Sylvain Fleury

Effects of mycophenolic acid on human immunodeficiency virus infection in vitro and in vivo.

Mycophenolic acid, a selective inhibitor of the de novo synthesis of guanosine nucleotides in T and B lymphocytes, has been proposed to inhibit human immunodeficiency virus (HIV) replication in vitro by depleting the substrate (guanosine nucleotides) for reverse transcriptase. Here we show that mycophenolic acid induced apoptosis and cell death in a large proportion of activated CD4+ T cells, thus indicating that it may inhibit HIV infection in vitro by both virological mechanisms and immunological mechanisms (depletion of the pool of activated CD4+ T lymphocytes). Administration of mycophenolate mophetil, the ester derivate of mycophenolic acid, to HIV-infected subjects treated with anti-retroviral therapy and with undetectable viremia resulted in the reduction of the number of dividing CD4+ and CD8+ T cells and in the inhibition of virus isolation from purified CD4+ T-cell populations. Based on these results, the potential use of mycophenolate mophetil in the treatment of HIV infection deserves further investigation in controlled clinical trials.

Multiply Attenuated, Self-Inactivating Lentiviral Vectors Efficiently Deliver and Express Genes for Extended Periods of Time in Adult Rat Cardiomyocytes In Vivo

Background—Among retroviral vectors, lentiviral vectors are unique in that they transduce genes into both dividing and nondividing cells. However, their ability to provide sustained myocardial transgene expression has not been evaluated. Methods and Results—Multiply attenuated, self-inactivating lentivectors based on human immunodeficiency virus-1 contained the enhanced green fluorescent protein (EGFP) gene under the transcriptional control of either the cytomegalovirus (CMV) immediate-early enhancer/promoter, the elongation factor-1&agr; (EF-1&agr;) promoter, or the phosphoglycerate-kinase (PGK) promoter. Lentivectors transduced adult rat cardiomyocytes in a dose-dependent manner (transduction rates, >90%; multiplicity of infection, ≈5). The CMV promoter achieved higher EGFP expression levels than the EF-1&agr; and PGK promoters. Insertion of the central polypurine tract pol sequence improved gene transfer efficiency by ≈2-fold. In vivo gene transfer kinetics was studied by measuring the copy number of integrated lentivirus DNA and EGFP concentrations in cardiac extracts by real-time polymerase chain reaction and ELISA, respectively. With CMV promoter-containing lentivectors, vector DNA peaked at day 3, declined by ≈4-fold at day 14, but then remained stable up to week 10. Similarly, EGFP expression peaked at day 7, decreased by ≈7-fold at day 14, but was essentially stable thereafter. In contrast, vector DNA and EGFP expression declined rapidly with EF-1&agr; promoter–containing lentivectors. Peak EGFP expression with titer-matched adenovectors was ≈35% higher than with CMV lentivectors but was lost rapidly over time. Conclusions—Lentivectors efficiently transduce and express genes for extended periods of time in cardiomyocytes in vivo. Lentivectors provide a useful tool for studying myocardial biology and a potential system for gene heart therapy.

Helper-dependent adenovirus vectors devoid of all viral genes cause less myocardial inflammation compared with first-generation adenovirus vectors.

Abstract.Background:First-generation, E1-deleted (ΔE1) adenovirus vectors currently used in cardiovascular gene therapy trials are limited by tissue inflammation, mainly due to immune responses to viral gene products. Recently, helper-dependent (HD; also referred to as “gutless”) adenovirus vectors devoid of all viral coding sequences have been shown to cause low inflammation when injected intravenously or into skeletal muscles. However, HD vectors have not been evaluated in cardiovascular tissues.Methods and results:HD and ΔE1 vectors containing a cytomegalovirus-driven expression cassette for the green fluorescent protein (GFP) gene were administered intramyocardially to adult rats (n = 54). GFP expression was measured by ELISA at varying time intervals after gene transfer. HD and ΔE1 vectors were equally efficient at transducing the myocardium. Tissue inflammation was assessed by immunostaining for leukocytes and quantitative real-time RT-PCR for cytokine mRNA expression. Monocyte/macrophages, CD4+ and CD8+ lymphocytes infiltrating the myocardium were less abundant with HD than ΔE1 vectors. Transcripts levels for pro-inflammatory cytokines such as IL-1β, tumor necrosis factor-α, and RANTES were decreased with HD vectors. However, both vectors were associated with a decline in GFP expression over time, although low-level expression was occasionally detectable 10 weeks after HD vector administration. The two vectors transduced endothelial cells in rat arteries (n = 11) with comparable efficiencies. Vascular GFP expression was not detectable at 10 weeks.Conclusions:HD vectors are as efficient as ΔE1 vectors at transducing the myocardium and vascular endothelium, while causing less myocardial inflammation. Thus, HD vectors may be superior to earlier-generation adenovirus vectors for cardiovascular gene therapy applications.

Virological and immunological characteristics of HIV treatment failure.

BackgroundResistance to antiretroviral treatment is prevalent. There is limited knowledge of the determinants of disease evolution in subjects infected with multidrug-resistant HIV (MDR-HIV). MethodsInfectivity, replication, chemokine receptor usage, and env, gag, protease and reverse transcriptase sequence analysis was performed for MDR-HIV isolates from 14 HIV-infected individuals and compared to wild-type HIV isolates from individuals naive to antiretroviral treatment. Expression of CD45RO/RA, Ki67 and interferon-γ and CD4 proliferative response to various antigens was determined for individuals infected with MDR-HIV and compared to that in individuals with optimal suppression of viral replication. ResultsInfectivity and replication are diminished for various MDR-HIV isolates, usually in the context of an increase in CD4 and CD4+CD45RA+ T-cell counts. However, a number of MDR-HIV isolates are associated with high in vivo viraemia and pronounced immunosuppression, and display in vitro levels of infectivity and replication comparable to those of wild-type strains. No specific genetic sequence or chemokine receptor usage predicted the fitness of an MDR isolate. ConclusionsDespite the biological diversity of resistant viruses and the range of host responses observed, our descriptive analysis indicates that viral factors play a role in determining the degree of immune damage observed in the context of MDR-HIV infection.

Lentiviral gene transfer of the chemokine antagonist RANTES 9-68 prolongs heart graft survival.

Background. Allograft tolerance might be achieved by expressing immunomodulatory proteins through gene therapy. We have evaluated the possibility of promoting significantly allograft survival in a vascularized cardiac allograft model by performing ex vivo gene transfer. We used a lentiviral vector encoding the chemokine antagonist RANTES 9-68 that is capable of competing with native RANTES. Methods. The Fisher donor/Lewis recipient rat strain combinations were used and all animals received for the first 5 days posttransplantation a subtherapeutic dose of cyclosporine A (1.5 mg/kg). Ex vivo gene transfer into heart allograft was performed by multiple injections of the SIN.cPPT lentiviral vector, which corresponds to the multiply attenuated, self-inactivating lentivector derived from the human immunodeficiency virus (HIV)-1. Results. About 6% of the cardiac tissue had integrated lentiviral vector, which closely matches the mean in vivo RANTES antagonist expression of 5% obtained by immunohistochemistry. In vivo RANTES 9-68 expression has significantly prolonged graft survival (median [25%–75%]: 20 [17–26] days), compared to the control 15 ([14-15] days; P=0.0007). Furthermore, hearts transduced with RANTES 9-68 showed a significant (P<0.05) reduction in cell infiltration and intragraft expression of TNF-&agr;, IFN-&ggr;, endogenous RANTES, and TGF-&bgr;. Conclusion. Lentiviral gene transfer of RANTES 9-68 antagonist attenuates significantly the inflammatory response and delays allograft rejection, despite low levels of transduction. Future improvement of heart transduction by lentiviral vectors, as it has been achieved with other vectors, might become an attractive alternative therapy for treating allografts that require sustained gene expression for better organ preservation.

Gene transfer of RANTES and MCP-1 chemokine antagonists prolongs cardiac allograft survival.

Vascularized organ allografts are rapidly destroyed by host immune cells that are recruited along chemokine gradients. Among chemokines, Regulated on Activation, Normal T-cell Expressed and Secreted (RANTES) CC chemokine ligand (CCL5) and monocyte chemoattractant protein (MCP)-1 (CCL2) are upregulated in rejecting cardiac allografts. To antagonize these chemokines, we constructed adenoviral vectors expressing NH2-terminal deletion (8ND) mutants of the respective genes. Using the F344-to-LEW rat model, intragraft gene transfer of chemokine analogs prolonged cardiac allograft survival from 10.1±0.7 and 10.4±0.7 days using non-coding adenovirus and vehicle alone, respectively, to 17.0±0.7 days for 8ND-RANTES (P<0.001) and 14.2±0.8 days for 8ND-MCP-1 (P<0.01). 8ND-RANTES reduced graft infiltration by monocytes/macrophages, cluster of differentiation (CD) 8α+ and T-cell receptor αβ+ cells, while 8ND-MCP-1 reduced monocytes/macrophages. In mixed leukocyte reactions in vitro, proliferation of host lymphocytes from regional lymph nodes in response to donor splenocytes was unaffected by 8ND-RANTES gene transfer. Using a two-gene approach, the contribution of 8ND-MCP-1 was negligible, consistent with available evidence that 8ND-RANTES inhibits both RANTES and MCP-1 activities. 8ND-RANTES gene transfer and a short course of low-dose cyclosporine A synergistically prolonged graft survival to 37.8±5.5 vs 15.4±0.5 days with cyclosporine alone (P<0.001). These results suggest a role for anti-chemokine gene therapy as an adjuvant therapy in heart transplantation.

CCR5, RANTES and CX3CR1 polymorphisms : Possible genetic links with acute heart rejection

Background. The inflammation response is modulated by the elaborated chemokine-chemokine receptor system, which also plays an important role in the development of acute rejection (AR). In this study, we hypothesized that functional genetic variants of some of these modulatory proteins might influence the outcome of AR. Methods. In a retrospective analysis of a cohort of heart transplanted patients (n=158), we examined eight polymorphisms in four genes implicated in this inflammatory process: RANTES, CCR5, CCR2 and CX3CR1. On the basis of timing occurrence, AR episodes (grade≥ 3A) were classified in “early” (0–3 months posttransplantation; EAR) or “late” outcomes (4–12 months posttransplantation; LAR). Results. The incidences of EAR and LAR were 57.6% and 41%, respectively. Number of LAR episodes was significantly higher in subjects that have already experienced one or more EAR episodes, as compared to subjects that had no EAR (median [25%-75%]: 4 (3–5) vs. 1 [1–2.5] respectively; P<0.0001). Statistical univariate analysis showed that none of the mentioned polymorphisms were correlated with EAR or LAR. However, allele-allele association analysis showed that subjects carrying both the CX3CR1 249I allele and CCR5 No-E haplotypes were significantly at lower risk of experiencing EAR (OR=0.2 [95%-CI=0.1–0.5], P=0.001). In contrast subjects carrying both the CCR5 E haplotype and the RANTES –403A allele were significantly at higher risk to develop LAR (OR=8.1 [95%-CI=2.3–28.7], P=0.002). Conclusions. This exploratory study in heart transplantation suggests that the outcomes of EAR and LAR episodes may be influenced by genetic variant interactions such as “CX3CR1 249I*CCR5 No-E” and “CCR5 E*RANTES –403A.”

Gene transfer of cytoprotective and immunomodulatory molecules for prevention of cardiac allograft rejection.

Current treatments of heart transplantation are limited by incomplete effectiveness, significant toxicity, and failure to prevent chronic rejection. Genetic manipulation of the donor heart at the time of removal offers the unique opportunity to produce a therapeutic molecule within the graft itself, while minimizing systemic effects. Cytoprotective approaches including gene transfer of heme oxygenase (HO)-1, endothelial nitric oxide synthase, and antisense oligodeoxynucleotides specific for nuclear factor (NF)-kappa B or intercellular adhesion molecule (ICAM)-1 reduced ischaemia-reperfusion injury and delayed cardiac allograft rejection in small animals. Exogenous overexpression of immunomodulatory cytokines such as interleukin (IL)-4, IL-10 and transforming growth factor-beta, as well as gene transfer of inhibitors of pro-inflammatory cytokines also delayed graft rejection. Gene transfer-based blockade of T-cell costimulatory activation with CTLA4-Ig or CD40-Ig resulted in long-lasting graft survival and donor-specific unresponsiveness, as manifested by acceptance of a second graft from the original donor strain but rejection of third-party grafts. Similar results were obtained with donor major histocompatibility complex class I gene transfer into bone marrow cells. Gene therapy approaches to chronic rejection included gene transfer of HO-1, soluble Fas, tissue plasminogen activator and antisense oligodeoxynucleotides specific for the anti-apoptotic mediator Bcl-x or the E2F transcription factor. Despite major experimental advances, however, gene therapy for heart transplantation has not entered the clinical arena yet. Fundamental questions regarding the most suitable vector, the best gene, and safety issues remain unanswered. Well-controlled studies that compare gene therapy with established treatments in non-human primates are needed before clinical trials can be started.

Sampling lymphoid tissue cells by ultrasound-guided fine needle aspiration of lymph nodes in Hiv-infected patients

OBJECTIVE To establish the feasibility of using ultrasound-guided lymph node needle aspiration as a means to obtain lymphoid tissue cells for the determination of a series of immunologic and virologic measures in HIV-infected patients. DESIGN First, a comparison of the characteristics of cell populations obtained by simultaneous needle aspiration and standard excisional biopsy in six patients. Second, use of lymph node needle aspiration to assess longitudinally T-cell subset changes in patients initiating highly effective antiretroviral treatment. METHODS T-cell subsets (CD4 and CD8) and percentage Ki67+ cycling T cells were measured in lymph node cell populations harvested by ultrasound-guided aspiration or standard biopsy by flow cytometry. Cellular RNA content was assessed by a modification of the Roche Amplicor HIV-1 Monitor test. RESULTS CD4 and CD8 T-cell percentage and HIV RNA cell content of lymph node cell suspensions obtained from the simultaneous performance of ultrasound-guided needle aspiration and excisional biopsy in the same patients were correlated (n = 6). Among the 87 aspiration sessions reported here, mononuclear cell suspensions were obtained in 100% of the sessions, in numbers ranging between 4x10(4) to 6.7x10(6) cells (median: 7x10(5)). This limited number of cells did not allow to perform all type of analyses in all patients. By prioritizing the cells for the determination of T-cell subsets and proliferation rate, this approach was instrumental for demonstrating the normalization of the T-cell subset ratio and the kinetic of normalization of proliferating rates of CD4 and CD8 T cells, as well as the decrease in HIV-1 viral load in the lymph node following HAART initiation. CONCLUSION Ultrasound-guided aspiration appears to be a non-invasive and ad libitum, safe and repeatable procedure for the longitudinal monitoring of changes in lymph nodes.

Gene transfer of interleukin-18-binding protein attenuates cardiac allograft rejection.

Interleukin (IL) 18 is a potent pro‐inflammatory Th1 cytokine that exerts pleiotropic effector functions in both innate and acquired immune responses. Increased IL‐18 production during acute rejection has been reported in experimental heart transplantation models and in kidney transplant recipients. IL‐18‐binding protein (IL‐18BP) binds IL‐18 with high affinity and neutralizes its biologic activity. We have analyzed the efficacy of an adenoviral vector expressing an IL‐18BP‐Ig fusion protein in a rat model of heart transplantation. IL‐18BP‐Ig gene transfer into Fisher (F344) rat donor hearts resulted in prolonged graft survival in Lewis recipients (15.8 ± 1.4 days vs. 10.3 ± 2.5 and 10.1 ± 2.1 days with control virus and buffer solution alone, respectively; P < 0.001). Immunohistochemical analysis revealed decreased intra‐graft infiltrates of monocytes/macrophages, CD4+, CD8α+ and T‐cell receptor αβ+ cells after IL‐18BP‐Ig versus mock gene transfer (P < 0.05). Real‐time reverse transcriptase polymerase chain reaction analysis showed decreased cytokine transcripts for the RANTES chemokine and transforming growth factor‐β after IL‐18BP‐Ig gene transfer (P < 0.05). IL‐18BP‐Ig gene transfer attenuates inflammatory cell infiltrates and prolongs cardiac allograft survival in rats. These results suggest a contributory role for IL‐18 in acute rejection. Further studies aiming at defining the therapeutic potential of IL‐18BP are warranted.