negative samples) were tested simultaneously using the 6 locally used RDTs and as gold standard the chemiluminescent assays for HBsAg and the RT-TMA for HCV.detection. The six evaluated RDTs demonstrated a sensitivity and a negative predictive value (NPV) of 100% whereas the specificity and positive predictive value (PPV) varied from 46% to 98.1%. SB BioLine HBsAg test performed best in this study with 100% of sensitivity, 97.1% of specificity,100% of NPV and 96.9% of PPV. Furthermore, sensitivity, specificity, NPV and PPV for SB BioLine HCV test were as follows: 100%, 98,1%, 100% and 93.9%. Therefore, SD BioLine tests (HBsAg, HCV) would be selected as the first line RDTs for the detection and the diagnostic of hepatitis B and C. They can prevent blood-borne transmission of HBV and HCV in areas with limited incomes as Lubumbashi

Correction: First evaluation of the Next-Generation Sequencing platform for the detection of HIV-1 drug resistance mutations in Belgium.

[This corrects the article DOI: 10.1371/journal.pone.0209561.]