RESUMO
OBJECTIVES: The National Health Service in England (NHS England) does not provide pre-exposure prophylaxis (PrEP) against HIV, forcing people to purchase generic versions on the internet. However, there are concerns about the authenticity of medicines purchased online. We established an innovative service offering plasma tenofovir (TFV) and emcitrabine (FTC) therapeutic drug monitoring for people buying generic PrEP online, to ensure that drug concentrations in vivo were consistent with those of propriety brands and previously published data. METHODS: TFV/FTC concentrations were measured by ultra-performance liquid chromatography ultraviolet detection. Evaluation of renal function and testing for HIV, hepatitis B virus (HBV) and hepatitis C virus (HCV) were also carried out, at baseline and every 3-6 months, with risk reduction advice. RESULTS: A total of 293 individuals presented having purchased PrEP on the internet: 85% were white, 84% were taking daily PrEP, and 16% were event-driven. Most were on generic TFV disoproxil fumarate (TDF)/FTC from Cipla Ltd. Median (range) TFV and FTC plasma concentrations were 104 (21-597) ng/mL and 140 (17-1876) ng/mL, respectively. All concentrations were above our established plasma TFV and FTC targets, based on previously published data. Renal function was normal in all evaluable individuals and no new cases of HIV, HBV or HCV infection were seen. CONCLUSIONS: In a population at high risk of HIV acquisition, who cannot yet access PrEP on the NHS, concentrations of TFV and FTC in generic formulations purchased over the internet were similar to (or slightly higher than) those measured in phase I studies with the original formulation from Gilead (Truvada™), which has demonstrated high levels of protection against HIV infection in previous PrEP clinical trials.
Assuntos
Fármacos Anti-HIV/administração & dosagem , Quimioprevenção/métodos , Transmissão de Doença Infecciosa/prevenção & controle , Emtricitabina/administração & dosagem , Infecções por HIV/prevenção & controle , Profilaxia Pré-Exposição/métodos , Tenofovir/administração & dosagem , Adolescente , Adulto , Idoso , Fármacos Anti-HIV/efeitos adversos , Fármacos Anti-HIV/farmacocinética , Cromatografia Líquida , Emtricitabina/efeitos adversos , Emtricitabina/farmacocinética , Feminino , Humanos , Londres , Masculino , Pessoa de Meia-Idade , Plasma/química , Tenofovir/efeitos adversos , Tenofovir/farmacocinética , Resultado do Tratamento , Adulto JovemRESUMO
In this study, we examined whether the proportion of tubal factor infertility (TFI) that is attributable to Chlamydia trachomatis, the population excess fraction (PEF), can be estimated from serological data using finite mixture modeling. Whole-cell inclusion immunofluorescence serum antibody titers were recorded among infertile women seen at St. Michael's Hospital in Bristol, United Kingdom, during the period 1985-1995. Women were classified as TFI cases or controls based on laparoscopic examination. Finite mixture models were used to identify the number of component titer distributions and the proportion of serum samples in each, from which estimates of PEF were derived. Four titer distributions were identified. The component at the highest titer was found only in samples from women with TFI, but there was also an excess of the second-highest titer component in TFI cases. Minimum and maximum estimates of the PEF were 28.0% (95% credible interval: 6.9, 50.0) and 46.8% (95% credible interval: 23.2, 64.1). Equivalent estimates based on the standard PEF formula from case-control studies were 0% and over 65%. Finite mixture modeling can be applied to serological data to obtain estimates of the proportion of reproductive damage attributable to C. trachomatis Further studies using modern assays in contemporary, representative populations should be undertaken.
Assuntos
Anticorpos Antibacterianos/sangue , Infecções por Chlamydia/complicações , Chlamydia trachomatis , Infertilidade Feminina/etiologia , Estudos de Casos e Controles , Infecções por Chlamydia/diagnóstico , Chlamydia trachomatis/imunologia , Chlamydia trachomatis/isolamento & purificação , Feminino , HumanosRESUMO
BACKGROUND: Treatment of chronic hepatitis C virus (HCV) infection in HIV-1-co-infected individuals remains challenging due to numerous factors, including drug-drug interactions. The aim of this study was to assess the safety and pharmacokinetic (PK) profile of raltegravir and ribavirin when dosed separately and together. METHODS: Fourteen healthy volunteers [mean (standard deviation) age 35 (10) years, 71% male] entered this phase 1 PK study and received single-dose ribavirin (800 mg) on day 1 (phase 1). Following a washout period, subjects received raltegravir (400 mg twice daily) on days 15-19 (phase 2) and single-dose ribavirin (800 mg) with raltegravir (400 mg) on day 20 (phase 3). Intensive PK sampling was undertaken on days 1, 19 and 20 and differences in geometric mean ratios (GMRs) for PK parameters between study periods were assessed. RESULTS: No statistically significant differences in PK parameters were observed for raltegravir between phases 2 and 3. A statistically significant decrease in maximum plasma concentration (C(max)) and an increase in time to maximum plasma concentration (T(max)) were observed for ribavirin in phase 3 compared with phase 1 [GMR (95% confidence interval) 0.79 (0.62-1.00) and 1.39 (1.08-1.78), respectively], whereas no significant differences in other ribavirin PK parameters were observed between study phases. No clinically significant safety concerns were reported. CONCLUSIONS: The PK profile of ribavirin is altered when administered with raltegravir (reduced C(max) and increased T(max)), with no safety concerns identified. This is unlikely to be of clinical significance or have an impact on the antiviral effects of ribavirin in HIV-1- and HCV-co-infected subjects.
Assuntos
Antivirais/efeitos adversos , Antivirais/farmacocinética , Pirrolidinonas/efeitos adversos , Pirrolidinonas/farmacocinética , Ribavirina/efeitos adversos , Ribavirina/farmacocinética , Adolescente , Adulto , Antivirais/administração & dosagem , Quimioterapia Combinada , Feminino , Voluntários Saudáveis , Humanos , Masculino , Pessoa de Meia-Idade , Pirrolidinonas/administração & dosagem , Raltegravir Potássico , Ribavirina/administração & dosagem , Adulto JovemRESUMO
Foamy viruses, distantly related to the major subfamily of Retroviruses, Orthoretroviruses that include oncoviruses (for example, murine leukemia virus (MLV)) and lentiviruses (human immunodeficiency virus (HIV)), are endemic in mammalian species, but not in human populations. Humans infected by accidental or occupational exposure remain well. The virus is not transmitted to others, nor is it associated with any disease. These features added to its broad host range, efficient transduction of progenitor cells and an integration profile less likely to induce insertional mutagenesis, make these viruses attractive as vectors. Long-term reversal of disease phenotype in dogs with the genetic defect, leukocyte adhesion deficiency, by foamy virus vector therapy strengthens the case for their clinical exploitation.
Assuntos
Doenças Genéticas Inatas/terapia , Vetores Genéticos/uso terapêutico , Spumavirus/genética , Animais , Técnicas de Transferência de Genes/normas , Técnicas de Transferência de Genes/tendências , Terapia Genética/métodos , Terapia Genética/normas , Terapia Genética/tendências , Vetores Genéticos/genética , Vetores Genéticos/normas , Humanos , Spumavirus/patogenicidadeRESUMO
Sequencing of the human genome has established that our DNA harbours many endogenous retrovirus (ERV) sequences, remnants of ancestral exogenous retroviral infections fixed in the germline DNA. In recent years, human ERVs (HERVs) have been implicated in melanomagenesis. Retrovirus-like particles and the expression of HERV mRNA and proteins have been demonstrated in melanoma tissue. In addition, antibodies to HERV proteins have been observed in patients with melanoma. In vitro and mouse models have provided fascinating insights into the potential mechanisms of HERVs in melanomagenesis. This review considers the evidence associating HERVs with melanoma.
Assuntos
Retrovirus Endógenos/genética , Melanoma/virologia , Infecções por Retroviridae/genética , Neoplasias Cutâneas/virologia , Integração Viral/genética , Animais , Retrovirus Endógenos/classificação , Medicina Baseada em Evidências , Regulação Viral da Expressão Gênica/genética , Genoma Humano/genética , Genoma Viral/genética , Humanos , Melanoma/genética , RNA Viral/genética , Infecções por Retroviridae/classificação , Neoplasias Cutâneas/genéticaRESUMO
AIM: In this paper we compare the transduction efficiency, toxicity, and safety of retroviral vectors [equine infectious anemia virus (EIAV), human immunodeficiency virus-1 (HIV-1), human foamy virus (PFV] and adenovirus (Ad) for potential use in gene therapy of corneal endothelial cells. METHOD: Murine corneal endothelial cells were transduced with EIAV, HIV-1, PFV, and Ad, resulting in the overexpression of a green fluorescent protein (eGFP) transgene marker. The transduction efficiency was assessed by flow cytometry, while cytotoxicity and apoptosis rate were detected by annexin V/propidium iodide (PI) stain. RESULTS: Ad had the highest transduction efficiency with 99% of the cells expressing the transgene, followed by EIAV (95%), HIV-1 (75%), and PFV (43%). However, the high transduction efficiency of Ad also resulted in the highest apoptosis rate (25%) in the corneal endothelial cells. There was no detectable difference in the toxicity between PFV and HIV-1 (10%). EIAV transduction had the lowest cytotoxicity, with only 3% of the cells being annexin V/PI positive. CONCLUSION: Compared to other vectors EIAV exhibited high transduction efficiency combined with low toxicity to corneal endothelial cells. Therefore, it is a powerful tool for gene therapy applications in selected corneal endothelial diseases.
Assuntos
Adenoviridae/genética , Epitélio Corneano/metabolismo , Proteínas de Fluorescência Verde/metabolismo , HIV-1/genética , Vírus da Anemia Infecciosa Equina/genética , Spumavirus/genética , Transfecção/métodos , Animais , Linhagem Celular , Doenças da Córnea/genética , Doenças da Córnea/terapia , DNA Viral/administração & dosagem , DNA Viral/genética , Perfilação da Expressão Gênica , Terapia Genética/métodos , Vetores Genéticos/genética , Proteínas de Fluorescência Verde/genética , Cavalos , Humanos , CamundongosRESUMO
Advances in immunoglobulin (Ig) sequencing technology are leading to new perspectives on immune system dynamics. Much research in this nascent field has focused on resolving immune responses to viral infection. However, the dynamics of B-cell diversity in early HIV infection, and in response to anti-retroviral therapy, are still poorly understood. Here, we investigate these dynamics through bulk Ig sequencing of samples collected over 2 years from a group of eight HIV-1 infected patients, five of whom received anti-retroviral therapy during the first half of the study period. We applied previously published methods for visualizing and quantifying B-cell sequence diversity, including the Gini index, and compared their efficacy to alternative measures. While we found significantly greater clonal structure in HIV-infected patients versus healthy controls, within HIV patients, we observed no significant relationships between statistics of B-cell clonal expansion and clinical variables such as viral load and CD4(+) count. Although there are many potential explanations for this, we suggest that important factors include poor sampling resolution and complex B-cell dynamics that are difficult to summarize using simple summary statistics. Importantly, we find a significant association between observed Gini indices and sequencing read depth, and we conclude that more robust analytical methods and a closer integration of experimental and theoretical work is needed to further our understanding of B-cell repertoire diversity during viral infection.
Assuntos
Diversidade de Anticorpos , Anticorpos Anti-HIV/genética , Infecções por HIV/genética , Infecções por HIV/imunologia , HIV-1 , Linfócitos B/imunologia , Estudos de Casos e Controles , Seleção Clonal Mediada por Antígeno , Variação Genética , Infecções por HIV/tratamento farmacológico , Humanos , Receptores de Antígenos de Linfócitos B/genética , Recombinação V(D)JRESUMO
OBJECTIVE: To assess the therapeutic response and investigate the significance of polymorphic codons in African patients receiving highly-active antiretroviral therapy (HAART). DESIGN AND METHODS: African patients were identified from the St Mary's Hospital HIV-1 database. Clinical outcome was assessed by viral load and CD4 cell count. Pre- and post-therapy sequences of RT and protease were analysed. The impact of subtype and individual polymorphic codons on therapeutic outcome was assessed statistically (Fishers exact and chi2 tests) and phylogenetically (Jukes and Cantor). RESULTS: Of 79 drug-naive African patients who were prescribed HAART, 60 remained undetectable for 1 year, with no differences detected in the clinical response to non-nucleoside reverse transcriptase inhibitor (NNRTI)- or protease inhibitor (PI)-containing regimes. Country of origin, sex and viral subtype had no impact on outcome of HAART. A total of 133 polymorphisms were identified in pol (37 in protease and 96 in RT), with a mean of 9.0 in protease and 22.3 in RT per patient. There was no significant difference in the overall numbers of polymorphisms per patient, and no single polymorphism had any impact on clinical outcome. Sequences from 'failing' patients experiencing viral rebound produced few mutations known to be associated with drug resistance, suggesting minimal drug pressure. CONCLUSIONS: The response of patients infected with African subtypes of HIV-1 to HAART appears to be independent of regime, HIV-1 clade and baseline polymorphisms. Non-B subtypes are fully sensitive to HAART and, accordingly, therapy should not be withheld from African patients for reasons of viral diversity.
Assuntos
Terapia Antirretroviral de Alta Atividade , Infecções por HIV/tratamento farmacológico , Protease de HIV/genética , Transcriptase Reversa do HIV/genética , Polimorfismo Genético , África , Contagem de Linfócito CD4 , Progressão da Doença , Resistência Microbiana a Medicamentos/genética , Feminino , Infecções por HIV/virologia , HIV-1/enzimologia , HIV-1/genética , Humanos , Masculino , Resultado do Tratamento , Carga ViralRESUMO
The envelope glycoproteins of HIV, gp120 and gp41, contain epitopes recognized by neutralizing antibodies. Studies of human sera from infected individuals indicate that group-specific neutralization antigens common to most isolates of HIV-1 exist, and that some HIV-2 antisera cross-neutralize HIV-1. Neutralization epitopes for HIV-1 have been identified and mapped, including a group-specific antigen on gp41, and a type-specific antigen on gp120. Neutralization "escape" mutants have been selected in vitro with a neutralizing mab to the type-specific antigenic loop. The CD4 antigen binds HIV-1 gp120 with high affinity and acts as the receptor on human and simian T-lymphocytes and monocytes for all strains of HIV-1, HIV-2, and SIV tested. Following binding to the CD4 receptor, HIV becomes internalized by a pH-independent process. The principle binding domain for gp120 is located in the N-terminal V domain of CD4. Anti-idiotypic sera to CD4 mabs recognizing the same site weakly neutralize HIVs of many strains, and soluble, recombinant forms of CD4 strongly neutralize HIV. Neither anti-CD4 mabs nor sCD4 inhibit the low level of plating of HIV observed on tumour cells in culture of glial (brain) and muscle origin, indicating that CD4 is not essential for infection of these cell types.
Assuntos
Antígenos de Diferenciação de Linfócitos T/imunologia , Antígenos HIV/imunologia , HIV/imunologia , Animais , Variação Antigênica , Epitopos/imunologia , Humanos , Testes de Neutralização , Receptores Imunológicos/imunologia , Receptores Virais/imunologia , Linfócitos T/imunologiaRESUMO
This study investigated the effects of a combination antiretroviral drug regimen (indinavir and two nucleoside analogs or ritonavir and saquinavir) on the levels of CD34+ colony-forming units (CFU-Cs) in the peripheral blood of HIV-1+ patients. Ten patients who were receiving combination antiretroviral drug therapy were studied and their peripheral blood CD34+ CFU-Cs were measured prior to, 1 month after, and 4 to 6 months after the commencement of therapy. The levels of CD4+ T cells increased significantly in these patients (paired t test, p = 0.0027) and plasma viral load became undetectable in all but one patient studied. Measurements of the CFU-Cs showed that their levels tended to increase on the commencement of therapy, and these levels became significantly higher than baseline by 4-6 months (paired t test, p = 0.0293). Analysis of the different colony phenotype demonstrated that the main contributor to this increase consisted of burst-forming unit erythroid (BFU-E) cells. These data also demonstrated that there was an inverse correlation between the rise in CFU-Cs at 4-6 months compared with CD4+ cell, CD8+ cell, and neutrophil counts, and hemoglobin concentration, at baseline. The demonstrated increase in the levels of CD34+ CFU-Cs suggests that HIV-1 may have an inhibitory effect on these cells in vivo, and that this inhibition may be abrogated by suppression of viral replication.
Assuntos
Fármacos Anti-HIV/uso terapêutico , Antígenos CD34 , Infecções por HIV/tratamento farmacológico , Infecções por HIV/imunologia , Inibidores da Protease de HIV/uso terapêutico , HIV-1 , Células-Tronco Hematopoéticas/imunologia , Adulto , Células Cultivadas , Quimioterapia Combinada , Infecções por HIV/sangue , Infecções por HIV/virologia , Humanos , Indinavir/uso terapêutico , Pessoa de Meia-Idade , Ritonavir/uso terapêutico , Saquinavir/uso terapêuticoRESUMO
Sulfated polysaccharides have been shown to inhibit human immunodeficiency virus (HIV) infection in vitro. Dextrin sulfate, fucoidan, and dextran sulfate fail to neutralize virions directly, but interact with target cells to inhibit virus entry. Ionic interactions of sulfated polyanions with oppositely charged cell surface components, including CD4, have been assumed to be the inhibitory mechanism. It is shown that the sulfated polysaccharides inhibit infection of both CD4+ and CD4- cell lines by HIV and also that they inhibit HTLV-1 and, to a lesser extent, the simian retrovirus, MPMV, which use receptors other than CD4. One binding site for radiolabeled fucoidan on the surface of human T cells is an 18 kD protein, but its significance is not yet clear.
Assuntos
Antivirais/farmacologia , Infecções por HIV/prevenção & controle , HIV-1/efeitos dos fármacos , Polissacarídeos/farmacologia , Antígenos CD4/efeitos dos fármacos , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linhagem Celular , Proteína gp120 do Envelope de HIV/efeitos dos fármacos , Infecções por HIV/tratamento farmacológico , Infecções por HIV/imunologia , Humanos , Receptores Virais/efeitos dos fármacosRESUMO
Located close to the crown of the V3 type-specific neutralization loop of the human immunodeficiency virus type 1 (HIV-1) (IIIB) SU glycoprotein gp120, are several potential sites that should be susceptible to proteolytic cleavage by enzymes of trypsinlike or chymotrypsinlike specificity, or by aspartic proteinases. The linkages potentially sensitive to chymotryptic/aspartic proteinase cleavage are retained also within the equivalent domain of HIV-2 (ROD) gp105. We show that thrombin and tryptase cleave HIV-1 gp120 specifically at the tryptic site (GPGR decreases AFVT), and that cathepsin E, an endosomal aspartic proteinase, cleaves at the chymotrypsinlike site (GPGRAF decreases VT). HIV-2 gp105 is also cut by cathepsin E at a site (QIML decreases MSGH) in its V3 loop. Cleavage of HIV-1 gp120 by thrombin is enhanced by sCD4 binding, but is prevented by transient exposure of gp120 to nonionic detergent. Thrombin treatment of HIV-1 gp120 destroys the binding sites for some neutralizing monoclonal antibodies (MAbs) on the V3 loop, but does not affect the affinity of gp120 for sCD4. Conversely, binding of neutralizing MAbs to the HIV-1 V3 loop prior to addition of thrombin or cathepsin E blocks the cleavage reactions, and the binding of some HIV-positive sera to gp120 blocks thrombin cleavage. Analysis of published sequences suggests that all HIV-1, HIV-2, and simian immunovirus (SIV) isolates contain potential proteolytic cleavage sites at similar positions in their V3 loops or equivalent domains. We suggest that cleavage of the V3 loop by a cell surface or endosomal proteinase occurs during the HIV-cell fusion reaction, and that neutralizing antibodies directed against the V3 loop might act by inhibition of this reaction.
Assuntos
Proteína gp120 do Envelope de HIV/metabolismo , HIV-1/metabolismo , HIV-2/metabolismo , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/metabolismo , Antígenos CD4/metabolismo , Catepsina E , Catepsinas/metabolismo , Linhagem Celular , Cricetinae , Eletroforese em Gel de Poliacrilamida , Endopeptidases/metabolismo , HIV-1/imunologia , HIV-1/fisiologia , HIV-2/fisiologia , Humanos , Insetos , Fusão de Membrana , Dados de Sequência Molecular , Testes de Neutralização , Peptídeo Hidrolases/metabolismo , Trombina/metabolismoRESUMO
The first human foamy virus (HFV) to be described was isolated from nasopharyngeal carcinoma tissue from a Kenyan patient. Early seroepidemiology concluded that there was a significant infection rate, particularly among Africans. Awareness of foamy viruses as potential vectors has stimulated interest in the natural seroprevalence of HFV infection. We, therefore, investigated the prevalence of HFV infection in more than 5000 human sera collected from diverse populations. To maximize the chances of including the major antigenic epitopes, recombinant proteins derived from the HFV gag and env genes divided into three (the 5' amino terminal, the 3' carboxy terminal, and an internal overlapping region) were used as antigens in an ELISA. In contrast to most other seroepidemiological investigations of HFV infection, highly reactive sera identified by ELISA were subjected to further analysis by additional serological assays and, where PBMCs were available, PCR. None of the serum samples were confirmed as positive. It is worth noting that with our ELISA, the highest level of serum reactivity to HFV was found in subjects from Pacific islands (17%), and in Central Africa (34% in Malawi), areas previously cited as having a high level of HFV infection. Taken together with sequence analysis endorsing the phylogenetic closeness of HFV to SFV-6/7, these data strongly suggest that HFV is not naturally found in the human population.
Assuntos
Infecções por Retroviridae/epidemiologia , Spumavirus/imunologia , Western Blotting , Ensaio de Imunoadsorção Enzimática , Soropositividade para HIV/epidemiologia , Inquéritos Epidemiológicos , Humanos , Neoplasias Nasofaríngeas/virologiaRESUMO
A survey of antibody responses to human herpesvirus 8 (HHV-8) was undertaken to examine the mode of transmission of this virus to children born to mothers with HIV. Methods. Serum samples from a cohort of 92 mother-infant pairs and a cross-sectional cohort of 100 children (median age, 4 years) were tested. In the cohort of mother-infant pairs, 14 infants were HIV-infected, 72 were not and the HIV status was unknown for 6. In the cohort of children 70 were HIV-infected and 30 were vertically exposed but uninfected. Serologic responses to two HHV-8 antigens, latency-associated nuclear antigen and the structural antigen encoded by open reading frame 65 were detected by immunofluorescent antibody test and enzyme-linked immunoassay. Results were confirmed by Western blot. Results. All HHV-8-seropositive mothers were African (17 of 92, 18.5%). Six of their infants were HHV-8-seronegative and 11 had at least 1 HHV-8-seropositive sample. One of the 11 infants tested only at birth had a lower antibody titer than the mother; the remaining 10 infants had decreasing titers up to 7 months of age and 6 became seronegative. No infants born to HHV-8-seronegative mothers had antibodies to the virus. The seroprevalence to HHV-8 was 6% in the cohort of children. All had African mothers and their median age was greater than that of the cohort (8.4 vs. 4.0 years). Five were coinfected with HIV. Conclusions. HHV-8 was not vertically transmitted by any of the HIV-coinfected mothers. Acquisition of antibody to HHV-8 occurred in older children, implying a horizontal route of transmission.
Assuntos
Transmissão de Doença Infecciosa , Infecções por Herpesviridae/transmissão , Herpesvirus Humano 8/imunologia , Complicações Infecciosas na Gravidez/virologia , Adulto , Anticorpos Antivirais/sangue , Criança , Pré-Escolar , Estudos de Coortes , Estudos Transversais , Ensaio de Imunoadsorção Enzimática , Feminino , Imunofluorescência , Infecções por HIV/complicações , Infecções por Herpesviridae/complicações , Infecções por Herpesviridae/virologia , Humanos , Lactente , Transmissão Vertical de Doenças Infecciosas , GravidezRESUMO
AIMS: To develop an indirect ELISA using the heat shock protein (hsp60) of Chlamydia trachomatis as antigen. METHODS: The hsp60 gene was amplified by PCR, expressed in the vector pDEV-107 and transformed into Escherichia coli. The recombinant protein, expressed as a beta-galactosidase fusion product, was captured onto a solid phase using a monoclonal antibody directed against beta-galactosidase. Following incubation with goat anti-human antibody conjugated to peroxidase and colour development on addition of peroxidase substrate, antibody recognition of antigen was quantified by optical density at 492 nm. RESULTS: A sensitive and relatively specific ELISA to detect hsp60 has been produced, which can be exploited to determine the antibody response to C trachomatis hsp60. CONCLUSIONS: This assay will permit the future investigation of the immunopathogenesis of persistent inflammation following C trachomatis infection.
Assuntos
Chaperonina 60/sangue , Infecções por Chlamydia/sangue , Chlamydia trachomatis/imunologia , Ensaio de Imunoadsorção Enzimática/métodos , Western Blotting , Humanos , Sensibilidade e Especificidade , beta-GalactosidaseAssuntos
Dermatologia , Retrovirus Endógenos/genética , RNA Mensageiro/análise , RNA Viral/análise , Preservação de Tecido , Pesquisa Biomédica/métodos , Dermatologia/educação , Expressão Gênica/genética , Humanos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise Serial de Tecidos/métodos , Preservação de Tecido/métodosRESUMO
A novel assay is described for the detection of HIV-1 drug resistance that is simple, cheap and sensitive. HIV-1 drug resistance in B and non-B HIV-1 subtypes was investigated using Mutagenically-Separated PCR (MS--PCR) --- a competitive semi-nested PCR which uses mutagenic primers. The assay was assessed for sensitivity, specificity and its ability to detect mutant virus within a mixed mutant--wild-type population. Gene sequencing was carried out simultaneously for comparison. MS--PCR detected five copies of HIV-1 RNA from laboratory isolates and 50 copies from patient samples. We demonstrate 100% specificity of detection for wild type or mutant virus for clades A, B, C, D and E. For mixed populations of virus, MS--PCR can detect at least a 10% mix of wild type:mutant, or vice-versa. When applied to African patient samples MS--PCR detected 91.6% of the codons tested. Concordance with sequencing data was 88.8% for protease and 97.2% for RT. MS--PCR is sensitive and specific for the detection of mutations in HIV-1, and can be adapted easily to test for resistance at any codon of interest.
Assuntos
Fármacos Anti-HIV/farmacologia , Resistência Microbiana a Medicamentos/genética , Infecções por HIV/virologia , HIV-1/efeitos dos fármacos , África , Terapia Antirretroviral de Alta Atividade , Resistência a Múltiplos Medicamentos , Infecções por HIV/tratamento farmacológico , HIV-1/genética , Humanos , Mutação Puntual/genética , Reação em Cadeia da Polimerase/métodos , Sensibilidade e EspecificidadeRESUMO
Retroviruses may cause diseases in their vertebrate hosts. They are distinguished by their common means of replication involving reverse transcription, a process inhibited by nucleoside reverse transcriptase inhibitors (NRTIs) and other compounds used in antiretroviral chemotherapy. Previous work on NRTIs has been limited to their effect on human immunodeficiency virus (HIV) (for review see Ho & Hitchcock, 1989; Weller, 1999) and little information exists regarding the efficacy and therapeutic potential of these drugs against other retroviruses. We have tested all six NRTIs licensed for HIV treatment [didanosine (ddI), zalcitabine (ddC), lamivudine (3TC), stavudine (d4T), zidovudine (AZT) and abacavir (ABC)] against seven retroviruses representative of the traditional subfamilies: Spumavirinae, Lentivirinae and the Oncovirinae. As expected, each drug showed a range of activities against the panel of retroviruses, some drugs inhibiting other viruses at concentrations well below those required for HIV. Overall, AZT was the most active inhibitor (IC50 range, 0.032-1.0 microM), being most active against the Spuma (foamy) viruses. Abacavir was inhibitory for HIV-1, MN strain (HIV-1 MN), amphotrophic murine leukemia virus (MLV-A) and simian foamy virus type 6 (SFV-6). The least effective inhibitor, 3TC (IC50 range, 0.32->100 microM), was most potent against simian retrovirus types 1 and 2 (SRV-1, SRV-2) and HIV-1, but did not inhibit foamy viruses and MLV-A. Additionally, there were differences in the concentration of drug required to inhibit closely related viruses. Taken together, these data suggest that NRTIs have a wide spectrum of antiretroviral activity and the activity of compounds, even against closely related retroviruses, cannot be predicted.
Assuntos
Antivirais/farmacologia , Nucleosídeos/farmacologia , Retroviridae/efeitos dos fármacos , Inibidores da Transcriptase Reversa/farmacologia , Animais , Antivirais/toxicidade , Linhagem Celular , Cricetinae , Didanosina/farmacologia , Didanosina/toxicidade , Relação Dose-Resposta a Droga , Humanos , Concentração Inibidora 50 , Lamivudina/farmacologia , Lamivudina/toxicidade , Vison , Nucleosídeos/toxicidade , Inibidores da Transcriptase Reversa/toxicidade , Estavudina/farmacologia , Estavudina/toxicidade , Especificidade por Substrato , Zalcitabina/farmacologia , Zalcitabina/toxicidade , Zidovudina/farmacologia , Zidovudina/toxicidadeRESUMO
Investigation of the biological activity of viruses in vitro necessitates some means of identifying their presence within cells and assaying their activity. Since virus particles themselves are metabolically inert, their detection and quantitation is dependent on their cellular effects or on synthesis of viral antigens, enzymes, and the like. Virus-induced cell fusion is a cytopathic cellular response easily recognizable by microscopic examination, which has been exploited to considerable advantage in virology, as will be briefly discussed.
RESUMO
This study set out to investigate whether plasma mannose-binding protein (MBP) deficiency caused by mutations in the MBP gene associates with pyogenic or opportunistic infections in HIV-infected patients. Plasma samples were selected randomly from 131 HIV-infected patients followed prospectively for a period not exceeding 12 months or until death. Plasma MBP concentrations were measured by an ELISA and genotyping was determined by amplification of exon 1 of the MBP gene by polymerase chain reaction (PCR) technology, followed by restriction enzyme analysis and Southern blotting using sequence-specific oligonucleotide probes. Neither MBP concentration nor genotype was found to associate with disease progression or opportunistic infection rate. There was an unexpected increased bacterial infection rate in patients with MBP levels greater than 100 ng/ml and wild type genotype. Thus, MBP does not appear to play a role in HIV infection. MBP is an acute phase reactant and this may explain the higher levels in those with more frequent pyogenic infections.