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International audience; BACKGROUND: The efficacy of raltegravir plus optimized background therapy (OBT) has been demonstrated for antiretroviral (ARV)-experienced HIV-1-infected patients in randomized clinical trials. We studied viro-immunological response, pharmacokinetic parameters and genotypic test results in an observational cohort of multiple ARV class-experienced patients starting a raltegravir-based regimen. METHODS: Already enrolled ANRS CO3 Aquitaine Cohort patients with virological failure were included in this study after starting a raltegravir-based regimen (400 mg twice a day, week 0). Virological success was defined by the plasma HIV-1 RNA level [viral load (VL)] or =1 and or =2 (P = 0.02), respectively. Raltegravir-related mutations emerged in 9 of 11 failing patients (82%): Q148H/R (n = 5), N155S/H (n = 3) and S230N (n = 1). Median CD4 increases from week 0 to week 4 and week 24 were 28 (-4, 85) and 57 (0, 156) cells/mm(3), respectively. A poor immune response was independently associated with a lower VL decline (week 0 to week 12) [odds ratio (OR): 3.5, 95% confidence interval (CI): 1.4, 8.4, for 1 log(10) less] and CD4+% at baseline (OR: 2.6, 95% CI: 0.97, 8.3, for 10% lower). CONCLUSIONS: Raltegravir plus OBT provided a good virological success rate in highly pre-treated patients under clinical routine conditions.

Background HIV type-1 (HIV-1) has been shown to be frequently transmitted by acutely infected patients. We investigated the relationship between the dynamics of HIV-1 transmission within recently infected patients, the HIV-1 variability and the transmission of antiretroviral drug resistance. Methods We included patients infected between 1996 and 2006, with a plasma sample obtained Results Genotypic resistance was detected in 37/263 (14.1%) patients. Patients were infected by HIV-1 clade B in 222 (84%) cases and with non-B subtypes in 41 (16%). A total of 80 (30.4%) RT sequences were segregated in 24 clusters with bootstrap values >98% for 22 clusters. The frequency of grouping in clusters was higher within B sequences compared with non-B sequences (35.1% versus 4.9%; P-4). Drug-resistant isolates were retrieved in only 3 clusters, but the prevalence of resistance in clustering viruses (10/80, 12.5%) was not different than in isolated sequences. Conclusions The segregation into clusters suggested frequent forward transmission events in patients infected with HIV-1 subtype B, including the possibility of transmission of drug-resistant isolates. These findings warrant increasing prevention efforts and serological screening in the at-risk populations.

Constitutively active Rac stimulates Akt activity in T lymphocytes cultured in suspension. This regulation contrasts with findings obtained in fibroblasts, endothelial or neuronal cells grown on substrate, where Akt stimulation occurs independently of Rac. We now show that V12Rac-mediated stimulation of Akt is not restricted to the hematopoietic lineage but is dependent on the adherence status of the cell. V12Rac-mediated stimulation of Akt as well as molecular association between Rac and Akt occurred exclusively in cells kept in suspension. Stimulation and complex formation are dependent on SHIP but in a manner that differs from its role in dephosphorylation of phosphoinositide lipids. Adherent cells lacking SHIP, but not those lacking PTEN, are able to activate Akt through the Rac pathway. Our data reveal the existence of a bona fide Rac to Akt signaling pathway, tightly regulated by SHIP and operational in suspended cells only. This pathway may point to an alternative survival signal that is called into action when cells lose contact with the substrate and/or with other cells.

The single antigen flow bead (SAFB) assay greatly improves the identification of antigenic specificity of anti-HLA alloantibodies. However, it may underestimate or miss high titer antibodies due to the prozone phenomenon caused by a competition between the fluorescent anti-IgG conjugate and serum complement, for the alloantibody. We explored this effect in our cohort of transplant candidates and transplanted recipients. Among a total of 292 and 269 patients with at least three different sera tested with class I and/or II SAFB assays respectively, we identified 9 patients (6 in class I and 3 in class II) who displayed a profound drop (≥ 75%) followed by a subsequent rise (≥ 100%), in strong (mean fluorescence intensity >8000) antibody levels, across an 18-month period. We postulated that such abrupt fluctuations were not explainable by naturally occurring transient desensitization. Sera were analysed with the SAFB assay using EDTA-treated serum and direct complement C1q staining, and with complement-dependent cytotoxicity and flow cytometry crossmatches (CDCXM and FCXM respectively). The prozone phenomenon was involved in all cases. Because it relies on complement activation, the CDCXM was not sensitive to this phenomenon, but the FCMXM was not either, although it resembles in its principle to the SAFB assay. Four additional anti-human conjugates targeting the IgG Fc fragment or the light chains did not circumvent the SAFB drawback. Therefore, a quick decrease in antibody strength must alert against a potential risk for recipients at the time of the transplant, using virtual crossmatch strategies. A prospective pre-transplant crossmatch still remains an ultimate safeguard.

International audience; BACKGROUND: To date, 11 HIV-1 subtypes and 48 circulating recombinant forms have been described worldwide. The underlying reason why their distribution is so heterogeneous is not clear. Host genetic factors could partly explain this distribution. The aim of this study was to describe HIV-1 strains circulating in an unexplored area of Mekong Delta, Vietnam, and to assess the impact of optimal epitope mutations on HLA binding. METHODS: We recruited 125 chronically antiretroviral-naive HIV-1-infected subjects from five cities in the Mekong Delta. We performed high-resolution DNA typing of HLA class I alleles, sequencing of Gag and RT-Prot genes and phylogenetic analysis of the strains. Epitope mutations were analyzed in patients bearing the HLA allele restricting the studied epitope. Optimal wild-type epitopes from the Los Alamos database were used as reference. T-cell epitope recognition was predicted using the immune epitope database tool according to three different scores involved in antigen processing (TAP and proteasome scores) and HLA binding (MHC score). RESULTS: All sequences clustered with CRF01_AE. HLA class I genotyping showed the predominance of Asian alleles as A*11:01 and B*46:01 with a Vietnamese specificity held by two different haplotypes. The percentage of homology between Mekong and B consensus HIV-1 sequences was above 85%. Divergent epitopes had TAP and proteasome scores comparable with wild-type epitopes. MHC scores were significantly lower in divergent epitopes with a mean of 2.4 (±0.9) versus 2 (±0.7) in non-divergent ones (p

International audience; BACKGROUND: Our aim was to study the in vivo viral genetic pathways for resistance to raltegravir, in antiretroviral-experienced patients with virological failure (VF) on raltegravir-containing regimens. METHODS: We set up a prospective study including antiretroviral-experienced patients receiving raltegravir-based regimens. Integrase (IN) genotypic resistance analysis was performed at baseline. IN was also sequenced at follow-up points in the case of VF, i.e. plasma HIV-1 RNA>400 copies/mL at month 3 and/or >50 copies/mL at month 6. For phenotyping, the IN region was recombined with an IN-deleted HXB2-based HIV-1 backbone. A titrated amount of IN recombinant viruses was used for antiviral testing against raltegravir and elvitegravir. RESULTS: Among 51 patients, 11 (21.6%) had VF. Four different patterns of IN mutations were observed: (i) emergence of Q148H/R with secondary mutations (n=5 patients); (ii) emergence of N155H, then replaced by a pattern including Y143C/H/R (n=3); (iii) selection of S230N (n=1); and (iv) no evidence of selection of IN mutations (n=2). The median raltegravir and elvitegravir fold changes (FCs) were 244 (154-647) and 793 (339-892), respectively, for the Q148H/R pattern, while the median raltegravir and elvitegravir FCs were 21 (6-52) and 3 (2-3), respectively, with Y143C/H/R. The median plasma raltegravir Cmin was lower in patients with selection of the N155H mutation followed by Y143C/H/R compared with patients with Q148H/R and with patients without emerging mutations or without VF. CONCLUSIONS: Diverse genetic profiles can be associated with VF on raltegravir-containing regimens, including the dynamics of replacement of mutational profiles. Pharmacokinetic parameters could be involved in this genetic evolution.

International audience; OBJECTIVES: Our aim was to analyse the evolution of HIV-1 2-long terminal repeat (2-LTR) circular DNA in vitro and ex vivo in the presence of raltegravir. PATIENTS AND METHODS: Twenty-five patients starting a raltegravir-based regimen were included. Total HIV-1 DNA and 2-LTR DNA were quantified at baseline and in follow-up samples up to month 12. The effect of raltegravir on the formation of 2-LTR circles was evaluated in HeLa P4 cells. The effect of raltegravir was also investigated by sequence analysis of the 2-LTR circle junctions. RESULTS: Among 21 patients with undetectable 2-LTR DNA at baseline, 7 had detectable 2-LTR DNA during the follow-up. Three of four patients with detectable 2-LTR DNA at baseline had undetectable 2-LTR DNA during the follow-up (P = 0.27). The mean 2-LTR level increased significantly (+0.07 log(10)/month, P = 0.02) in raltegravir-treated patients, and a 2-LTR increase was also observed in raltegravir-treated HeLa P4 cells, with a peak at 3 days post-infection. 2-LTR DNA showed a high prevalence of deletions ex vivo (64.5%) and in vitro (50%) in the presence of raltegravir, which was not statistically different from the prevalence in untreated patients or cells. CONCLUSIONS: In antiretroviral-experienced patients receiving raltegravir, 2-LTR DNA increased while total HIV-1 DNA decreased over time. The frequent rearrangements found in 2-LTR sequences warrant further investigations to determine the dynamics of evolution of unintegrated HIV-1 DNA.

Ectopic expression of a constitutive active mutant of the GTPase Cdc42 (V12Cdc42) in vascular endothelial cells triggers the dissolution of stress fibres and focal adhesion contacts and causes the repolymerisation of actin into dots. Each punctate structure consists of an F-actin core surrounded by a vinculin ring, consistent with the definition of podosomes. We now report further analysis of these complexes and show the presence of established podosomal markers such as cortactin, gelsolin, dynamin, N-WASP, and Arp2/3 which are absent in focal adhesions. Endothelial podosomes appear as randomly distributed conical structures, distributed on, but restricted to, the ventral membrane and confined to contact sites between cells and their substratum. The nature of the extracellular matrix does not influence podosome formation nor their spatial organisation. Induction of podosomes in response to V12Cdc42 is not associated with a migratory nor with a proliferative phenotype. These results add endothelial cells to the list of cell types endowed with the ability to form podosomes in vitro and raise the possibility that endothelial cells could form such structures under certain physiological or pathological conditions.