RESUMEN
Background and objectives: To enter the target cell, HIV-1 binds not only CD4 but also a co-receptor ß-chemokine receptor 5 (CCR5) or α chemokine receptor 4 (CXCR4). Limited information is available on the impact of co-receptor usage on HIV-1 replication in monocyte-derived macrophages (MDM) and on the homeostasis of this important cellular reservoir. Materials and Methods: Replication (measured by p24 production) of the CCR5-tropic 81A strain increased up to 10 days post-infection and then reached a plateau. Conversely, the replication of the CXCR4-tropic NL4.3 strain (after an initial increase up to day 7) underwent a drastic decrease becoming almost undetectable after 10 days post-infection. The ability of CCR5-tropic and CXCR4-tropic strains to induce cell death in MDM was then evaluated. While for CCR5-tropic 81A the rate of apoptosis in MDM was comparable to uninfected MDM, the infection of CXCR4-tropic NL4.3 in MDM was associated with a rate of 14.3% of apoptotic cells at day 6 reaching a peak of 43.5% at day 10 post-infection. Results: This suggests that the decrease in CXCR4-tropic strain replication in MDM can be due to their ability to induce cell death in MDM. The increase in apoptosis was paralleled with a 2-fold increase in the phosphorylated form of p38 compared to WT. Furthermore, microarray analysis showed modulation of proapoptotic and cancer-related genes induced by CXCR4-tropic strains starting from 24 h after infection, whereas CCR5 viruses modulated the expression of genes not correlated with apoptotic-pathways. Conclusions: In conclusion, CXCR4-tropic strains can induce a remarkable depletion of MDM. Conversely, MDM can represent an important cellular reservoir for CCR5-tropic strains supporting the role of CCR5-usage in HIV-1 pathogenesis and as a pharmacological target to contribute to an HIV-1 cure.
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VIH-1/efectos de los fármacos , VIH-1/crecimiento & desarrollo , Compuestos Heterocíclicos/farmacología , Macrófagos/efectos de los fármacos , Fármacos Anti-VIH/farmacología , Bencilaminas , Ciclamas , Fragmentación del ADN/efectos de los fármacos , VIH-1/aislamiento & purificación , Humanos , Receptores CCR5/efectos de los fármacos , Receptores CCR5/genética , Receptores CXCR4/efectos de los fármacos , Receptores CXCR4/genéticaRESUMEN
A paucity of information is available on the activity of protease inhibitors (PI) in chronically-infected monocyte-derived macrophages (MDM) and on the kinetics of viral-rebound after PI removal in vitro. To fill this gap, the activity of different concentrations of amprenavir (AMP) was evaluated in chronically-infected MDM by measuring p24-production every day up to 12 days after drug administration and up to seven days after drug removal. Clinically-relevant concentrations of AMP (4 and 20 µM) drastically decreased p24 amount released from chronically-infected MDM from Day 2 up to Day 12 after drug administration. The kinetics of viral-rebound after AMP-removal (4 and 20 µM) showed that, despite an initial increase, p24-production over time never reached the level observed for untreated-MDM, suggesting a persistent intracellular drug activity. In line with this, after AMP-removal, human immunodeficiency virus 1 (HIV-1) infectivity and intracellular the p24/p55 ratio (reflecting virion-maturation) were remarkably lower than observed for untreated MDM. Overall, AMP shows high efficacy in blocking HIV-1 replication in chronically-infected MDM, persisting even after drug-removal. This highlights the role of protease inhibitors in preventing the establishment of this important HIV-1 reservoir, thus reducing viral-dissemination in different anatomical compartments.
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Carbamatos/farmacología , Inhibidores de la Proteasa del VIH/farmacología , VIH-1/efectos de los fármacos , Macrófagos/virología , Monocitos/virología , Sulfonamidas/farmacología , Replicación Viral/efectos de los fármacos , Células Cultivadas , Furanos , VIH-1/fisiología , HumanosRESUMEN
BACKGROUND: An impaired HBsAg-secretion can increase HBV oncogenic-properties. Here, we investigate genetic-determinants in HBsAg correlated with HBV-induced hepatocellular carcinoma (HCC), and their impact on HBsAg-secretion and cell-proliferation. METHODS: This study included 128 chronically HBV-infected patients: 23 with HCC (73.9% D; 26.1% A HBV-genotype), and 105 without cirrhosis/HCC (72.4% D, 27.6% A) as reference-group. The impact of mutations on HBsAg-secretion was assessed by measuring the ratio [secreted/intracellular HBsAg] until day 5 post-transfection. The impact of mutations on cell-cycle advancement was assessed by flow-cytometry. RESULTS: Two HBsAg mutations significantly correlated with HCC: P203Q (17.4% [4/23] in HCC vs 1.0% [1/105] in non-HCC, P=0.004); S210R (34.8% [8/23] in HCC vs 3.8% [4/105] in non-HCC, P <0.001); P203Q+S210R (17.4% [4/23] in HCC vs 0% [0/110] in non-HCC, P=0.001). Both mutations reside in trans-membrane C-terminal domain critical for HBsAg-secretion. In in-vitro experiments, P203Q, S210R and P203Q+S210R significantly reduced the ratio [secreted/intracellular HBsAg] compared to wt at each time-point analysed (P <0.05), supporting an impaired HBsAg-secretion. Furthermore, P203Q and P203Q+S210R increased the percentage of cells in S-phase compared to wt, indicating cell-cycle progression (P203Q:26±13%; P203Q+S210R:29±14%; wt:18%±9, P <0.01. Additionally, S210R increased the percentage of cells in G2/M-phase (26±8% for wt versus 33±6% for S210R, P <0.001). CONCLUSIONS: Specific mutations in HBsAg C-terminus significantly correlate with HBV-induced HCC. They hamper HBsAg-secretion and are associated with increased cellular proliferation, supporting their involvement in HCC-development. The identification of viral genetic markers associated with HCC is critical to identify patients at higher HCC-risk that may deserve intensive liver monitoring, and/or early anti-HBV therapy.
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Carcinoma Hepatocelular/patología , Antígenos de Superficie de la Hepatitis B/genética , Virus de la Hepatitis B/genética , Hepatitis B Crónica/patología , Neoplasias Hepáticas/patología , Mutación , Adulto , Anciano , Carcinoma Hepatocelular/virología , Ciclo Celular , Proliferación Celular , Femenino , Frecuencia de los Genes , Genotipo , Antígenos de Superficie de la Hepatitis B/metabolismo , Virus de la Hepatitis B/metabolismo , Virus de la Hepatitis B/fisiología , Hepatitis B Crónica/virología , Interacciones Huésped-Patógeno , Humanos , Neoplasias Hepáticas/virología , Masculino , Persona de Mediana Edad , Análisis Multivariante , Factores de RiesgoRESUMEN
BACKGROUND: This study characterizes and defines the clinical value of hepatitis B virus (HBV) quasispecies with reverse transcriptase and HBV surface antigen (HBsAg) heterogeneity in patients with acute HBV infection. METHODS: Sixty-two patients with acute HBV infection (44 with genotype D infection and 18 with genotype A infection) were enrolled from 2000 to 2010. Plasma samples obtained at the time of the first examination were analyzed by ultradeep pyrosequencing. The extent of HBsAg amino acid variability was measured by Shannon entropy. RESULTS: Median alanine aminotransferase and serum HBV DNA levels were 2544 U/L (interquartile range, 1938-3078 U/L) and 5.88 log10 IU/mL (interquartile range, 4.47-7.37 log10 IU/mL), respectively. Although most patients serologically resolved acute HBV infection, only 54.1% developed antibody to HBsAg (anti-HBs). A viral population with ≥1 immune-escape mutation was found in 53.2% of patients (intrapatient prevalence range, 0.16%-100%). Notably, by Shannon entropy, higher genetic variability at HBsAg amino acid positions 130, 133, and 157 significantly correlated with no production of anti-HBs in individuals infected with genotype D (P < .05). Stop codons were detected in 19.3% of patients (intrapatient prevalence range, 1.6%-47.5%) and occurred at 11 HBsAg amino acid positions, including 172 and 182, which are known to increase the oncogenic potential of HBV.Finally, ≥1 drug resistance mutation was detected in 8.1% of patients (intrapatient prevalence range, 0.11%-47.5% for primary mutations and 10.5%-99.9% for compensatory mutations). CONCLUSIONS: Acute HBV infection is characterized by complex array of viral quasispecies with reduced antigenicity/immunogenicity and enhanced oncogenic potential. These viral variants may induce difficult-to-treat HBV forms; favor HBV reactivation upon iatrogenic immunosuppression, even years after infection; and potentially affect the efficacy of the current HBV vaccination strategy.
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Antígenos de Superficie de la Hepatitis B/genética , Virus de la Hepatitis B/genética , Hepatitis B/virología , ADN Polimerasa Dirigida por ARN/genética , Enfermedad Aguda , Adulto , Sustitución de Aminoácidos , Estudios de Cohortes , Farmacorresistencia Viral/genética , Femenino , Variación Genética , Genotipo , Hepatitis B/epidemiología , Hepatitis B/inmunología , Antígenos de Superficie de la Hepatitis B/inmunología , Virus de la Hepatitis B/clasificación , Virus de la Hepatitis B/inmunología , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Italia/epidemiología , Masculino , Persona de Mediana Edad , Mutación , Prevalencia , Análisis de Secuencia de ADNRESUMEN
BACKGROUND: Thymosin α-1 (Tα1) exploits a specific action on lymphoid cells and is able to induce in peripheral blood mononuclear cells (PBMCs) a strong transcriptional response. CD8 antiviral factor activity plays a role in the control or prevention of HIV-1 infection by a non-cytolytic mechanism. The ability of Tα1 to modulate the release of antiretroviral soluble factors by CD8(+) cells was investigated. METHODS: Supernatants from lipopolysaccharide (LPS) stimulated CD8(+)-isolated cells treated with Tα1 were screened on in vitro infection of human monocyte-derived macrophages (MDMs) and PBMCs with HIV-1, and of PBMCs with human T lymphotropic virus 1 (HTLV-1). In CD8(+) cells, as well as in PBMCs of healthy donors as from HIV(+) individuals, a microarray analysis to assess the transcriptional response after treatment was performed. RESULTS: Tα1 potentiates the release, in LPS-stimulated CD8(+) cells, of soluble factors able to inhibit both in vitro HIV-1 infection of MDMs and PBMCs and in vitro HTLV-1 infection of PBMCs. A distinctive transcriptional profile was induced by Tα1 in PBMCs from HIV(+) donors. CONCLUSIONS: These findings suggest that Tα1 would represent a re-evaluated approach to antiretroviral therapy in combination with innovative treatments and with vaccine administration.
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Antígenos CD8/metabolismo , VIH-1/efectos de los fármacos , Virus Linfotrópico T Tipo 1 Humano/efectos de los fármacos , Timosina/análogos & derivados , Antivirales/farmacología , Antivirales/uso terapéutico , Antígenos CD8/inmunología , Infecciones por Deltaretrovirus/tratamiento farmacológico , Infecciones por Deltaretrovirus/inmunología , Infecciones por Deltaretrovirus/metabolismo , Sinergismo Farmacológico , Infecciones por VIH/inmunología , VIH-1/inmunología , Virus Linfotrópico T Tipo 1 Humano/inmunología , Virus Linfotrópico T Tipo 1 Humano/metabolismo , Humanos , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/metabolismo , Timalfasina , Timosina/farmacología , Timosina/uso terapéuticoRESUMEN
UNLABELLED: Hepatitis B virus (HBV) reactivation during immunosuppression can lead to severe acute hepatitis, fulminant liver failure, and death. Here, we investigated hepatitis B surface antigen (HBsAg) genetic features underlying this phenomenon by analyzing 93 patients: 29 developing HBV reactivation and 64 consecutive patients with chronic HBV infection (as control). HBsAg genetic diversity was analyzed by population-based and ultradeep sequencing (UDS). Before HBV reactivation, 51.7% of patients were isolated hepatitis B core antibody (anti-HBc) positive, 31.0% inactive carriers, 6.9% anti-HBc/anti-HBs (hepatitis B surface antibody) positive, 6.9% isolated anti-HBs positive, and 3.4% had an overt HBV infection. Of HBV-reactivated patients, 51.7% were treated with rituximab, 34.5% with different chemotherapeutics, and 13.8% with corticosteroids only for inflammatory diseases. In total, 75.9% of HBV-reactivated patients (vs. 3.1% of control patients; P<0.001) carried HBsAg mutations localized in immune-active HBsAg regions. Of the 13 HBsAg mutations found in these patients, 8 of 13 (M103I-L109I-T118K-P120A-Y134H-S143L-D144E-S171F) reside in a major hydrophilic loop (target of neutralizing antibodies [Abs]); some of them are already known to hamper HBsAg recognition by humoral response. The remaining five (C48G-V96A-L175S-G185E-V190A) are localized in class I/II-restricted T-cell epitopes, suggesting a role in HBV escape from T-cell-mediated responses. By UDS, these mutations occurred in HBV-reactivated patients with a median intrapatient prevalence of 73.3% (range, 27.6%-100%) supporting their fixation in the viral population as a predominant species. In control patients carrying such mutations, their median intrapatient prevalence was 4.6% (range, 2.5%-11.3%; P<0.001). Finally, additional N-linked glycosylation (NLG) sites within the major hydrophilic loop were found in 24.1% of HBV-reactivated patients (vs. 0% of chronic patients; P<0.001); 5 of 7 patients carrying these sites remained HBsAg negative despite HBV reactivation. NLG can mask immunogenic epitopes, abrogating HBsAg recognition by Abs. CONCLUSION: HBV reactivation occurs in a wide variety of clinical settings requiring immune-suppressive therapy, and correlates with HBsAg mutations endowed with enhanced capability to evade immune response. This highlights the need for careful patient monitoring in all immunosuppressive settings at reactivation risk and of establishing a prompt therapy to prevent HBV-related clinical complications.
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Antígenos de Superficie de la Hepatitis B/genética , Hepatitis B Crónica/inmunología , Evasión Inmune , Terapia de Inmunosupresión , Activación Viral , Adulto , Anciano , Farmacorresistencia Viral , Femenino , Glicosilación , Anticuerpos contra la Hepatitis B/sangre , Virus de la Hepatitis B/fisiología , Hepatitis B Crónica/tratamiento farmacológico , Hepatitis B Crónica/virología , Humanos , Masculino , Persona de Mediana Edad , MutaciónRESUMEN
BACKGROUND: To define HBsAg-mutations correlated with different serum HBV-DNA levels in HBV chronically-infected drug-naive patients. METHODS: This study included 187 patients stratified into the following ranges of serum HBV-DNA:12-2000 IU/ml, 2000-100,000 IU/ml, and >100,000 IU/ml. HBsAg-mutations were associated with HBV-DNA levels by applying a Bayesian-Partitional-Model and Fisher-exact test. Mutant and wild-type HBV genotype-D genomes were expressed in Huh7 cells and HBsAg-production was determined in cell-supernatants at 3 days-post-transfection. RESULTS: Specific HBsAg-mutations (M197T,-S204N-Y206C/H-F220L) were significantly correlated with serum HBV-DNA <2000 IU/ml (posterior-probability>90%, P < 0.05). The presence of Y206C/H and/or F220L was also associated with lower median (IQR) HBsAg-levels and lower median (IQR) transaminases (for HBsAg:250[115-840] IU/ml for Y206C/H and/or F220L versus 4300[640-11,838] IU/ml for wild-type, P = 0.023; for ALT:28[21-40] IU/ml versus 53[34-90] IU/ml, P < 0.001). These mutations were localized in the HBsAg C-terminus, known to be involved in virion and/or HBsAg secretion. The co-occurrence of Y206C + F220L was found significant by cluster-analysis, (P = 0.02). In addition, in an in-vitro model Y206C + F220L determined a 2.8-3.3 fold-reduction of HBsAg-amount released in supernatants compared to single mutants and wt (Y206C + F220L = 5,679 IU/ml; Y206H = 16,305 IU/ml; F220L = 18,368 IU/ml; Y206C = 18,680 IU/ml; wt = 14,280 IU/ml, P < 0.05). CONCLUSIONS: Specific HBsAg-mutations (compartmentalized in the HBsAg C-terminus) correlated with low-serum HBV-DNA and HBsAg-levels. These findings can be important to understand mechanisms underlying low HBV replicative potential including the inactive-carrier state.
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ADN Viral/sangre , Antígenos de Superficie de la Hepatitis B/genética , Virus de la Hepatitis B/genética , Hepatitis B Crónica/virología , Adulto , Teorema de Bayes , Portador Sano/virología , Femenino , Genotipo , Antígenos de Superficie de la Hepatitis B/sangre , Antígenos de Superficie de la Hepatitis B/química , Humanos , Masculino , Persona de Mediana Edad , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación , Transaminasas/sangreRESUMEN
OBJECTIVES: To evaluate the replication capacity and phenotypic susceptibility to dolutegravir and raltegravir of wild-type and raltegravir-resistant HIV-1 strains in several cellular systems. METHODS: The antiviral activities of dolutegravir and raltegravir were evaluated in human primary monocyte-derived macrophages (MDMs), peripheral blood mononuclear cells (PBMCs) and C8166 T lymphocytic cells. The following raltegravir resistance mutations were analysed: N155H, Y143C, N155Hâ+âY143C and G140S+Q148H. RESULTS: In the absence of drug, the replication capacity of raltegravir-resistant viruses was strongly reduced compared with wild-type in all cellular models analysed. In MDMs and PBMCs, a dramatic decrease in viral replication was observed for the double mutants N155Hâ+âY143C and G140Sâ+âQ148H (ranging from 0.1% to 2.5% compared with wild-type). In MDMs, dolutegravir exhibited high potency, with EC50 and EC90 values of 1.1 ± 0.9 and 5.5 ± 3.4 nM, respectively (comparable to raltegravir). These values (particularly for EC90) were significantly lower than those observed in PBMCs (EC50: 2.7 ± 1.5 nM; EC90: 14.8 ± 0.9 nM) and C8166 cells (EC50: 5.5 ± 0.8 nM; EC90: 64.8 ± 5.8 nM). In all cellular models analysed, dolutegravir showed full efficacy against N155H and Y143C mutants (dolutegravir fold-change resistance ranging from 0.1 to 1.4; raltegravir fold-change resistance ranging from 0.1 to 10.3). In C8166 (the only cell model in which replication capacity was sufficient to perform the test) dolutegravir showed full efficacy against mutations N155Hâ+âY143C (dolutegravir fold-change resistance: 0.6) and a slightly lower activity against G140S+Q148H (dolutegravir fold-change resistance: 2.1). CONCLUSIONS: Dolutegravir is effective in different HIV cellular targets and against raltegravir-resistant mutants. The high efficacy of dolutegravir in MDMs (cells with limited metabolism) has relevant clinical implications in light of the role of MDMs in the transmission of HIV infection and dissemination in different body compartments.
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Farmacorresistencia Viral , Inhibidores de Integrasa VIH/farmacología , VIH-1/efectos de los fármacos , Compuestos Heterocíclicos con 3 Anillos/farmacología , Linfocitos/virología , Macrófagos/virología , Replicación Viral/efectos de los fármacos , Células Cultivadas , Integrasa de VIH/genética , Integrasa de VIH/metabolismo , VIH-1/fisiología , Humanos , Pruebas de Sensibilidad Microbiana , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Oxazinas , Piperazinas , Piridonas , Pirrolidinonas/farmacología , Raltegravir PotásicoRESUMEN
Determination of hepatitis C virus genotype is crucial for establishing the duration of antiviral therapy and predicts response to treatment. In this study, consecutive serum samples collected from two patients with chronic hepatitis C infection were tested by two assays used widely, the Abbott RealTime HCV Genotype II and the Versant HCV Genotype 2.0 assays, in order to assign a genotype to the virus. The obtained results were verified by phylogenetic analysis of the NS5B region and sequencing of the 5'-UTR of the viral genome. Testing of the serum samples from both patients gave an indeterminate result with the Abbott assay. By contrast, the Versant assay gave an indeterminate result for one patient and identified an HCV-2b subtype in the other patient. Phylogenetic analysis of the NS5B region confirmed the presence of HCV-2b in this latter patient and disclosed the presence of HCV-3h in the other patient. Sequencing of the 5'-UTR revealed the presence of nucleotide changes at positions -166 and -119 of HCV-2b, and at positions -138, -108 and -99 of HCV-3h. Nucleotide mutations located in the 5'-untraslated region of hepatitis C virus may impair the ability of commercial assays to assign an HCV genotype.
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Regiones no Traducidas 5' , Hepacivirus/clasificación , Hepacivirus/genética , Hepatitis C Crónica/virología , Técnicas de Diagnóstico Molecular/métodos , Polimorfismo Genético , Virología/métodos , Anciano , Femenino , Genotipo , Hepatitis C Crónica/diagnóstico , Humanos , Masculino , Persona de Mediana Edad , Filogenia , Análisis de Secuencia de ADN , Proteínas no Estructurales Virales/genéticaRESUMEN
The activity of raltegravir and 4 other integrase inhibitors (MK-2048, L870,810, IN2, and IN5) was investigated in primary human macrophages, PBMC and C8166-lymphocytic T cells, in order to determine their relative potency and efficacy in different cellular systems of HIV infection. Raltegravir showed better protective efficacy in all cell types; MK-2048, L870,810 and IN5 showed a potent anti-HIV-1 activity in macrophages, while in lymphocytes only MK-2048 and L870,810 showed an inhibitory effect comparable to raltegravir. IN2 was a poorly effective anti-HIV-1 compound in all cellular systems. All effective integrase inhibitors exhibited a potent antiviral activity against both X4 and R5 HIV-1 strains. In general, raltegravir, MK-2048, L870,810 and IN5 showed anti HIV activity similar or slightly higher in macrophages compared to PBMC and C8166 T cells: for MK-2048, the EC(50) was 0.4, 0.9, 11.5 nM in macrophages, in PBMCs and T cells, respectively; for L870,810, the EC(50) was 1.5, 14.3, and 10.6 nM, respectively; for IN5 the EC(50) was 0.5, 13.7, and 5.7 nM, respectively.
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Inhibidores de Integrasa VIH/farmacología , VIH-1/efectos de los fármacos , Linfocitos/virología , Macrófagos/virología , Apoptosis , Células Cultivadas , Proteína p24 del Núcleo del VIH/análisis , Inhibidores de Integrasa VIH/toxicidad , VIH-1/crecimiento & desarrollo , Humanos , Pruebas de Sensibilidad MicrobianaRESUMEN
The goal of this study was to explore the presence of integrase strand transfer inhibitor (InSTI) resistance mutations in HIV-1 quasispecies present in InSTI-naïve patients and to evaluate their in vitro effects on phenotypic susceptibility to InSTIs and their replication capacities. The RT-RNase H-IN region was PCR amplified from plasma viral RNA obtained from 49 HIV-1 subtype B-infected patients (21 drug naïve and 28 failing highly active antiretroviral therapy [HAART] not containing InSTIs) and recombined with an HXB2-based backbone with RT and IN deleted. Recombinant viruses were tested against raltegravir and elvitegravir and for replication capacity. Three-hundred forty-four recombinant viruses from 49 patients were successfully analyzed both phenotypically and genotypically. The majority of clones were not phenotypically resistant to InSTIs: 0/344 clones showed raltegravir resistance, and only 3 (0.87%) showed low-level elvitegravir resistance. No primary resistance mutations for raltegravir and elvitegravir were found as major or minor species. The majority of secondary mutations were also absent or rarely present. Secondary mutations, such as T97A and G140S, found rarely and only as minority quasispecies, were present in the elvitegravir-resistant clones. A novel mutation, E92G, although rarely found in minority quasispecies, showed elvitegravir resistance. Preexisting genotypic and phenotypic raltegravir resistance was extremely rare in InSTI-naïve patients and confined to only a restricted minority of secondary variants. Overall, these results, together with others based on population and ultradeep sequencing, suggest that at this point IN genotyping in all patients before raltegravir treatment may not be cost-effective and should not be recommended until evidence of transmitted drug resistance to InSTIs or the clinical relevance of IN minor variants/polymorphisms is determined.
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Inhibidores de Integrasa VIH/uso terapéutico , Integrasa de VIH/genética , Pirrolidinonas/uso terapéutico , Quinolonas/uso terapéutico , Farmacorresistencia Viral/genética , Genotipo , Humanos , Mutación , Fenotipo , Raltegravir PotásicoRESUMEN
BACKGROUND: Oxidative stress plays a key role in the neuropathogenesis of Human Immunodeficiency Virus-1 (HIV-1) infection causing apoptosis of astroglia cells and neurons. Recent data have shown that oxidative stress is also responsible for the acceleration of human fibroblast telomere shortening in vitro. In the present study we analyzed the potential relations occurring between free radicals formation and telomere length during HIV-1 mediated astroglial death. RESULTS: To this end, U373 human astrocytoma cells have been directly exposed to X4-using HIV-1IIIB strain, for 1, 3 or 5 days and treated (where requested) with N-acetylcysteine (NAC), a cysteine donor involved in the synthesis of glutathione (GSH, a cellular antioxidant) and apoptosis has been evaluated by FACS analysis. Quantitative-FISH (Q-FISH) has been employed for studying the telomere length while intracellular reduced/oxidized glutathione (GSH/GSSG) ratio has been determined by High-Performance Liquid Chromatography (HPLC). Incubation of U373 with HIV-1IIIB led to significant induction of cellular apoptosis that was reduced in the presence of 1 mM NAC. Moreover, NAC improved the GSH/GSSG, a sensitive indicator of oxidative stress, that significantly decreased after HIV-1IIIB exposure in U373. Analysis of telomere length in HIV-1 exposed U373 showed a statistically significant telomere shortening, that was completely reverted in NAC-treated U373. CONCLUSION: Our results support the role of HIV-1-mediated oxidative stress in astrocytic death and the importance of antioxidant compounds in preventing these cellular damages. Moreover, these data indicate that the telomere structure, target for oxidative damage, could be the key sensor of cell apoptosis induced by oxidative stress after HIV infection.
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Apoptosis/fisiología , Astrocitoma/patología , VIH-1/metabolismo , Estrés Oxidativo/fisiología , Telómero/patología , Acetilcisteína/farmacología , Análisis de Varianza , Antivirales/farmacología , Apoptosis/efectos de los fármacos , Astrocitoma/ultraestructura , Línea Celular Tumoral , Ensayo de Inmunoadsorción Enzimática , Glutatión/metabolismo , Disulfuro de Glutatión/metabolismo , Humanos , Microscopía Electrónica/métodos , Estrés Oxidativo/efectos de los fármacos , Telómero/efectos de los fármacos , Factores de TiempoRESUMEN
Human immunodeficiency virus (HIV-1) is the responsible agent of acquired immunodeficiency syndrome (AIDS), a multi system disorder including the central nervous system (CNS). The CNS is an immunological privileged site providing a sanctuary and reservoir for HIV-1. Monocytes derived macrophages (MDM) and microglia play a critical role in the development of HIV-associated dementia (HAD). Although the use of highly active antiretroviral therapy (HAART) has led to a strong reduction of HAD incidence, the prevalence of minor HIV-1 associated cognitive impairment appears rising among AIDS patients. Various factors including toxicity, insurgence of drug resistance and sometimes limited access to HAART, contribute to this phenomenon. Independent evolution of drug resistance mutations in several areas of the CNS may emerge as consequence of incomplete suppression of HIV-1, probably related to poor penetration of antiretroviral drugs into CNS. The emergence of resistant virus in the CNS may considerable influence the outcome of neurological disease and also the reseeding of HIV-1 in the systemic circulation upon failure of therapy. In this review, we outline the current state of knowledge regarding the pathophysiology of CNS injury in HIV-1 infection and will focus on the effects of HAART on CNS.
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Complejo SIDA Demencia/terapia , Terapia Antirretroviral Altamente Activa/métodos , VIH-1 , Complejo SIDA Demencia/etiología , Animales , Infecciones por VIH/complicaciones , HumanosRESUMEN
Monocytes/macrophages (M/M) are strategic reservoirs of HIV-1, spreading the virus to other cells and inducing apoptosis in T-lymphocytes, astrocytes and neurons. M/M are commonly infected by R5 HIV-1 strains, which use the chemokine receptor CCR5. D-Ala-peptide T-amide (DAPTA), or Peptide T, named for its high threonine content (ASTTTNYT), is a synthetic peptide comprised of eight amino acids (185-192) of the gp120 V2 region and functions as a viral entry inhibitor by targeting selectively CCR5. The anti-HIV-1 activity of DAPTA was evaluated in M/M infected with R5 HIV-1 strains. DAPTA at 10(-9) M inhibited HIV-1 replication in M/M by > 90%. PCR analysis of viral cDNA in M/M showed that DAPTA blocks HIV entry and in this way prevents HIV-1 infection. Moreover, DAPTA acts as a strong inhibitor and was more active than the non-peptidic CCR5 antagonist TAK-779 in inhibiting apoptosis (mediated by RS HIV-1 strains produced and released by infected M/M) on a neuroblastoma cell line. Our results suggest that antiviral compounds which interfere with receptor mechanisms such as CCR5 could be important, either alone or in combination with other antiretroviral treatments, in preventing HIV infection in the central nervous system and the consequential neuronal damage that leads to neuronal AIDS.
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Apoptosis/efectos de los fármacos , Antagonistas de los Receptores CCR5 , VIH-1/efectos de los fármacos , Macrófagos/efectos de los fármacos , Monocitos/efectos de los fármacos , Neuronas/efectos de los fármacos , Péptido T/farmacología , Fármacos Anti-VIH/síntesis química , Fármacos Anti-VIH/química , Fármacos Anti-VIH/farmacología , Anticuerpos Monoclonales/efectos de los fármacos , Anticuerpos Monoclonales/inmunología , Apoptosis/fisiología , Sitios de Unión , Línea Celular , ADN Viral/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Proteína gp120 de Envoltorio del VIH/antagonistas & inhibidores , Proteína gp120 de Envoltorio del VIH/farmacología , Humanos , Macrófagos/inmunología , Macrófagos/virología , Pruebas de Sensibilidad Microbiana , Monocitos/virología , Neuronas/fisiología , Neuronas/virología , Péptido T/síntesis química , Péptido T/química , Receptores CCR5/fisiología , Relación Estructura-Actividad , Replicación Viral/efectos de los fármacosRESUMEN
BACKGROUND: Monocytes/Macrophages (M/M) play a pivotal role as a source of virus during the whole course of HIV-1 infection. Enhanced oxidative stress is involved in the pathogenesis of HIV-1 infection. HIV-1 regulatory proteins induce a reduction of the expression and the activity of MnSOD, the mitochondrial isoform leading to a sustained generation of superoxide anions and peroxynitrite that represent important mediators of HIV-1 replication in M/M. MnTBAP (Mn(III)tetrakis(4-benzoic acid)porphrin chloride), a synthetic peroxynitrite decomposition catalyst, reduced oxidative stress subsequent to peroxynitrite generation. RESULTS: Virus production was assessed by p24 ELISA, western blot, and electron microscopy during treatment with MnTBAP. MnTBAP treatment showed a reduction of HIV-1 replication in both acutely and chronically infected M/M: 99% and 90% inhibition of p24 released in supernatants compared to controls, respectively. Maturation of p55 and p24 was strongly inhibited by MnTBAP in both acutely and chronically infected M/M. EC50 and EC90 are 3.7 (+/- 0.05) microM and 19.5 (+/- 0.5) microM, in acutely infected M/M; 6.3 (+/- 0.003) microM and 30 (+/- 0.6) microM, in chronically infected M/M. In acutely infected peripheral blood limphocytes (PBL), EC50 and EC90 are 7.4 (+/- 0.06) microM and of 21.3 (+/- 0.6) microM, respectively. Treatment of acutely-infected M/M with MnTBAP inhibited the elevated levels of malonildialdehyde (MDA) together with the nitrotyrosine staining observed during HIV-1 replication. MnTBAP strongly reduced HIV-1 particles in infected M/M, as shown by electron microscopy. Moreover, in presence of MnTBAP, HIV-1 infectivity was reduced of about 1 log compared to control. CONCLUSION: Results support the role of superoxide anions in HIV-1 replication in M/M and suggest that MnTBAP may counteract HIV-1 replication in combination with other antiretroviral treatments.
Asunto(s)
VIH-1/fisiología , Macrófagos/metabolismo , Metaloporfirinas/metabolismo , Ácido Peroxinitroso/metabolismo , Replicación Viral/fisiología , Células Cultivadas , Infecciones por VIH/metabolismo , Infecciones por VIH/virología , VIH-1/metabolismo , Humanos , Macrófagos/inmunología , Macrófagos/virologíaRESUMEN
Human immunodeficiency virus type 1 (HIV-1) infection is characterized by a progressive decrease of CD4(+) T cells accompanied by other immune dysfunctions. Telomerase is transiently activated in lymphocytes during activation and is able to compensate for the progressive telomeric loss that occurs at each cell division, contributing to ensure the telomere length necessary for multiple proliferative events. The effect of HIV-1 infection on telomerase activity and on the expression of some of the factors involved in its regulation in CD4(+) T cells was investigated. Telomerase was found to be downregulated in both nuclear and cytoplasmic compartments, together with an impairment of human telomerase reverse transcriptase (hTERT) expression and of the cell machinery involved in hTERT phosporylation.
Asunto(s)
Linfocitos T CD4-Positivos/microbiología , Infecciones por VIH/metabolismo , VIH-1 , Telomerasa/metabolismo , Núcleo Celular/metabolismo , Células Cultivadas , Citoplasma/metabolismo , Regulación hacia Abajo , Humanos , FosforilaciónRESUMEN
Two novel 6-desfluoroquinolone derivatives, HM-12 and HM-13, were evaluated for anti-human immunodeficiency virus (anti-HIV) activity in acutely, chronically, and latently HIV type 1 (HIV-1)-infected cell cultures and were found to behave as potent HIV-1 transcription inhibitors. In order to extend this result in vivo, we developed an artificial hu-SCID mouse model for HIV-1 latency based on SCID mice engrafted with latently HIV-1-infected promyelocytic OM-10.1 cells in which HIV-1 can be reactivated in vivo by the administration of human tumor necrosis factor alpha (hTNF-alpha). Treating these SCID mice with HM-12 or HM-13 prior to hTNF-alpha stimulation resulted in a pronounced suppressive effect on viral reactivation. Since both quinolone derivatives were able to inhibit the reactivation of HIV-1 from this artificial viral reservoir in vivo, we provide encouraging evidence for the use of quinolones in the control of HIV-1 infections.
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Fármacos Anti-VIH/farmacología , Modelos Animales de Enfermedad , VIH-1/efectos de los fármacos , VIH-1/fisiología , Quinolonas/farmacología , Animales , Fármacos Anti-VIH/uso terapéutico , Línea Celular , Infecciones por VIH/tratamiento farmacológico , VIH-1/genética , Humanos , Ratones , Ratones SCID , Modelos Biológicos , Quinolonas/uso terapéuticoRESUMEN
Monocyte-derived macrophages (M/M) are considered the second cellular target of HIV-1 and a crucial virus reservoir. M/M are widely distributed in all tissues and organs, including the CNS, where they represent the most common HIV-infected cells. Differently from activated CD4+ T lymphocytes, M/M are resistant to the cytopathic effect of HIV and survive HIV infection for a long time. Moreover, HIV-1 replication in M/M is a key pathogenetic event during the course of HIV-1 infection. Overall findings strongly support the clinical relevance of anti-HIV drugs in M/M. Nucleoside RT inhibitors (NRTIs) are more active against HIV in M/M than in CD4+ T lymphocytes. Their activity is further boosted by the presence of an additional monophosphate group (i.e., a phosphonate group, as in the case of Tenofovir), thus overcoming the bottleneck of the low phosphorylation ability of M/M. In contrast, the antiviral activity of non-NRTIs (not affecting the DNA chain elongation) in M/M is similar to that in CD4+ T lymphocytes. Protease inhibitors are the only clinically approved drugs acting at a late stage of the HIV lifecycle. They are able to interfere with HIV replication in HIV-1 chronically infected M/M, even if at concentrations greater than those observed in HIV-1 chronically infected CD4+ T lymphocytes. Finally, several new drugs have been shown to interfere efficiently with HIV replication in M/M, including entry inhibitors. A better understanding of the activity of the anti-HIV drugs in M/M may represent a key element for the design of effective anti-HIV chemotherapy.
Asunto(s)
Fármacos Anti-VIH/farmacología , Infecciones por VIH/inmunología , VIH-1/efectos de los fármacos , Macrófagos/efectos de los fármacos , Fármacos Anti-VIH/uso terapéutico , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD4-Positivos/virología , Infecciones por VIH/tratamiento farmacológico , Humanos , Macrófagos/virología , Pruebas de Sensibilidad Microbiana , Relación Estructura-Actividad , Replicación Viral/efectos de los fármacosRESUMEN
It is widely recognized that macrophages (M/M) represent a crucial target of HIV-1 in the body and play a pivotal role in the pathogenic progression of HIV-1 infection. This strongly supports the clinical relevance of therapeutic strategies able to interfere with HIV-1 replication in M/M. In vitro studies showed that nucleoside analogue inhibitors of HIV-1 reverse transcriptase have potent antiviral activity in M/M, although the limited penetration of these compounds in sequestered body compartments and low phosphorylation ability of M/M, suggest that a phosphonate group linked to NRTIs may confer greater anti-HIV-1 activity in M/M. Differently, the antiviral activity of non-nucleoside reverse transcriptase inhibitors in M/M is similar to that found in CD4+ lymphocytes. Interestingly, protease inhibitors, acting at a post-integrational stage of HIV-1 life-cycle are the only drugs active in chronically infected M/M. A careful analysis of the distribution of antiviral drugs, and the assessment of their activity in M/M, represent key factors in the development of therapeutic strategies aimed to the treatment of HIV-1-infected patients. Moreover, testing new and promising antiviral compounds in such cells may provide crucial hints about their efficacy in patients infected by HIV.
Asunto(s)
Fármacos Anti-VIH/farmacología , Infecciones por VIH/tratamiento farmacológico , VIH-1/efectos de los fármacos , Macrófagos/virología , Replicación Viral/efectos de los fármacos , Fármacos Anti-VIH/química , Fármacos Anti-VIH/uso terapéutico , Infecciones por VIH/virología , VIH-1/fisiología , Humanos , Lectinas de Plantas/química , Lectinas de Plantas/farmacología , Lectinas de Plantas/uso terapéutico , Inhibidores de la Transcriptasa Inversa/uso terapéuticoRESUMEN
The entry of human immunodeficiency virus (HIV) into the central nervous system (CNS) causes both the establishment of a lifelong viral reservoir in the brain and symptoms of neurological dysfunction that have an AIDS dementia complex (ADC) clinical appearance. Neurological dysfunction in ADC patients still remains an unresolved problem. However, ADC pathogenesis may be a multistep process that starts with HIV invasion of CNS by crossing the blood-brain barrier (BBB). It progresses by developing a chronic inflammatory status that can cause dysfunction in neurons and astrocytes that result in apoptotic death. Monocytes-macrophages (M/M) may play an important role by concealing the HIV transfer across the BBB. Furthermore, HIV-infected M/M could produce and release neurotoxic factors. In this review the main mediators and cells involved in pathogenesis and development of ADC are highlighted. A better understanding of the mechanisms involved in this process may help in a successful therapeutic approach to the neuropathogenesis of HIV infection.