RESUMO
The bioavailability of ivermectin is modulated by lipid-based formulations and membrane efflux transporters such as Breast Cancer Resistance Protein and P-glycoprotein (BCRP and P-gp). We have investigated the effect of oleic acid on the uptake of ivermectin in vitro using Caco-2 cells and in vivo in the intestines of wild-type mice. Complex micelles (M) with oleic acid induced a significant increase (e. g. for M3 was 7-fold, p≤0.001) in the uptake of the drug in a time-dependent manner with no involvement of cholesterol in the mechanism. In vivo results showed a significant increase in the concentration of plasma and intestinal mucosa ivermectin (p≤0.01) in mice receiving oleic acid-based drug formulation. We also examined the expression of the drug efflux transporter, BCRP and P-gp in Caco-2 cells and found a significant decrease (p≤0.001) in their level in the presence of 5 mM oleic acid. Treatment of mice with oleic acid-based formulation showed a significant decrease in the activity of P-gp in the intestinal mucosa (p≤0.01). This study highlighted the effect of oleic acid in decreasing the expression and the activity of P-gp-mediated ivermectin efflux and in limiting the drug absorption by increasing its uptake and bioavailability in Caco-2 cells and intestine, respectively.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Ivermectina/farmacocinética , Ácido Oleico/farmacologia , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Disponibilidade Biológica , Linhagem Celular Tumoral , Interações Medicamentosas , Humanos , Absorção Intestinal/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Ivermectina/sangue , CamundongosRESUMO
Heterosexual contact is the primary mode of human immunodeficiency virus (HIV) type 1 (HIV-1) transmission worldwide. The chemokine receptor CCR5 is the major coreceptor that is associated with the mucosal transmission of R5-tropic HIV-1 during sexual intercourse. The CCR5 molecule is thus a target for antibody-based therapeutic strategies aimed at blocking HIV-1 entry into cells. We have previously demonstrated that polyreactive natural antibodies (NAbs) from therapeutic preparations of immunoglobulin G and from human breast milk contain NAbs directed against CCR5. Such antibodies inhibit the infection of human macrophages and T lymphocytes by R5-tropic isolates of HIV in vitro. In the present study, we demonstrate that human immunoglobulins from the cervicovaginal secretions of HIV-seronegative or HIV-seropositive women contain NAbs directed against the HIV-1 coreceptor CCR5. Natural affinity-purified anti-CCR5 antibodies bound to CCR5 expressed on macrophages and dendritic cells and further inhibited the infection of macrophages and dendritic cells with primary and laboratory-adapted R5-tropic HIV but not with X4-tropic HIV. Natural anti-CCR5 antibodies moderately inhibited R5-tropic HIV transfer from monocyte-derived dendritic cells to autologous T cells. Our results suggest that mucosal anti-CCR5 antibodies from healthy immunocompetent donors may hamper the penetration of HIV and may be suitable for use in the development of novel passive immunotherapy regimens in specific clinical settings of HIV infection.
Assuntos
Células Dendríticas/virologia , Anticorpos Anti-HIV/farmacologia , HIV-1/efeitos dos fármacos , Macrófagos/virologia , Receptores CCR5/imunologia , Sítios de Ligação de Anticorpos/imunologia , Antagonistas dos Receptores CCR5 , Colo do Útero , Células Dendríticas/imunologia , Feminino , Anticorpos Anti-HIV/isolamento & purificação , Anticorpos Anti-HIV/metabolismo , Humanos , Macrófagos/imunologia , Receptores CCR5/biossíntese , Receptores CCR5/metabolismo , VaginaRESUMO
BACKGROUND: Plant lectins such as Galanthus nivalis agglutinin (GNA) and Hippeastrum hybrid agglutinin (HHA) are natural proteins able to link mannose residues, and therefore inhibit HIV-target cell interactions. Plant lectins are candidate for microbicide development. OBJECTIVE: To evaluate the activity against HIV of the mannose-specific plant lectins HHA and GNA at the cellular membrane level of epithelial cells and monocyte-derived dendritic cells (MDDC), two potential target cells of HIV at the genital mucosal level. METHODS: The inhibitory effects of HHA and GNA were evaluated on HIV adsorption to genital epithelial HEC-1A cell line, on HIV transcytosis throughout a monolayer of polarized epithelial HEC-1A cells, on HIV adsorption to MDDC and on transfer of HIV from MDDC to autologous T lymphocytes. RESULTS: HHA faintly inhibited attachment to HEC-1A cells of the R5-tropic HIV-1Ba-L strain, in a dose-dependent manner, whereas GNA moderately inhibited HIV adsorption in the same context, but only at high drug doses. Only HHA, but not GNA, inhibited HIV-1JR-CSF transcytosis in a dose-dependent manner. By confocal microscopy, HHA, but not GNA, was adsorbed at the epithelial cell surface, suggesting that HHA interacts specifically with receptors mediating HIV-1 transcytosis. Both plant lectins partially inhibited HIV attachment to MDDC. HHA inhibited more efficiently the transfer of HIV from MDDC to T cell, than GNA. Both HHA and GNA lacked toxicity below 200 microg/ml irrespective the cellular system used and do not disturb the monolayer integrity of epithelial cells. CONCLUSION: These observations demonstrate higher inhibitory activities of the lectin plant HHA by comparison to GNA, on HIV adsorption to HEC-1A cell line, HIV transcytosis through HEC-1A cell line monolayer, HIV adsorption to MDDC and HIV transfer from MDDC to T cells, highlighting the potential interest of HHA as effective microbicide against HIV.
Assuntos
Anti-Infecciosos/farmacologia , Desenho de Fármacos , HIV-1/efeitos dos fármacos , Lectinas de Ligação a Manose/química , Lectinas de Ligação a Manose/farmacologia , Lectinas de Plantas/química , Lectinas de Plantas/farmacologia , Adulto , Linhagem Celular , Células Dendríticas/citologia , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/virologia , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/virologia , HIV-1/fisiologia , Humanos , Lectinas de Ligação a Manose/toxicidade , Monócitos/citologia , Fitoterapia , Lectinas de Plantas/toxicidade , Ligação Viral/efeitos dos fármacosRESUMO
BACKGROUND: Necrosis is a frequent condition during AIDS, notably in organs targetted by opportunistic infections. Soluble factors released by necrotic cells are important for signalling cell damage, but little is known concerning their effect on HIV-1 replication. We focused on HMGB1, an abundant component of the chromatin that is released from necrotic cells and can act as a pro-inflammatory mediator. MATERIALS AND METHODS: A native form of HMGB1 was obtained from necrotic Hela cells, whereas a purified recombinant HMGB1 was generated in Escherichia coli. ACH-2 and U1 cells were used as models of persistent HIV-1 infection in lymphocytes and monocytes. Reactivation from latency was also investigated ex vivo using peripheral blood mononuclear cells (PBMC) collected from HIV-1-infected patients controlled by HAART. HIV-1 expression was quantified by enzyme-linked immunosorbent assay, real-time reverse transcription-polymerase chain reaction and branched DNA techniques. Flow cytometry and blocking experiments were used to identify the receptor used by HMGB1. Chromatin immunoprecipitation was used to investigate long-terminal repeat activation upon stimulation by HMGB1. RESULTS: HMGB1 increased HIV-1 transcription in chronically infected cells, a process that did not require de-novo protein synthesis. HIV-1 induction relied on HMGB1 interaction with the receptor for advanced glycation end-products. The activation pathway involved p38 and extracellular signal-related kinase as well as nuclear factor kappa B binding to the HIV-1 promoter. Finally, HMGB1 reactivated HIV-1 from latently infected PBMC collected in aviraemic HIV-infected patients. CONCLUSION: This work establishes for the first time a link between necrosis and HIV-1 replication, which involves HMGB1, a soluble mediator released by damaged cells.
Assuntos
Infecções por HIV/virologia , HIV-1/efeitos dos fármacos , Proteína HMGB1/farmacologia , Ativação Viral/efeitos dos fármacos , Terapia Antirretroviral de Alta Atividade , Células Cultivadas , Citocinas/biossíntese , Relação Dose-Resposta a Droga , MAP Quinases Reguladas por Sinal Extracelular/fisiologia , Infecções por HIV/tratamento farmacológico , Infecções por HIV/patologia , HIV-1/fisiologia , Proteína HMGB1/fisiologia , Células HeLa , Humanos , NF-kappa B/fisiologia , Necrose , Receptor para Produtos Finais de Glicação Avançada , Receptores Imunológicos/fisiologia , Proteínas Recombinantes/farmacologia , Ativação Transcricional/efeitos dos fármacos , Fator de Necrose Tumoral alfa/biossíntese , Ativação Viral/fisiologia , Latência Viral , Proteínas Quinases p38 Ativadas por Mitógeno/fisiologiaRESUMO
In the present study, we demonstrated that opsonization of primary HIV-1 with human complement enhances infection of immature monocyte-derived dendritic cells (iDC) and transmission in trans of HIV to autologous CD4(+) T lymphocytes. Infection of iDC by opsonized primary R5- and X4-tropic HIV was increased 3- to 5-fold as compared with infection by the corresponding unopsonized HIV. Enhancement of infection was dependent on CR3 as demonstrated by inhibition induced by blocking Abs. The interaction of HIV with CCR5 and CXCR4 on iDC was affected by opsonization. Indeed, stromal-derived factor-1 was more efficient in inhibiting infection of iDC with opsonized R5-tropic HIV-1(BaL) (45%) than with heat-inactivated complement opsonized virus and similarly RANTES inhibited more efficiently infection of iDC with opsonized X4-tropic HIV-1(NDK) (42%) than with heat-inactivated complement opsonized virus. We also showed that attachment of complement-opsonized virus to DC-specific ICAM-grabbing nonintegrin (DC-SIGN) molecule on iDC and HeLa DC-SIGN(+) CR3(-) cells was 46% and 50% higher compared with heat-inactivated complement opsonized virus, respectively. Hence, Abs to DC-SIGN suppressed up to 80% and 60% the binding of opsonized virus to HeLa cells and iDC, respectively. Furthermore, Abs to DC-SIGN inhibited up to 70% of the infection of iDC and up to 65% of infection in trans of autologous lymphocytes with opsonized virus. These results further demonstrated the role of DC-SIGN in complement opsonized virus uptake and infection. Thus, the virus uses complement to its advantage to facilitate early steps leading to infection following mucosal transmission of HIV.
Assuntos
Linfócitos T CD4-Positivos/metabolismo , Moléculas de Adesão Celular/metabolismo , Transformação Celular Viral/fisiologia , Proteínas do Sistema Complemento/metabolismo , Células Dendríticas/metabolismo , HIV/fisiologia , Lectinas Tipo C/metabolismo , Antígeno de Macrófago 1/metabolismo , Receptores de Superfície Celular/metabolismo , Diferenciação Celular , Células Cultivadas , Células Dendríticas/citologia , Humanos , Monócitos/citologia , Monócitos/metabolismo , Fenótipo , Receptores CCR5/metabolismo , Receptores CXCR4/metabolismoRESUMO
The mechanism of viral transmission across the mucosal barrier is poorly understood. Using the endometrial epithelium-derived cell line HEC-1A, we found that the cells are capable of sequestering large numbers of HIV-1 particles but are refractory to cell-free viral infection. The removal of heparan sulfate moieties of cell-surface proteoglycans (HSPG) from the apical pole of HEC-1A accounted for at least 60% of both R5- and X4-HIV-1 attachment, showing their important implication in viral attachment. HEC-1A cells also have the capacity to endocytose a weak proportion of the attached virus and pass it along to underlying cells. Fucose, N-acetylglucosamine and mannosylated-residues inhibited the transcytosis of some virus isolates, suggesting that mannose receptors can be implicated on the both R5- and X4-HIV-1 transcytosis. The inhibition of HIV transcytosis by blocking CCR5 mAb suggests the implication of specific interaction between the viral gp120 and sulfated moiety of syndecans during the transcytosis of mostly R5- and X4-HIV-1. At the basolateral pole of HEC-1A, HSPG sequestered X4- and not R5-HIV-1, highlighting the important role of HEC-1A as an X4 virus reservoir. The cell-free virus particles that have transcytosed could infect activated T cells but with a weaker efficiency than virus that had not transcytosed. The specific stimulation of HEC-1A by R5-HIV-1 increased the release of monocytes/chemokines-attracting chemokines (IL-8 and GR0) and proinflammatory cytokines (TNF-beta and IL-1alpha) that enhanced the production of virus by activated T cells. This study suggests that R5 and X4 viruses can differentially use epithelial cells to ensure their own spread.
Assuntos
Endométrio/virologia , Células Epiteliais/virologia , HIV-1/fisiologia , Receptores CCR5/fisiologia , Receptores CXCR4/fisiologia , Acetilglucosamina/farmacologia , Fármacos Anti-HIV/farmacologia , Antagonistas dos Receptores CCR5 , Linhagem Celular , Quimiocina CXCL1 , Quimiocinas CXC/biossíntese , Endocitose/efeitos dos fármacos , Feminino , Fucose/farmacologia , Proteína do Núcleo p24 do HIV/análise , Infecções por HIV/transmissão , Infecções por HIV/virologia , HIV-1/imunologia , HIV-1/patogenicidade , Proteoglicanas de Heparan Sulfato/fisiologia , Humanos , Interleucina-1alfa/biossíntese , Interleucina-8/biossíntese , Lectinas Tipo C/fisiologia , Manose/análogos & derivados , Manose/farmacologia , Receptor de Manose , Lectinas de Ligação a Manose/fisiologia , Receptores de Superfície Celular/fisiologia , Fator de Necrose Tumoral alfa/biossíntese , Virulência , Ligação ViralRESUMO
The ability of macrophages to adapt to changing cytokine environments results in the dominance of a particular functional phenotype of macrophages, which would play a significant role in HIV pathogenesis. In comparison with untreated macrophages (M0), we examined the role of macrophages derived from IFN-gamma-activated monocytes (M1) in the HIV spread. We show that M0 and M1 bind with the same efficiency HIV-1 with a predominant role of C-type lectins in the R5-HIV attachment and of the heparan sulfate proteoglycans in the X4-HIV attachment. Despite similar levels of R5- and X4-HIV DNA, M1 replicates and weakly transmits the virus to activated T cells by releasing CXCR4- and CCR5-interacting chemokines. The blockade of dendritic cell-specific ICAM-3-grabbing nonintegrin expressed on M1 by mAb does not interfere with the viral transfer. Uninfected M1 recruits HIV-sensitive T cells efficiently and releases soluble factors, enhancing the viral production by these recruited cells. This study highlights the role of IFN-gamma to induce a population of macrophages that archive HIV-1 within a latent stage and cause the persistence of the virus by favoring the recruitment of T cells or enhancing the viral replication in infected CD4(+) T cells.