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1.
Virol J ; 19(1): 29, 2022 02 10.
Artigo em Inglês | MEDLINE | ID: mdl-35144624

RESUMO

Ultraviolet (UV) light has previously been established as useful method of disinfection, with demonstrated efficacy to inactivate a broad range of microorganisms. The advent of ultraviolet light-emitting diodes provides advantages in ease of disinfection, in that there can be delivery of germicidal UV with the same light unit that delivers standard white light to illuminate a room. Herein we demonstrate the efficacy and feasibility of ultraviolet light-emitting diodes as a means of decontamination by inactivating two distinct virus models, human coronavirus 229E and human immunodeficiency virus. Importantly, the same dose of ultraviolet light that inactivated human viruses also elicited complete inactivation of ultraviolet-resistant bacterial spores (Bacillus pumilus), a gold standard for demonstrating ultraviolet-mediated disinfection. This work demonstrates that seconds of ultraviolet light-emitting diodes (UV-LED) exposure can inactivate viruses and bacteria, highlighting that UV-LED could be a useful and practical tool for broad sanitization of public spaces.


Assuntos
Coronavirus Humano 229E , Desinfecção , HIV-1 , Raios Ultravioleta , Inativação de Vírus/efeitos da radiação , Coronavirus Humano 229E/efeitos da radiação , Desinfecção/métodos , HIV-1/efeitos da radiação , Humanos
2.
Viruses ; 12(8)2020 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-32823598

RESUMO

HIV-1 infects 39.5 million people worldwide, and cART is effective in preventing viral spread by reducing HIV-1 plasma viral loads to undetectable levels. However, viral reservoirs persist by mechanisms, including the inhibition of autophagy by HIV-1 proteins (i.e., Nef and Tat). HIV-1 reservoirs can be targeted by the "shock and kill" strategy, which utilizes latency-reversing agents (LRAs) to activate latent proviruses and immunotarget the virus-producing cells. Yet, limitations include reduced LRA permeability across anatomical barriers and immune hyper-activation. Ionizing radiation (IR) induces effective viral activation across anatomical barriers. Like other LRAs, IR may cause inflammation and modulate the secretion of extracellular vesicles (EVs). We and others have shown that cells may secrete cytokines and viral proteins in EVs and, therefore, LRAs may contribute to inflammatory EVs. In the present study, we mitigated the effects of IR-induced inflammatory EVs (i.e., TNF-α), through the use of mTOR inhibitors (mTORi; Rapamycin and INK128). Further, mTORi were found to enhance the selective killing of HIV-1-infected myeloid and T-cell reservoirs at the exclusion of uninfected cells, potentially via inhibition of viral transcription/translation and induction of autophagy. Collectively, the proposed regimen using cART, IR, and mTORi presents a novel approach allowing for the targeting of viral reservoirs, prevention of immune hyper-activation, and selectively killing latently infected HIV-1 cells.


Assuntos
Citocinas/imunologia , Vesículas Extracelulares/imunologia , HIV-1/efeitos da radiação , Radiação Ionizante , Serina-Treonina Quinases TOR/antagonistas & inibidores , Latência Viral/efeitos dos fármacos , Antivirais/farmacologia , Autofagia/efeitos dos fármacos , Benzoxazóis/farmacologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/efeitos da radiação , Linfócitos T CD4-Positivos/virologia , Vesículas Extracelulares/virologia , Feminino , HIV-1/efeitos dos fármacos , Humanos , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/virologia , Masculino , Células Mieloides/efeitos dos fármacos , Células Mieloides/efeitos da radiação , Células Mieloides/virologia , Pirimidinas/farmacologia , Sirolimo/farmacologia , Células U937 , Ativação Viral/efeitos da radiação
3.
Nucl Med Biol ; 82-83: 80-88, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32113033

RESUMO

BACKGROUND: cART has significantly improved the life expectancy of people living with HIV (PLWH). However, it fails to eliminate the long-lived reservoir of latent HIV-infected cells. Radioimmunotherapy (RIT) relies on antigen-specific monoclonal antibodies (mAbs) for targeted delivery of lethal doses of ionizing radiation to cells. Previously, we have demonstrated that human mAb 2556 against HIV gp41 conjugated with 213Bismuth radioisotope (t1/2 = 46 min, alpha-emitter) selectively killed HIV-infected cells. 225Actinium (t1/2 = 9.92 d, alpha-emitter) and 177Lutetium (t1/2 = 6.7 d, beta-emitter) are two long-lived clinically proven radioisotopes for cancer treatment which might be more effective in killing infected cells systemically and in CNS. METHODS: In this study we have conjugated 2556 mAb with 213Bi, 225Ac and 177Lu, and compared their ability to kill HIV-infected human peripheral blood mononuclear cells (PBMCs) and monocytes. PBMCs and monocytes from healthy donors were infected with HIVp49.5 and treated in vitro with increasing concentrations of 213Bi (4-20 µCi)-, 225Ac (20-100 nCi)- and 177Lu (4-50 µCi)-2556 mAb. RESULTS: After three days post-treatment of infected PBMCs and monocytes, 213Bi- and 177Lu-conjugated 2556 mAb reduced virus production measured by p24 level in a dose-dependent manner, whereas, 225Ac-2556 showed minimal effect. However, seven days post-treatment all three radioisotopes showed significantly more pronounced reduction of virus replication as compared to control labeled mAb with 225Ac-2556 showing the least non-specific killing. CONCLUSION: These results indicate that RIT holds promise as a novel treatment option for the eradication of HIV-infected cells that merits further study in combination with cART and reactivation drugs.


Assuntos
Anticorpos Monoclonais/imunologia , HIV-1/fisiologia , Glicoproteínas de Membrana/imunologia , Anticorpos Monoclonais/química , Linhagem Celular , Quebras de DNA de Cadeia Dupla/efeitos da radiação , HIV-1/efeitos da radiação , Humanos , Marcação por Isótopo , Leucócitos Mononucleares/virologia , Monócitos/virologia
4.
J Virol ; 91(15)2017 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-28490588

RESUMO

The envelope glycoproteins (Envs) on the surfaces of HIV-1 particles are targeted by host antibodies. Primary HIV-1 isolates demonstrate different global sensitivities to antibody neutralization; tier-1 isolates are sensitive, whereas tier-2 isolates are more resistant. Single-site mutations in Env can convert tier-2 into tier-1-like viruses. We hypothesized that such global change in neutralization sensitivity results from weakening of intramolecular interactions that maintain Env integrity. Three strategies commonly applied to perturb protein structure were tested for their effects on global neutralization sensitivity: exposure to low temperature, Env-activating ligands, and a chaotropic agent. A large panel of diverse tier-2 isolates from clades B and C was analyzed. Incubation at 0°C, which globally weakens hydrophobic interactions, causes gradual and reversible exposure of the coreceptor-binding site. In the cold-induced state, Envs progress at isolate-specific rates to unstable forms that are sensitive to antibody neutralization and then gradually lose function. Agents that mimic the effects of CD4 (CD4Ms) also induce reversible structural changes to states that exhibit isolate-specific stabilities. The chaotropic agent urea (at low concentrations) does not affect the structure or function of native Env. However, urea efficiently perturbs metastable states induced by cold and CD4Ms and increases their sensitivity to antibody neutralization and their inactivation rates Therefore, chemical and physical agents can guide Env from the stable native state to perturbation-sensitive forms and modulate their stability to bestow tier-1-like properties on primary tier-2 strains. These concepts can be applied to enhance the potency of vaccine-elicited antibodies and microbicides at mucosal sites of HIV-1 transmission.IMPORTANCE An effective vaccine to prevent transmission of HIV-1 is a primary goal of the scientific and health care communities. Vaccine-elicited antibodies target the viral envelope glycoproteins (Envs) and can potentially inhibit infection. However, the potency of such antibodies is generally low. Single-site mutations in Env can enhance the global sensitivity of HIV-1 to neutralization by antibodies. We found that such a hypersensitivity phenotype can also be induced by agents that destabilize protein structure. Exposure to 0°C or low concentrations of Env-activating ligands gradually guides Env to metastable forms that expose cryptic epitopes and that are highly sensitive to neutralization. Low concentrations of the chaotropic agent urea do not affect native Env but destabilize perturbed states induced by cold or CD4Ms and increase their neutralization. The concept of enhancing antibody sensitivity by chemical agents that affect the structural stability of proteins can be applied to increase the potency of topical microbicides and vaccine-elicited antibodies.


Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Anti-HIV/imunologia , HIV-1/imunologia , Produtos do Gene env do Vírus da Imunodeficiência Humana/imunologia , Biomimética , Antígenos CD4/metabolismo , Temperatura Baixa , Epitopos/imunologia , Infecções por HIV/virologia , HIV-1/efeitos dos fármacos , HIV-1/genética , HIV-1/isolamento & purificação , HIV-1/efeitos da radiação , Humanos , Testes de Neutralização , Ureia/metabolismo , Produtos do Gene env do Vírus da Imunodeficiência Humana/genética
5.
JCI Insight ; 2(4): e91230, 2017 02 23.
Artigo em Inglês | MEDLINE | ID: mdl-28239658

RESUMO

The conditioning regimen used as part of the Berlin patient's hematopoietic cell transplant likely contributed to his eradication of HIV infection. We studied the impact of conditioning in simian-human immunodeficiency virus-infected (SHIV-infected) macaques suppressed by combination antiretroviral therapy (cART). The conditioning regimen resulted in a dramatic, but incomplete depletion of CD4+ and CD8+ T cells and CD20+ B cells, increased T cell activation and exhaustion, and a significant loss of SHIV-specific Abs. The disrupted T cell homeostasis and markers of microbial translocation positively correlated with an increased viral rebound after cART interruption. Quantitative viral outgrowth and Tat/rev-induced limiting dilution assays showed that the size of the latent SHIV reservoir did not correlate with viral rebound. These findings identify perturbations of the immune system as a mechanism for the failure of autologous transplantation to eradicate HIV. Thus, transplantation strategies may be improved by incorporating immune modulators to prevent disrupted homeostasis, and gene therapy to protect transplanted cells.


Assuntos
Linfócitos T CD4-Positivos/efeitos da radiação , Linfócitos T CD8-Positivos/efeitos da radiação , Infecções por HIV/imunologia , HIV-1/efeitos da radiação , Transplante de Células-Tronco Hematopoéticas/métodos , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Vírus da Imunodeficiência Símia/efeitos da radiação , Condicionamento Pré-Transplante/métodos , Irradiação Corporal Total , Animais , Antirretrovirais/uso terapêutico , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Infecções por HIV/tratamento farmacológico , Homeostase/efeitos da radiação , Infecções por Lentivirus/tratamento farmacológico , Infecções por Lentivirus/imunologia , Macaca nemestrina , Síndrome de Imunodeficiência Adquirida dos Símios/tratamento farmacológico , Transplante Autólogo , Carga Viral/efeitos da radiação
6.
Sci Rep ; 6: 36619, 2016 11 18.
Artigo em Inglês | MEDLINE | ID: mdl-27857152

RESUMO

Hepatitis C virus (HCV) and human immunodeficiency virus (HIV-1) transmissions among people who inject drugs (PWID) continue to pose a challenging global health problem. Here, we aimed to analyse a universally applicable inactivation procedure, namely microwave irradiation, as a safe and effective method to reduce the risk of viral transmission. The exposure of HCV from different genotypes to microwave irradiation resulted in a significant reduction of viral infectivity. Furthermore, microwave irradiation reduced viral infectivity of HIV-1 and of HCV/HIV-1 suspensions indicating that this inactivation may be effective at preventing co-infections. To translate microwave irradiation as prevention method to used drug preparation equipment, we could further show that HCV as well as HIV-1 infectivity could be abrogated in syringes and filters. This study demonstrates the power of microwave irradiation for the reduction of viral transmission and establishment of this safety strategy could help reduce the transmission of blood-borne viruses.


Assuntos
Infecções por HIV/prevenção & controle , HIV-1/efeitos da radiação , Hepacivirus/efeitos da radiação , Hepatite C/prevenção & controle , Micro-Ondas , Abuso de Substâncias por Via Intravenosa/complicações , Filtração/instrumentação , Genótipo , Infecções por HIV/complicações , Infecções por HIV/transmissão , HIV-1/patogenicidade , Hepacivirus/genética , Hepacivirus/patogenicidade , Hepatite C/complicações , Hepatite C/transmissão , Humanos
7.
Transfusion ; 56(9): 2256-66, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27282889

RESUMO

BACKGROUND: Current pathogen reduction systems for platelet concentrates (PCs) require addition of chemical compounds and/or reduction of plasma content in PCs. We have investigated a new method using xenon (Xe) flash-pulse light without additional compounds or plasma replacement. STUDY DESIGN AND METHODS: An aliquot of apheresis platelets (PLTs) in plasma inoculated with bacteria or human immunodeficiency virus Type 1 (HIV-1) was irradiated with Xe flash-pulse light (Xe flash phototreatment). Bacterial growth was monitored up to 6 days of storage, whereas HIV-1 infectivity was assayed just after treatment. Pairs of Xe flash-phototreated and untreated PCs were examined for PLT lesion during the storage period. RESULTS: Under the current conditions, a low titer (1.8 colony-forming units [CFUs]/mL) of Staphylococcus aureus did not proliferate during the 6-day storage period, but grew in some cases at high-titer (24.0 CFUs/mL) inoculation. HIV-1 infectivity was reduced by 1.8 log. PLT recovery of the treated PCs was lower than untreated ones. An increase of mean PLT volume and glucose consumption, together with a decrease of hypotonic shock response and pH, were enhanced by the treatment. CD62P- and PAC-1-positive PLTs increased after the treatment, indicating the induction of PLT activation. Among biologic response modifiers, soluble CD40 ligand was significantly increased in the treated PCs on Day 6. CONCLUSIONS: Xe flash phototreatment could prevent bacterial proliferation and reduce HIV-1 infectivity in 100% plasma PCs without any additional compounds, but enhanced PLT storage lesions. Further improvement is required to increase the potency of pathogen inactivation with reducing PLT damage.


Assuntos
Plaquetas/efeitos dos fármacos , Plaquetas/efeitos da radiação , HIV-1/efeitos dos fármacos , HIV-1/efeitos da radiação , Staphylococcus/efeitos dos fármacos , Staphylococcus/efeitos da radiação , Raios Ultravioleta , Xenônio , Plaquetas/microbiologia , Plaquetas/virologia , Preservação de Sangue/métodos , Desinfecção/métodos , Humanos , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/efeitos da radiação
8.
BMC Womens Health ; 16: 25, 2016 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-27169666

RESUMO

BACKGROUND: The burden of cervical cancer remains huge globally, more so in sub-Saharan Africa. Effectiveness of screening, rates of recurrence following treatment and factors driving these in Africans have not been sufficiently studied. The purpose of this study therefore was to investigate factors associated with recurrence of cervical intraepithelial lesions following thermo-coagulation in HIV-positive and HIV-negative Nigerian women using Visual Inspection with Acetic Acid (VIA) or Lugol's Iodine (VILI) for diagnosis. METHODS: A retrospective cohort study was conducted, recruiting participants from the cervical cancer "see and treat" program of IHVN. Data from 6 sites collected over a 4-year period was used. Inclusion criteria were: age ≥18 years, baseline HIV status known, VIA or VILI positive and thermo-coagulation done. Logistic regression was performed to examine the proportion of women with recurrence and to examine factors associated with recurrence. RESULTS: Out of 177 women included in study, 67.8 % (120/177) were HIV-positive and 32.2 % (57/177) were HIV-negative. Recurrence occurred in 16.4 % (29/177) of participants; this was 18.3 % (22/120) in HIV-positive women compared to 12.3 % (7/57) in HIV-negative women but this difference was not statistically significant (p-value 0.31). Women aged ≥30 years were much less likely to develop recurrence, adjusted OR = 0.34 (95 % CI = 0.13, 0.92). Among HIV-positive women, CD4 count <200cells/mm(3) was associated with recurrence, adjusted OR = 5.47 (95 % CI = 1.24, 24.18). CONCLUSION: Recurrence of VIA or VILI positive lesions after thermo-coagulation occurs in a significant proportion of women. HIV-positive women with low CD4 counts are at increased risk of recurrent lesions and may be related to immunosuppression.


Assuntos
Eletrocoagulação/normas , Infecções por HIV/complicações , Infecções por HIV/terapia , Displasia do Colo do Útero/fisiopatologia , Adulto , Estudos de Coortes , Eletrocoagulação/métodos , Feminino , Infecções por HIV/epidemiologia , HIV-1/patogenicidade , HIV-1/efeitos da radiação , Humanos , Terapia a Laser/métodos , Terapia a Laser/normas , Pessoa de Meia-Idade , Nigéria/epidemiologia , Estudos Retrospectivos , Neoplasias do Colo do Útero/diagnóstico , Neoplasias do Colo do Útero/radioterapia , Displasia do Colo do Útero/complicações , Displasia do Colo do Útero/epidemiologia
9.
AIDS Res Hum Retroviruses ; 32(2): 120-4, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26765533

RESUMO

The use of highly active antiretroviral therapy against HIV-1 for last two decades has reduced mortality of patients through extension of nonsymptomatic phase of infection. However, HIV-1 can be preserved in long-lived resting CD4(+) T cells, which form a viral reservoir in infected individuals, and potentially in macrophages and astrocytes. Reactivation of viral replication is critical since the host immune response in combination with antiretroviral therapy may eradicate the virus (shock and kill strategy). In this opinion piece, we consider potential application of therapeutic doses of irradiation, the well-known and effective stress signal that induces DNA damage and activates cellular stress response, to resolve two problems: activate HIV-1 replication and virion production in persistent reservoirs under cART and deplete infected cells through selective cell killing using DNA damage responses.


Assuntos
Dano ao DNA/efeitos da radiação , Infecções por HIV/radioterapia , HIV-1/efeitos da radiação , Ativação Viral/efeitos da radiação , Latência Viral/efeitos da radiação , Replicação Viral/efeitos da radiação , Fármacos Anti-HIV/uso terapêutico , Terapia Antirretroviral de Alta Atividade , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/virologia , Reparo do DNA/efeitos da radiação , Infecções por HIV/tratamento farmacológico , Infecções por HIV/virologia , HIV-1/efeitos dos fármacos , Humanos
10.
Transfusion ; 56(4): 831-6, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26585542

RESUMO

BACKGROUND: The risk of transfusion-transmitted infection (TTI) has been minimized by introduction of nucleic acid testing (NAT) and pathogen inactivation (PI). This case report describes transmission of human immunodeficiency virus Type 1 (HIV-1) to two recipients despite these measures. STUDY DESIGN AND METHODS: In March 2009 a possible TTI of HIV-1 was identified in a patient that had received pooled buffy coat platelet concentrate (BC-PLT) in November 2005. The subsequent lookback study found two more patients who had received methylene blue (MB)-treated fresh-frozen plasma (FFP) and red blood cells (RBCs) from the same donation. In November 2005 the donor had tested negative for both HIV antibodies and HIV-1 RNA by 44 minipool (44 MP) NAT. Repository samples of this donation and samples from the recipients were used for viral load (VL) and sequence analysis. RESULTS: HIV-1 RNA was detectable by individual donation (ID)-NAT in the repository sample from the 2005 window period donation and a VL of 135 copies/mL was measured. HIV-1 infection was confirmed in both recipients of both BC-PLT (65 mL of plasma) and MB-FFP (261 mL of plasma), but not in the patient that had received 4-week-old RBCs (20 mL of plasma). The sequence analysis revealed a close phylogenetic relationship between the virus strains isolated from the donor and recipients, compatible with TTI. CONCLUSIONS: Approximately 17,600 and 4400 virions in the MB-FFP and BC-PLT were infectious, but 1350 virions in the RBCs were not. ID-NAT would have prevented this transmission, but the combination of MP-NAT and MB-PI did not.


Assuntos
Transfusão de Componentes Sanguíneos/efeitos adversos , Infecções por HIV/transmissão , HIV-1 , Luz , Azul de Metileno/farmacologia , Plasma/virologia , Inativação de Vírus , Adulto , Doadores de Sangue , Infecções por HIV/sangue , HIV-1/efeitos dos fármacos , HIV-1/genética , HIV-1/isolamento & purificação , HIV-1/efeitos da radiação , Humanos , Masculino , Plasma/efeitos dos fármacos , Plasma/efeitos da radiação , RNA Viral/sangue , Falha de Tratamento , Inativação de Vírus/efeitos dos fármacos , Inativação de Vírus/efeitos da radiação , Adulto Jovem
11.
Virology ; 485: 1-15, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26184775

RESUMO

The highly active antiretroviral therapy reduces HIV-1 RNA in plasma to undetectable levels. However, the virus continues to persist in the long-lived resting CD4(+) T cells, macrophages and astrocytes which form a viral reservoir in infected individuals. Reactivation of viral transcription is critical since the host immune response in combination with antiretroviral therapy may eradicate the virus. Using the chronically HIV-1 infected T lymphoblastoid and monocytic cell lines, primary quiescent CD4(+) T cells and humanized mice infected with dual-tropic HIV-1 89.6, we examined the effect of various X-ray irradiation (IR) doses (used for HIV-related lymphoma treatment and lower doses) on HIV-1 transcription and viability of infected cells. Treatment of both T cells and monocytes with IR, a well-defined stress signal, led to increase of HIV-1 transcription, as evidenced by the presence of RNA polymerase II and reduction of HDAC1 and methyl transferase SUV39H1 on the HIV-1 promoter. This correlated with the increased GFP signal and elevated level of intracellular HIV-1 RNA in the IR-treated quiescent CD4(+) T cells infected with GFP-encoding HIV-1. Exposition of latently HIV-1infected monocytes treated with PKC agonist bryostatin 1 to IR enhanced transcription activation effect of this latency-reversing agent. Increased HIV-1 replication after IR correlated with higher cell death: the level of phosphorylated Ser46 in p53, responsible for apoptosis induction, was markedly higher in the HIV-1 infected cells following IR treatment. Exposure of HIV-1 infected humanized mice with undetectable viral RNA level to IR resulted in a significant increase of HIV-1 RNA in plasma, lung and brain tissues. Collectively, these data point to the use of low to moderate dose of IR alone or in combination with HIV-1 transcription activators as a potential application for the "Shock and Kill" strategy for latently HIV-1 infected cells.


Assuntos
Apoptose/efeitos da radiação , Raios gama/uso terapêutico , Infecções por HIV/radioterapia , HIV-1/efeitos da radiação , Transcrição Gênica/efeitos da radiação , Animais , Fármacos Anti-HIV/farmacologia , Briostatinas/farmacologia , Linfócitos T CD4-Positivos , Linhagem Celular Tumoral , Sobrevivência Celular , Feminino , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Infecções por HIV/tratamento farmacológico , Infecções por HIV/genética , Infecções por HIV/virologia , HIV-1/fisiologia , Histona Desacetilase 1/genética , Histona Desacetilase 1/metabolismo , Humanos , Masculino , Metiltransferases/genética , Metiltransferases/metabolismo , Camundongos , Monócitos , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , RNA Viral/agonistas , RNA Viral/genética , RNA Viral/metabolismo , Proteínas Repressoras/agonistas , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Proteína Supressora de Tumor p53/agonistas , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Ativação Viral/efeitos da radiação , Replicação Viral/efeitos da radiação
12.
Nat Commun ; 6: 6461, 2015 Mar 09.
Artigo em Inglês | MEDLINE | ID: mdl-25751579

RESUMO

HIV protease (PR) is required for proteolytic maturation in the late phase of HIV replication and represents a prime therapeutic target. The regulation and kinetics of viral polyprotein processing and maturation are currently not understood in detail. Here we design, synthesize, validate and apply a potent, photodegradable HIV PR inhibitor to achieve synchronized induction of proteolysis. The compound exhibits subnanomolar inhibition in vitro. Its photolabile moiety is released on light irradiation, reducing the inhibitory potential by 4 orders of magnitude. We determine the structure of the PR-inhibitor complex, analyze its photolytic products, and show that the enzymatic activity of inhibited PR can be fully restored on inhibitor photolysis. We also demonstrate that proteolysis of immature HIV particles produced in the presence of the inhibitor can be rapidly triggered by light enabling thus to analyze the timing, regulation and spatial requirements of viral processing in real time.


Assuntos
Aminocumarinas/farmacologia , Carbamatos/farmacologia , Inibidores da Protease de HIV/farmacologia , Protease de HIV/química , HIV-1/efeitos dos fármacos , Precursores de Proteínas/antagonistas & inibidores , Valina/análogos & derivados , Aminocumarinas/síntese química , Sítios de Ligação , Carbamatos/síntese química , Células HEK293 , Protease de HIV/metabolismo , Inibidores da Protease de HIV/síntese química , HIV-1/fisiologia , HIV-1/efeitos da radiação , Humanos , Cinética , Luz , Modelos Moleculares , Fotólise , Ligação Proteica , Precursores de Proteínas/química , Precursores de Proteínas/metabolismo , Proteólise/efeitos dos fármacos , Fatores de Tempo , Valina/síntese química , Valina/farmacologia , Replicação Viral
13.
J Virol Methods ; 189(1): 125-8, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23384676

RESUMO

A method is described for achieving repeatable, complete inactivation of HIV, based on photo-inactivation of HIV reverse transcriptase (RT) with a non-nucleoside reverse transcriptase inhibitor (NNRTI), photoactive 4-[[4-[(4-azido-2,6-dimethylphenyl) amino]-2-pyrimidinyl]amino]benzonitrile (PA-DAPYa). These results show that PA-DAPYa inactivated completely a suspension of cell-free HIV-1 viral particles in a dose and time-dependent manner. Using an ELISA assay for p24, it is demonstrated that a 500nM concentration of PA-DAPYa is able to inactivate 500 TCID50 of HIV viral particles in suspension when irradiated with non-microbicidal wavelength UV light for 30min. No active p24 was detected on days 7, 14, and 21 days after culturing the inactivated HIV in peripheral blood mononuclear cells (PBMCs). Several batches of large quantities of HIV viral particles were demonstrated to be inactivated completely and repeatedly by this method. Therefore, a reliable method has been developed to inactivate HIV viral particles in a reproducible manner using an optimal concentration of PA-DAPYa and duration of UV exposure time of the treated particles. The inactivation of viral particles in suspension allows for large-scale production of an injectable formulation of inactivated HIV viral particles for vaccine development which should preserve the conformational and antigenic integrity of viral surface proteins.


Assuntos
Azidas/farmacologia , Proteína do Núcleo p24 do HIV/análise , HIV-1/efeitos dos fármacos , Pirimidinas/farmacologia , Inibidores da Transcriptase Reversa/farmacologia , Inativação de Vírus , Vacinas contra a AIDS , Células Cultivadas , Infecções por HIV/terapia , Infecções por HIV/virologia , Transcriptase Reversa do HIV/antagonistas & inibidores , HIV-1/fisiologia , HIV-1/efeitos da radiação , Humanos , Leucócitos Mononucleares/virologia , Raios Ultravioleta
14.
J Med Virol ; 85(2): 187-93, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23172701

RESUMO

The postnatal transmission of human immunodeficiency virus (HIV) from mothers to children occurs through breastfeeding. Although heat treatment of expressed breast milk is a promising approach to make breastfeeding safer, it is still not popular, mainly because the recommended procedures are difficult to follow, or time-consuming, or because mothers do not know which temperature is sufficient to inactivate HIV without destroying the nutritional elements of milk. To overcome these drawbacks, a simple and rapid method of heat treatment that a mother could perform with regular household materials applying her day-to-day art of cooking was examined. This structured experiment has demonstrated that both cell-free and cell-associated HIV type 1 (HIV-1) in expressed breast milk could be inactivated once the temperature of milk reached 65°C. Furthermore, a heating method as simple as heating the milk in a pan over a stove to 65°C inhibited HIV-1 transmission retaining milk's nutritional key elements, for example, total protein, IgG, IgA, and vitamin B(12) . This study has highlighted a simple, handy, and cost-effective method of heat treatment of expressed breast milk that mothers infected with HIV could apply easily and with more confidence.


Assuntos
Desinfecção/métodos , Infecções por HIV/virologia , HIV-1/efeitos da radiação , Calefação , Transmissão Vertical de Doenças Infecciosas/prevenção & controle , Viabilidade Microbiana/efeitos da radiação , Leite Humano/virologia , Desinfecção/economia , Feminino , Infecções por HIV/transmissão , HIV-1/fisiologia , Humanos
16.
Virology ; 417(1): 221-8, 2011 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-21726886

RESUMO

Previously we reported that hydrophobic aryl azides partition into hydrophobic regions of the viral membrane of enveloped viruses and inactivate the virus upon UVA irradiation for 2 min. Prolonged irradiation (15 min) resulted in viral protein aggregation as visualized via Western blot analysis, due to reactive oxygen species (ROS) formation, with preservation of the surface antigenic epitopes. Herein, we demonstrate that these aggregates show detergent resistance and that this property may be useful towards the creation of a novel orthogonal virus inactivation strategy for use in preparing experimental vaccines. When ROS-modified HIV virus preparations were treated with 1% Triton X-100, there was an increase in the percent of viral proteins (gp41, p24) in the viral pellet after ultracentrifugation through sucrose. Transmission electron microscopy (TEM) of these detergent-resistant pellets shows some recognizable virus fragments, and immunoprecipitation studies of the gp41 aggregates suggest the aggregation is covalent in nature, involving short-range interactions.


Assuntos
Azidas/farmacologia , HIV-1/efeitos dos fármacos , HIV-1/efeitos da radiação , Espécies Reativas de Oxigênio/farmacologia , Raios Ultravioleta , Inativação de Vírus , Fármacos Anti-HIV/química , Fármacos Anti-HIV/farmacologia , Anticorpos Neutralizantes , Anticorpos Antivirais , Azidas/química , Testes de Neutralização , Solubilidade
17.
J Med Assoc Thai ; 94 Suppl 2: S88-93, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21717885

RESUMO

OBJECTIVE: To describe effects of radiation therapy (RT) on immunological status (CD4 cell counts) and disease progression among HIV-positive cancer patients. MATERIAL AND METHOD: This prospective observational study was conducted among HIV-positive cancer patients who received RT for curative intention of cancer in five selected hospitals in Thailand. All subjects received external beam radiation therapy, according to standard clinical practice guidelines of RT. Blood samples were taken 4 times for complete blood count, CD4 cell count and plasma HIV RNA viral load (HIV-VL) assays before and in the last week of RT, then three and six months after completion of RT. RESULTS: This preliminary study reported immunological status and HIV-VL before and the last week of RT, among 29 HIV-positive female cancer patients enrolled from August 22, 2009 to June 30, 2010. The median age was 38 years (range 30-54). 27 patients (93 percent) had invasive cervical cancer. 26 patients (90 percent) were on antiretroviral treatment (ART). The mean baseline white blood cell (WBC) count, lymphocyte percentage were 6,771.7 cells/microL and 31.7 percent respectively. The mean baseline CD4 cell count and CD4%, 387.8 cells/microL and 17.5 percent respectively. In the last week of RT, 25 subjects (86 percent) had CD4 count less than 200 cells/microL. The last week, mean WBC count, and mean lymphocyte percentage decreased to 3,902.8 cells/microL and 17.5 percent respectively. Mean CD4 count number decreased to 157.7 cells/microL, but the mean CD4 % did not change. Four patients (14 percent) had increased HIV-VL after RT, of these two were not on ART and two were on ART for more than 1 year. CONCLUSION: The CD4 cell count was not a good surrogate for prediction of immunologic status of HIV-positive cancer patients during RT.


Assuntos
Infecções por HIV/imunologia , HIV-1/efeitos da radiação , Neoplasias/radioterapia , RNA Viral/efeitos da radiação , Carga Viral/efeitos da radiação , Adolescente , Adulto , Fármacos Anti-HIV/uso terapêutico , Terapia Antirretroviral de Alta Atividade , Contagem de Linfócito CD4 , Progressão da Doença , Feminino , Infecções por HIV/complicações , Infecções por HIV/tratamento farmacológico , HIV-1/imunologia , Humanos , Pessoa de Meia-Idade , Neoplasias/complicações , Estudos Prospectivos , RNA Viral/sangue , Tailândia , Adulto Jovem
18.
Breastfeed Med ; 6(3): 111-6, 2011 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-21091243

RESUMO

BACKGROUND AND OBJECTIVES: The World Health Organization recommends human immunodeficiency virus (HIV)-positive mothers in resource-poor regions heat-treat expressed breastmilk during periods of increased maternal-to-child transmission risk. Flash-heat, a "low tech" pasteurization method, inactivates HIV, but effects on milk protein bioactivity are unknown. The objectives were to measure flash-heat's effect on antimicrobial properties of lactoferrin, lysozyme, and whole milk and on the digestive resistance of lactoferrin and lysozyme. METHODS: Flash-heated and unheated breastmilk aliquots from HIV-positive mothers in South Africa were "spiked" with Staphylococcus aureus and Escherichia coli and then cultured for 0, 3, and 6 hours. Lysozyme and lactoferrin activities were determined by lysis of Micrococcus luteus cells and inhibition of enteropathogenic E. coli, respectively, measured spectrophotometrically. Percentages of proteins surviving in vitro digestion, lactoferrin and lysozyme activity, and bacteriostatic activity of whole milk in heated versus unheated samples were compared. RESULTS: There was no difference in rate of growth of E. coli or S. aureus in flash-heated versus unheated whole milk (p = 0.61 and p = 0.96, respectively). Mean (95% confidence interval) antibacterial activity of lactoferrin was diminished 11.1% (7.8%, 14.3%) and that of lysozyme by up to 56.6% (47.1%, 64.5%) by flash-heat. Digestion of lysozyme was unaffected (p = 0.12), but 25.4% less lactoferrin survived digestion (p < 0.0001). CONCLUSIONS: In summary, flash-heat resulted in minimally decreased lactoferrin and moderately decreased lysozyme bioactivity, but bacteriostatic activity of whole milk against representative bacteria was unaffected. This suggests flash-heated breastmilk likely has a similar profile of resistance to bacterial contamination as that of unheated milk. Clinical significance of the decreased bioactivity should be tested in clinical trials.


Assuntos
Aleitamento Materno , Infecções por HIV/transmissão , Temperatura Alta/uso terapêutico , Lactoferrina/efeitos da radiação , Leite Humano , Muramidase/efeitos da radiação , Anti-Infecciosos/metabolismo , Anti-Infecciosos/efeitos da radiação , Países em Desenvolvimento , HIV-1/efeitos da radiação , Humanos , Transmissão Vertical de Doenças Infecciosas/prevenção & controle , Lactoferrina/metabolismo , Testes de Sensibilidade Microbiana , Leite Humano/enzimologia , Leite Humano/efeitos da radiação , Leite Humano/virologia , Muramidase/metabolismo , Fatores de Risco , Esterilização/métodos
19.
Photochem Photobiol ; 86(5): 1099-108, 2010.
Artigo em Inglês | MEDLINE | ID: mdl-20630026

RESUMO

Hydrophobic UV-activatable compounds have been shown to partition into the hydrophobic region of biological membranes to selectively label transmembrane proteins, and to inactivate enveloped viruses. Here, we analyze various UV-activatable azido- and iodo-based hydrophobic compounds for their ability to inactivate a model-enveloped virus, human immunodeficiency virus (HIV-1 MN). Treatment of HIV-1 with 1,5-diazidonapthalene (DAN), 1-iodo, 5-azidonaphthalene (INA), 1-azidonaphthalene (AzNAP) or 4,4'-diazidobiphenyl (DABIPH) followed by UVA irradiation for 2 min resulted in complete viral inactivation, whereas treatment using analogous non-azido-containing controls had no effect. Incorporation of an azido moiety within these hydrophobic compounds to promote photoinduced covalent reactions with proteins was found to be the primary mechanism of viral inactivation for this class of compounds. Prolonged UVA irradiation of the virus in the presence of these azido compounds resulted in further modifications of viral proteins, due to the generation of reactive oxygen species, leading to aggregation as visualized via Western blot analysis, providing additional viral modifications that may inhibit viral infectivity. Furthermore, inactivation using these compounds resulted in the preservation of surface antigenic structures (recognized by neutralizing antibodies b12, 2g12 and 4e10), which is favorable for the creation of vaccines from these inactivated virus preparations.


Assuntos
Antivirais/farmacologia , Azidas/farmacologia , HIV-1/efeitos dos fármacos , HIV-1/efeitos da radiação , Raios Ultravioleta , Proteínas Virais/efeitos dos fármacos , Proteínas Virais/efeitos da radiação , Azidas/química , Linhagem Celular , HIV-1/patogenicidade , Humanos , Interações Hidrofóbicas e Hidrofílicas , Microscopia Eletrônica de Varredura , Estrutura Molecular
20.
Vaccine ; 27(44): 6137-42, 2009 Oct 19.
Artigo em Inglês | MEDLINE | ID: mdl-19715783

RESUMO

We describe a new method for the development of a preventive inactivated-HIV vaccine, based on photo-inactivation of HIV reverse transcriptase (RT), which preserves both the conformational and functional integrity of viral surface proteins. The RT of HIV-1 was selectively targeted for inactivation using a photo-labeled compound with specific affinity for HIV-1 RT. The photo-labeled virions were then exposed to UV light causing the photo-labeled compound to form a covalent bond cross-linking the photo-active compound to RT. Replication capacity of the treated virions was significantly reduced when compared to controls suggesting that exposure of treated virions to UV light had caused a stable interaction of RT and the photo-labeling compound.


Assuntos
Vacinas contra a AIDS/química , Transcriptase Reversa do HIV/efeitos da radiação , HIV-1/efeitos da radiação , Inibidores da Transcriptase Reversa/química , Inativação de Vírus , Linhagem Celular Tumoral , Sobrevivência Celular , Reagentes de Ligações Cruzadas , Proteína do Núcleo p24 do HIV/análise , Infecções por HIV , Humanos , Concentração Inibidora 50 , Marcadores de Fotoafinidade , Especificidade por Substrato , Raios Ultravioleta
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