Cross-resistance to elvitegravir and dolutegravir in 502 patients failing on raltegravir: a French national study of raltegravir-experienced HIV-1-infected patients

OBJECTIVES: The objectives of this study were to determine the prevalence and patterns of resistance to integrase strand transfer inhibitors (INSTIs) in patients experiencing virological failure on raltegravir-based ART and the impact on susceptibility to INSTIs (raltegravir, elvitegravir and dolutegravir). PATIENTS AND METHODS: Data were collected from 502 treatment-experienced patients failing a raltegravir-containing regimen in a multicentre study. Reverse transcriptase, protease and integrase were sequenced at failure for each patient. INSTI resistance-associated mutations investigated were those included in the last ANRS genotypic algorithm (v23). RESULTS: Among the 502 patients, at failure, median baseline HIV-1 RNA (viral load) was 2.9 log10 copies/mL. Patients had been previously exposed to a median of five NRTIs, one NNRTI and three PIs. Seventy-one percent harboured HIV-1 subtype B and the most frequent non-B subtype was CRF02_AG (13.3%). The most frequent mutations observed were N155H/S (19.1%), Q148G/H/K/R (15.4%) and Y143C/G/H/R/S (6.7%). At failure, viruses were considered as fully susceptible to all INSTIs in 61.0% of cases, whilst 38.6% were considered as resistant to raltegravir, 34.9% to elvitegravir and 13.9% to dolutegravir. In the case of resistance to raltegravir, viruses were considered as susceptible to elvitegravir in 11% and to dolutegravir in 64% of cases. High HIV-1 viral load at failure (P < 0.001) and low genotypic sensitivity score of the associated treatment with raltegravir (P < 0.001) were associated with the presence of raltegravir-associated mutations at failure. Q148 mutations were selected more frequently in B subtypes versus non-B subtypes (P = 0.004). CONCLUSIONS: This study shows that a high proportion of viruses remain susceptible to dolutegravir in the case of failure on a raltegravir-containing regimen

Implication of small GTPases Rho in endothelial response to high dose of ionizing radiation.

Microvasculature plays an important role in normal and tumoral tissue responses to high dose of irradiation (IR) as endothelial cells apoptotic death is a pre-requisite to deleterious effets of IR on surrounding tissues. Molecular mechanisms involved in this apoptotic pathway, despite being clearly independant of DNA damage, are still poorly understood. Small GTPases of the Rho family are crucial membrane-linked signalling proteins involved in many cellular functions, especially in actin cytoskeleton organisation but also in control of migration, proliferation and cell death. Their involvement in cellular response to ionizing radiation remains unclear, particularly in the endothelial compartment. Our study aims at studying 1) the regulation of activity of RhoA and Rac1, two main small Rho G proteins expressed in endothelial cells and 2) the possible role of these proteins in endothelial cellular functions critically affected by ionizing radiation like cytoskeleton reorganisation, cell death and migration. Using the microvascular endothelial cell line HMEC1 irradiated at 15 Gy, we show a rapid activation of RhoA concomitantly to an inactivation of Rac1. Analysis of actin cytoskeleton by confocal microscopy in HMEC1 cells indicate that 15 Gy-irradiation induces deep reorganisation of HMEC1 cell morphology, characterized by induction of stress fibers and decrease of lamellipodia, structures of polymerized actin respectively induced by RhoA and Rac1. We are currenly investigating the role of RhoA and Rac1 in induction of apoptotic cell death and in regulation of migration in 15 Gy-irradiated HMEC1, by the use of pharmacological specific inhibitors (Y-27632 for the RhoA pathway and NSC23766 for Rac1) and by invalidation of RhoA and Rac1 expression by stable RNA interference. Identifying Rho proteins as potential actors in endothelium damage to IR will permit a better understanding of molecular pathways involved and may lead to development of new strategies to modulate radiosensitization of this cellular compartment

WHY IMMUNOLIPOSOME AND NOT ADC TO EXTEND TRASTUZUMAB TO HER2 BREAST CANCER MODELS: A PROOF OF CONCEPT STUDY

International audienc

From 3D spheroids to tumor bearing mice: efficacy and distribution studies of trastuzumab-docetaxel immunoliposome in breast cancer

Anne Rodallec,1 Guillaume Sicard,1 Sarah Giacometti,1 Manon Carré,1 Bertrand Pourroy,2 Fanny Bouquet,3 Ariel Savina,3 Bruno Lacarelle,1 Joseph Ciccolini,1 Raphaelle Fanciullino1 1SMARTc Unit, Laboratory of Pharmacokinetics and Toxicology UFR Pharmacy, Center for Research on Cancer of Marseille, Inserm UMR1068, CNRS UMR7258, Aix Marseille University, Marseille, France; 2Pharmacy Department, APHM La Conception, Marseille, France; 3Roche Institute, Boulogne Billancourt, France Purpose: Nanoparticles are of rising interest in cancer research, but in vitro canonical cell monolayer models are not suitable to evaluate their efficacy when prototyping candidates. Here, we developed three-dimensional (3D) spheroid models to test the efficacy of trastuzumab-docetaxel immunoliposomes in breast cancer prior to further testing them in vivo. Materials and methods: Immunoliposomes were synthesized using the standard thin film method and maleimide linker. Two human breast cancer cell lines varying in Her2 expression were tested: Her2+ cells derived from metastatic site: mammary breast MDA-MB-453 and triple-negative MDA-MB-231 cells. 3D spheroids were developed and tested with fluorescence detection to evaluate viability. In vivo efficacy and biodistribution studies were performed on xenograft bearing nude mice using fluorescent and bioluminescent imaging. Results: In vitro, antiproliferative efficacy was dependent upon cell type, size of the spheroids, and treatment scheduling, resulting in subsequent changes between tested conditions and in vivo results. Immunoliposomes performed better than free docetaxel + free trastuzumab and ado-trastuzumab emtansine (T-DM1). On MDA-MB-453 and MDA-MB-231 cell growth was reduced by 76% and 25%, when compared to free docetaxel + free trastuzumab and by 85% and 70% when compared to T-DM1, respectively. In vivo studies showed tumor accumulation ranging from 3% up to 15% of the total administered dose in MDA-MB-453 and MDA-MB-231 bearing mice. When compared to free docetaxel + free trastuzumab, tumor growth was reduced by 89% (MDA-MB-453) and 25% (MDA-MB-231) and reduced by 66% (MDA-MB-453) and 29% (MDA-MB-231) when compared to T-DM1, an observation in line with data collected from 3D spheroids experiments. Conclusion: We demonstrated the predictivity of 3D in vitro models when developing and testing nanoparticles in experimental oncology. In vitro and in vivo data showed efficient drug delivery with higher efficacy and prolonged survival with immunoliposomes when compared to current anti-Her2 breast cancer strategies. Keywords: docetaxel, trastuzumab, breast cancer, immunoliposome, spheroids, distribution, tumor xenograf

The Th1 immune response against HIV-1 Gag p24-derived peptides in mice expressing HLA-A02.01 and HLA-DR1

Using HLA-DR1-transgenic H-2 class II knockout mice, we identified two new HLA-DR1-restricted HIV-1 Gag p24-derived epitopes (Gag321–340 and Gag331–350) and confirmed the immunogenicity of seven that have been previously described. The human relevance was confirmed for the two new ones (Gag321–340 and Gag331–350) assaying peripheral blood mononuclear cells from HLA-DR1+ HIV-1-infected long-term asymptomatic subjects and showing that Gag331–350 could prime CD4+ T cells from two HLA-DR1+ HIV-1 seronegative donors in vitro. Seven of these epitopes, structurally conserved among HIV-1 clade B isolates, were selected for a comparative evaluation of their Th1 helper potential by immunizing HLA-A02.01/HLA-DR1-transgenic, H-2 class I/class II knockout mice with recombinant mouse invariant chain constructs in which each helper epitope was inserted in association with two reporter HIV-1-derived HLA-A02.01-restricted CD8+ T cell epitopes. A T helper effect was demonstrated in all cases, and was particularly strong with epitopes Gag301–320, Gag321–340 and Gag271–290, which should, therefore, be considered in the design of new vaccines

Séquences IRM « SWAN, SWI et VenoBOLD » exploitant le phénomène de susceptibilité magnétique : principes techniques et applications cliniques

International audienc

Clin Exp Immunol

Natural Killer (NK) cell functions are regulated by diverse inhibitory and activating receptors including Killer cell Immunoglobulin-like receptors (KIR) which interact with HLA class I molecules. Some KIR/HLA genetic combinations were reported associated with spontaneous clearance (SC) of hepatitis C virus (HCV) but with discordant results, possibly reflecting KIR and/or HLA gene polymorphism according to populations. KIR/HLA genetic combinations associated with both an exhaustive NK and T cell repertoire were investigated in a cohort of HIV-HCV co-infected individuals with either SC (n=68) or chronic infection (CI, n=163) compared to uninfected blood donors (Ctrl, n=100). Multivariate analysis showed that the HLA C2C2 environment was associated with SC only in European HIV-HCV co-infected individuals (OR=4.30[1.57-12.25], p=0.005). KIR2D(+) NK cell repertoire and potential of degranulation of KIR2DL1/S1(+) NK cells were similar in SC European cohort compared to uninfected individuals. In contrast, decreased frequencies of KIR2DS1(+) and KIR2DL2(+) NK cells were detected in CI group of Europeans compared to SC and a decreased frequency of KIR2DL1/S1(+) NK cells compared to controls. On the T cell side, higher frequencies of DNAM-1(+) and CD57(+) T cells were observed in SC in comparison to controls. Interestingly, SC subjects emphasized increased frequencies of KIR2DL2/L3/S2(+) T cells compared to CI subjects. Our study underlines that the C2 environment may activate efficient KIR2DL1(+) NK cells in viral context and maintain KIR2DL2/L3/S2(+) mature T cell response in the absence of KIR2DL2 engagement with its cognate ligands in SC group of HCV-HIV co-infected European patients