RESUMO
BACKGROUND: Opportunities to improve emergency surgery outcomes exist through guided better practice and reduced variability. Few attempts have been made to define optimal care in emergency surgery, and few clinically derived key performance indicators (KPIs) have been published. A summit was therefore convened to look at resources for optimal care of emergency surgery. The aim of the Donegal Summit was to set a platform in place to develop guidelines and KPIs in emergency surgery. METHODS: The project had multidisciplinary global involvement in producing consensus statements regarding emergency surgery care in key areas, and to assess feasibility of producing KPIs that could be used to monitor process and outcome of care in the future. RESULTS: Forty-four key opinion leaders in emergency surgery, across 7 disciplines from 17 countries, composed evidence-based position papers on 14 key areas of emergency surgery and 112 KPIs in 20 acute conditions or emergency systems. CONCLUSIONS: The summit was successful in achieving position papers and KPIs in emergency surgery. While position papers were limited by non-graded evidence and non-validated KPIs, the process set a foundation for the future advancement of emergency surgery.
Assuntos
Lesões Encefálicas Traumáticas/cirurgia , Pediatria/métodos , Acidentes por Quedas/mortalidade , Acidentes por Quedas/estatística & dados numéricos , Acidentes de Trânsito/mortalidade , Acidentes de Trânsito/estatística & dados numéricos , Adolescente , Mundo Árabe , Lesões Encefálicas Traumáticas/epidemiologia , Criança , Pré-Escolar , Técnica Delphi , Feminino , Humanos , Lactente , Masculino , Oriente Médio/epidemiologia , Pediatria/tendências , Estudos Retrospectivos , Centros de Traumatologia/organização & administração , Centros de Traumatologia/estatística & dados numéricos , Resultado do TratamentoRESUMO
Preventing virally induced liver disease begins with an understanding of the host factors that define susceptibility to infection. Hepatitis C virus (HCV) is a global health issue, with an estimated 170 million infected individuals at risk of developing liver disease including fibrosis and hepatocellular carcinoma. The liver is the major reservoir supporting HCV replication and this hepatocellular tropism is defined by HCV engagement of cellular entry receptors. Hepatocytes are polarized in vivo and this barrier function limits HCV entry. We previously reported that activated macrophages promote HCV entry into polarized hepatocytes via a TNF-α-dependent process; however, the underlying mechanism was not defined. In this study, we show that several TNF superfamily members, including TNF-α, TNF-ß, TWEAK and LIGHT, promote HCV entry via NF-κB-mediated activation of myosin light chain kinase (MLCK) and disruption of tight junctions. These observations support a model where HCV hijacks an inflammatory immune response to stimulate infection and uncovers a role for NF-κB-MLCK signalling in maintaining hepatocellular tight junctions.
Assuntos
Hepacivirus/fisiologia , Hepatite C/virologia , Fígado/virologia , Quinase de Cadeia Leve de Miosina/metabolismo , NF-kappa B/metabolismo , Fatores de Necrose Tumoral/metabolismo , Internalização do Vírus , Carcinoma Hepatocelular/virologia , Ativação Enzimática , Hepatite C/metabolismo , Hepatócitos/virologia , Humanos , Fígado/metabolismo , Cirrose Hepática/virologia , Neoplasias Hepáticas/virologia , Transdução de Sinais , Junções Íntimas/metabolismo , Junções Íntimas/virologia , Fator de Transcrição RelA/metabolismoRESUMO
BACKGROUND: Undergoing an endoscopy is a stressful experience for patients. AIMS: To audit the endoscopy pathway to improve patient satisfaction. METHODS: A prospective survey of endoscopy patients to identify system improvements that were then implemented. RESULTS: The survey was performed before (N = 71) and after (N = 60) process improvements identified by the initial survey. Information provision and staff communication skills were identified for optimisation. Patient anxiety at home was significantly reduced (median 2 vs. 1, p < 0.01). Education of endoscopy staff significantly improved the quality of information provided before and after the procedure with regard to sedation (median 4 vs. 5, p < 0.01), discomfort (median 4 vs. 5, p < 0.01), complications (28 vs. 82 %, p < 0.01), findings (89 vs. 100 %, p < 0.01) and follow-up (73 vs. 90 %, p = 0.015). Gloucester Comfort Scores during endoscopy improved (median 1 vs. 0, p < 0.01) without increasing sedation levels. Patient feelings of invasion/trauma significantly decreased. Overall 95 % of patients were satisfied. CONCLUSION: Structured information leaflets and improved staff communication skills reduce anxiety and enhance patients' experiences. They are now standard operating procedures.
Assuntos
Ansiedade/prevenção & controle , Ansiedade/psicologia , Endoscopia Gastrointestinal/psicologia , Educação em Saúde/estatística & dados numéricos , Satisfação do Paciente , Adulto , Idoso , Colonoscopia/psicologia , Colonoscopia/estatística & dados numéricos , Endoscopia Gastrointestinal/estatística & dados numéricos , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Medição da Dor , Estudos ProspectivosRESUMO
Hepatitis B virus (HBV) is one of the world's major unconquered infections, resulting in progressive liver disease, and current treatments rarely cure infection. A limitation to discovering new therapies is our limited knowledge of HBV entry and dissemination pathways that hinders the development of in vitro culture systems. To address this gap in our understanding we optimized the genesis of infectious lentiviral pseudoparticles (HBVpps). The recent discovery that the bile salt transporter sodium taurocholate co-transporting polypeptide (NTCP) acts as a receptor for HBV enabled us to assess the receptor dependency of HBVpp infection. HBVpps preferentially infect hepatoma cells expressing NTCP, whereas other non-liver cells engineered to express NTCP do not support infection, suggesting that additional hepatocyte-specific factors are required for HBVpp internalization. These results highlight the value of the HBVpp system to dissect the pathways of HBV entry and dissemination.
Assuntos
Vírus da Hepatite B/fisiologia , Hepatócitos/virologia , Interações Hospedeiro-Patógeno , Lentivirus/fisiologia , Transportadores de Ânions Orgânicos Dependentes de Sódio/metabolismo , Receptores Virais/metabolismo , Simportadores/metabolismo , Internalização do Vírus , Linhagem Celular , Vírus da Hepatite B/genética , Humanos , Lentivirus/genéticaRESUMO
INTRODUCTION: The aim of this study is to determine if simulated 3D vision improves the speed and accuracy of laparoscopic phantom tasks in laparoscopically naïve subjects. METHODS: Thirty laparoscopically naïve subjects were divided into matched groups according to age, sex, hand dominance and initial scores on a standardised visio-spatial test. Laprotrain(©) laparoscopic simulators were used, one attached to the standard 2D monitor and the other to a simulated 3D monitor and 3D glasses were worn by the subjects in this group. Five standardised laparoscopic tasks were developed and the subjects underwent testing on four separate occasions with more than 24 h between sessions. The subjects were timed for each task and errors were recorded by two independent observers. In the second part of the study, subjects switched to the opposite group and task times and errors were again recorded. Statistical differences between groups were calculated using student t-test and Fisher's exact test. RESULTS: There were fifteen subjects in each group with no significant difference in demographic or psychometric variables. The mean time to complete the tasks was faster in the 3D group compared with the 2D group. There was a lower rate of errors noted in the 3D group compared with the 2D group but this only reached statistical significance in two of the five laparoscopic tasks. In the crossover study, subjects who had trained on simulated 3D had better task times and fewer errors compared to those who had trained on 2D simulators. DISCUSSION & CONCLUSION: Training on a simulated 3D model (compared to standard 2D) allows trainees to reach proficiency sooner.
Assuntos
Competência Clínica , Percepção de Profundidade , Laparoscopia/educação , Desempenho Psicomotor , Adulto , Estudos Controlados Antes e Depois , Estudos Cross-Over , Feminino , Humanos , Imageamento Tridimensional , Laparoscopia/métodos , Masculino , Análise e Desempenho de Tarefas , Adulto JovemRESUMO
Many viruses target the polarized epithelial apex during host invasion. In contrast, hepatitis C virus (HCV) engages receptors at the basal surface of hepatocytes in the polarized liver parenchyma. Hepatocyte polarization limits HCV entry by undefined mechanism(s). Given the recent reports highlighting a role for receptor mobility in pathogen entry, we studied the effect(s) of hepatocyte polarization on viral receptor and HCV pseudoparticle (HCVpp) dynamics using real-time fluorescence recovery after photobleaching and single particle tracking. Hepatoma polarization reduced CD81 and HCVpp dynamics at the basal membrane. Since cell polarization is accompanied by changes in the actin cytoskeleton and CD81 links to actin via its C-terminus, we studied the dynamics of a mutant CD81 lacking a C-terminal tail (CD81(ΔC)) and its effect(s) on HCVpp mobility and infection. CD81(ΔC) showed an increased frequency of confined trajectories and a reduction of Brownian diffusing molecules compared to wild-type protein in non-polarized cells. However, these changes were notobserved in polarized cells. HCVpp showed a significant reduction in Brownian diffusion and infection of CD81(ΔC) expressing non-polarized cells. In summary, these data highlight the dynamic nature of CD81 and demonstrate a role for CD81 lateral diffusion to regulate HCV infection in a polarization-dependent manner.
Assuntos
Polaridade Celular , Hepacivirus/fisiologia , Hepatócitos/fisiologia , Receptores Virais/metabolismo , Tetraspanina 28/metabolismo , Internalização do Vírus , Células Hep G2 , Hepatócitos/imunologia , Hepatócitos/virologia , Humanos , Microscopia de FluorescênciaRESUMO
Laparoscopic appendicectomy has become standard in the treatment of acute appendicitis in most hospitals in Ireland. Studies have shown that it is a safe procedure for trainees to perform. However, these studies were conducted in university teaching hospitals whereas a significant proportion of training in Ireland takes place in peripheral hospitals which provide a different training environment. The aim of this study was to determine whether laparoscopic appendicectomy is a safe procedure for surgical trainees to perform in a peripheral hospital setting. A retrospective analysis was performed of appendicectomies carried out at a peripheral hospital over a 12 month period. Comparisons were made between consultant surgeons and trainees for a variety of outcomes. Of 155 appendicectomies, 129 (83.2%) were performed laparoscopically, of which 10 (7.75%) were converted to open. Consultants performed 99 (77%) laparoscopic appendicectomies. There were no statistically significant differences between consultants and trainees in complication rates (19 (19.2%) vs. 4 (13.3%), p = 0.46), mean length of hospital stay (4.7 +/- 4.0 vs. 3.4 +/- 3.3 days, p = 0.13), or rate of conversion to open operation (9 (9.1%) vs. 1 (3.3%), p = 0.45). For cases of complicated appendicitis there were no significant differences between consultants and trainees in complication rates (12 vs. 2, p = 0.40) or length of hospital stay (6.4 +/- 3.9 vs. 4.7 +/- 5.6 days, p = 0.27). We conclude that laparoscopic appendicectomy is a safe procedure for trainees to perform in the peripheral hospital setting and should be incorporated into surgical training programs at an early stage of training.
Assuntos
Apendicectomia/métodos , Cirurgia Geral/educação , Internato e Residência , Laparoscopia/educação , Adulto , Apendicectomia/estatística & dados numéricos , Feminino , Humanos , Irlanda , Laparoscopia/estatística & dados numéricos , Tempo de Internação/estatística & dados numéricos , Masculino , Complicações Pós-Operatórias , Estudos RetrospectivosRESUMO
INTRODUCTION: The management of gastric lymphoma is controversial and a wide variety of unimodality or multimodality approaches have been used. The aim of this report is to highlight the variety of treatment regimens deployed, the outcomes achieved and to present a modern management approach for this enigmatic tumour. RESULTS: 42 cases of primary gastric lymphoma managed at one centre over a 15-year period were reviewed. Weight loss (52%), pain (41%) and anorexia (33%) were the most common presenting symptoms. Most patients (86%) had high-grade lymphoma. Primary treatment modalities included surgery (36%), chemotherapy (40%), supportive care only (22%) and H. pylori eradication (2%). Adjuvant therapies included chemotherapy (17%), radiotherapy (7%) and combined chemoradiotherapy (5%). The overall median survival was 53 months, with a five year survival of 46%. In the curative group, the median survival was 75 months and five year survival 58%. Curative surgery or chemotherapy +/- radiotherapy were similarly effective for stage IE and IIE disease. CONCLUSIONS: The prognosis for gastric lymphoma is grade- and stage-dependent. With equivalent outcomes for cure in localised gastric lymphoma for surgery and chemotherapy, the latter is preferred in this unit because of gastric preservation, with surgery being reserved for failed medical management or presentations with haemorrhage, perforation or obstruction refractive to steroid therapy.
Assuntos
Linfoma/terapia , Neoplasias Gástricas/terapia , Adulto , Idoso , Idoso de 80 Anos ou mais , Terapia Combinada , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Cuidados Paliativos , Taxa de Sobrevida , Resultado do TratamentoRESUMO
A second estrogen receptor, estrogen receptor-beta, was identified in 1996 and has led to an intensive re-evaluation of the role of estrogens in normal physiological and disease processes. While much has been learnt about this new receptor, there remain many outstanding questions, particularly regarding its prognostic significance and therapeutic implications.
Assuntos
Neoplasias da Mama/fisiopatologia , Receptor beta de Estrogênio/fisiologia , Neoplasias da Mama/terapia , Receptor beta de Estrogênio/genética , Feminino , Humanos , Prognóstico , Resultado do TratamentoRESUMO
We recently reported that retroviral pseudotypes bearing the hepatitis C virus (HCV) strain H and Con1 glycoproteins, genotype 1a and 1b, respectively, require CD81 as a coreceptor for virus-cell entry and infection. Soluble truncated E2 cloned from a number of diverse HCV genotypes fail to interact with CD81, suggesting that viruses of diverse origin may utilize different receptors and display altered cell tropism. We have used the pseudotyping system to study the tropism of viruses bearing diverse HCV glycoproteins. Viruses bearing these glycoproteins showed a 150-fold range in infectivity for hepatoma cells and failed to infect lymphoid cells. The level of glycoprotein incorporation into particles varied considerably between strains, generally reflecting the E2 expression level within transfected cells. However, differences in glycoprotein incorporation were not associated with virus infectivity, suggesting that infectivity is not limited by the absolute level of glycoprotein. All HCV pseudotypes failed to infect HepG2 cells and yet infected the same cells after transduction to express human CD81, confirming the critical role of CD81 in HCV infection. Interestingly, these HCV pseudotypes differed in their ability to infect HepG2 cells expressing a panel of CD81 variants, suggesting subtle differences in the interaction of CD81 residues with diverse viral glycoproteins. Our current model of HCV infection suggests that CD81, together with additional unknown liver specific receptor(s), mediate the virus-cell entry process.
Assuntos
Antígenos CD/metabolismo , Hepacivirus/classificação , Hepacivirus/patogenicidade , Hepatócitos/virologia , Proteínas do Envelope Viral/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Linhagem Celular Tumoral , Células Cultivadas , HIV-1/genética , HIV-1/metabolismo , Hepacivirus/genética , Hepacivirus/fisiologia , Humanos , Fígado , Linfócitos/virologia , Camundongos , Dados de Sequência Molecular , Especificidade de Órgãos , Tetraspanina 28 , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/genéticaRESUMO
BACKGROUND: The identification of a second estrogen receptor (ER-beta) has significant implications for therapeutic strategy in breast cancer management arising from the potential agonist effect of Tamoxifen at estrogen receptor sites and as such, antiestrogen therapy may be inappropriate in patients with a dominance of ER-beta. METHODS: To determine the proportion of breast cancer patients who may be so at risk, we developed a novel multiplexed RT-PCR technique to establish the relative ER-alpha and ER-beta levels in 53 primary breast cancers, 11 normal breast tissues and six cell lines. We further assessed the prognostic significance of receptor status relative to the Nottingham prognostic index (NPI). The ER-alpha and ER-beta status was also determined by immunohistochemistry using previously published and 'in-house' scoring systems. RESULTS: Using RT-PCR analysis, 46 tumours were hormone receptor positive (ER+) with 42 displaying ER-alpha predominance. Comparison with immunohistochemistry demonstrated 44/53 (ER-alpha) and 27/50 (ER-beta) concordance rates. There was no significant difference in the NPI between ER-alpha and ER-beta predominant cohorts or between ER+ and ER- cohorts. CONCLUSION: This study identifies the existence of a subgroup of ER+ patients in whom Tamoxifen therapy may be inappropriate and has significant implications for adjuvant therapy of primary breast cancer.
Assuntos
Biomarcadores Tumorais/genética , Neoplasias da Mama/genética , Carcinoma Ductal de Mama/genética , Carcinoma Lobular/genética , Receptores de Estrogênio/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias da Mama/epidemiologia , Carcinoma Ductal de Mama/epidemiologia , Carcinoma Lobular/epidemiologia , Receptor alfa de Estrogênio , Receptor beta de Estrogênio , Feminino , Perfilação da Expressão Gênica , Humanos , Imuno-Histoquímica , Pessoa de Meia-Idade , Estudos Prospectivos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Risco , Saúde da MulherRESUMO
Dendritic cells (DC) are required to initiate primary cellular immune responses. Human immunodeficiency virus type 1 (HIV-1) infection of DC may be central to transmission and persistence of virus and in the pathogenesis of AIDS. In symptomatic HIV-1-infected patients the proportion of DC in the mononuclear cell population was reduced. Provirus load in the T cells was 3-100 times higher than in DC and there was no correlation between the levels of infection in the two cell types. Phylogenetic analysis of amino acids in the V3 loop and flanking regions indicated intermingling of sequences and thus provides the first evidence for transfer of virus between DC and T cells in vivo. In one of three patients analysed there were significant differences in amino acid residues in the V3 region. This may reflect reduced interactions between DC and T cells in infected individuals and for the existence of variants with a stronger tropism for DC, which could play a role in transmission by initiating infection in mucosal DC.
Assuntos
Síndrome da Imunodeficiência Adquirida/imunologia , Síndrome da Imunodeficiência Adquirida/virologia , Células Dendríticas/virologia , Produtos do Gene env/genética , Variação Genética , HIV-1/genética , Linfócitos T/virologia , Sequência de Aminoácidos , Primers do DNA , Evolução Molecular , Citometria de Fluxo , Produtos do Gene env/química , HIV-1/classificação , HIV-1/patogenicidade , Humanos , Dados de Sequência Molecular , Filogenia , Reação em Cadeia da Polimerase , Provírus/genética , Valores de Referência , Alinhamento de Sequência , Linfócitos T Auxiliares-Indutores/virologiaRESUMO
The aim of this study was to identify the HIV types and subtypes prevalent in Israel among different populations in terms of risk or geographic origin of the HIV infection. A total of 149 blood samples were collected from HIV-positive persons from different risk groups for HIV infection who were living in Israel. HIV subtyping was performed by a V3-based peptide enzyme immunoassay, supplemented by direct sequencing of polymerase chain reaction products from the V3 region. Multiple HIV-1 subtypes were shown to circulate in Israel; whereas most of the infections among Israelis and Palestinians were of subtype B, infections among the large Ethiopian population in Israel were caused by HIV-1 subtype C. Occasionally, we found HIV-1 subtypes A and D and a putative B/C recombinant. No HIV-2 infection was identified. Sequence comparisons and phylogenetic tree analyses point at multiple introductions of HIV into the country. The presence of mainly two different HIV-1 subtypes, B and C, in two separated populations in Israel may result in two distinct epidemiologic patterns among HIV-infected individuals in Israel. Subtype C infection among the Ethiopians in Israel opens new research avenues toward better understanding the natural history of infection with HIV-1 subtype C in Ethiopians living in a Western society compared with those living in Ethiopia.
Assuntos
Infecções por HIV/epidemiologia , HIV/classificação , Sequência de Aminoácidos , Etiópia/epidemiologia , Genótipo , HIV/genética , Infecções por HIV/virologia , Humanos , Técnicas Imunoenzimáticas , Israel/epidemiologia , Oriente Médio/epidemiologia , Dados de Sequência Molecular , SorotipagemRESUMO
We constructed a number of HXB2 viruses chimeric for the gp 120 glycoprotein derived from a number of viable molecular clones obtained from a primary isolate. Comparative biological characterization of the parental primary viruses with the gp 120.HXB2 chimeras demonstrated identical patterns of cell tropism and cytopathicity. Furthermore, both parental and chimeric viruses were insensitive to neutralization by sCD4 and a panel of conformation-dependent monoclonal antibodies, demonstrating that transfer of the gp 120 protein alone was sufficient to confer a "neutralization-resistant" phenotype to the T-cell-adapted clone HXB2. We assessed the contribution of gp 120 epitopes to the neutralizing immune response by comparing the sensitivity of these viruses to neutralization by a panel of sera from HIV-infected individuals. Seven of eleven sera tested were able to neutralize HXB2 and two or more of the chimeric viruses; in contrast, only one serum neutralized more than one of the parental primary virus clones. The association of gp 120-gp41 envelope at the surface of infected PBMC cultures was measured in the presence or absence of soluble CD4. No differences in CD4-induced gp 120 dissociation were seen between the chimeric and parental virus-infected cultures. Since gp 120 conformation appeared the same between primary and chimeric viruses, we suggest that the ability of human sera to neutralize the chimeric viruses may be mediated by epitopes within gp41.
Assuntos
Anticorpos Anti-HIV/imunologia , Proteína gp120 do Envelope de HIV/imunologia , HIV-1/imunologia , Vírus Reordenados/imunologia , Sequência de Aminoácidos , Bacteriófago lambda , Sequência de Bases , Antígenos CD4/imunologia , Células Cultivadas , DNA Viral , Proteína gp120 do Envelope de HIV/genética , Proteína gp41 do Envelope de HIV/imunologia , HIV-1/genética , Células HeLa , Humanos , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/virologia , Dados de Sequência Molecular , Testes de Neutralização , Vírus Reordenados/genética , Proteínas Recombinantes/imunologia , Células Tumorais CultivadasRESUMO
Seven infectious molecular clones were obtained from a human immunodeficiency virus type 1 isolate with rapid/high replicative capacity. Biological characterization of progeny viruses obtained after transfection of clones into peripheral blood mononuclear cells showed that six clones yielded virus with restricted cell tropism, whereas one clone yielded virus able to replicate in cell lines. Although transfection of each of the clones 12, 13, and 82 individually gave rise to viruses with restricted tropism, viruses recovered from cotransfection of the mixtures of these clones exhibited altered phenotype, inasmuch as they were able to replicate in cell lines. To test whether recombination and/or complementation has taken place in the mixture of clones 12 + 13 + 82, the progeny virus was diluted to end point in 15 parallel series. Viruses with diverse biological phenotypes were recovered. With the help of distinctive restriction enzyme markers in regions comprising the vpu/env junction and variable regions 4 and 5 (V4/V5) of the env gene, recombinant genotypes could be identified with high frequency. No particular biological phenotype could be linked to a certain genotype in this study. The results show that different coexisting variants may interact and thereby influence the biological phenotype of a viral population.
Assuntos
HIV-1/genética , Recombinação Genética , Transfecção , Sequência de Bases , Linhagem Celular , Células Cultivadas , Clonagem Molecular , DNA Viral , HIV-1/fisiologia , Humanos , Técnicas de Diluição do Indicador , Dados de Sequência Molecular , Monócitos/microbiologia , Fenótipo , Células Tumorais Cultivadas , Replicação Viral/genéticaRESUMO
Direct sequencing of segments of the envelope gene of human immunodeficiency virus type 1 proviruses in peripheral blood mononuclear cells has revealed that a cohort of hemophiliacs who were infected after exposure to a single common batch of factor VIII share closely related virus strains. Seventy-four sequences extending from hypervariable regions V4 through V5 from nine patients yielded a mean intrapatient nucleotide distance of 5.5%, while a mean of 4.2% was observed in 39 sequences of the V3 loop (six patients). Phylogenetic analysis revealed that sequences of six Edinburgh patients were particularly closely related and those from a patient infected in the United States were very distinct. The mean nucleotide distance among these six was 8.3%, while the mean distance from the U.S.-derived sequences was 25.5% in the V4-V5 region. The rate of sequence change across this patient group has been estimated to be 0.4% per year in the V4-V5 region and 0.5% per year in the V3 region, with at least a twofold range across patients. Only two inactivating nucleotide substitutions have been observed in a total of 42 kb of sequence obtained from the env and gag genes during this study.
Assuntos
Evolução Biológica , Genes Virais , Proteína gp120 do Envelope de HIV/genética , HIV-1/genética , Mutação , Proteínas Estruturais Virais/genética , Sequência de Bases , Estudos de Coortes , Fator VIII/uso terapêutico , Variação Genética , Soropositividade para HIV , HIV-1/isolamento & purificação , Hemofilia A/complicações , Hemofilia A/microbiologia , Hemofilia A/terapia , Humanos , Dados de Sequência Molecular , Sondas de Oligonucleotídeos , Filogenia , Reação em Cadeia da Polimerase , Provírus/genética , Provírus/isolamento & purificaçãoRESUMO
The presence of HIV-1 DNA sequences in DNA from peripheral blood mononuclear cells (PBMCs) was investigated in a two-stage polymerase chain reaction ('double' PCR) using four sets of nested primers. The PBMCs tested were obtained from 46 children born to HIV-seropositive mothers, seven 'control' children born to HIV-seronegative mothers and seropositive fathers, and 45 healthy adult blood donors who were HIV seronegative. Nine of the children had symptomatic HIV infection and other laboratory features characteristic of HIV infection: all nine were PCR-positive with each set of primers in each of their 22 blood samples tested. The remaining 44 children had no clinical or laboratory evidence of HIV infection, and each of their 50 samples was PCR-negative with each set of primers, as were all blood donor samples. PCR-positive samples were tested in more detail using two of the sets of primers, which spanned hypervariable regions in the env gene. Polyacrylamide gel electrophoresis of DNA amplified from these regions yielded patterns of amplified DNA length variation which were characteristic for each child, and which changed little with time (in serial samples obtained over periods of 3-7 months). This excluded contamination as a cause of PCR positivity. This is the first report of the use of a double PCR for the diagnosis of HIV infection. The results demonstrate the specificity of this PCR method in diagnosis, with failure to reveal in this cohort any cases of vertically transmitted HIV-1 infection in addition to those already confirmed by conventional laboratory techniques.
Assuntos
DNA Viral/análise , Infecções por HIV/diagnóstico , Complicações Infecciosas na Gravidez , Pré-Escolar , Feminino , Produtos do Gene gag/análise , Proteína do Núcleo p24 do HIV , Infecções por HIV/transmissão , Humanos , Lactente , Reação em Cadeia da Polimerase , Gravidez , Proteínas do Core Viral/análiseRESUMO
In human immunodeficiency virus (HIV)-infected individuals, the proportion of circulating mononuclear cells (PBMCs) which carry HIV provirus and the number of HIV proviral sequences per infected PBMC have been matters for conjecture. Using a double polymerase chain reaction which allows the detection of single molecules of provirus and a method of quantifying the provirus molecules, we have measured provirus frequencies in infected individuals down to a level of one molecule per 10(6) PBMCs. As a general rule, only a small proportion of PBMCs contain provirus (median value of samples from 12 patients, one per 8,000 cells), and most if not all of the infected cells carry a single provirus molecule. The frequency of provirus-carrying cells correlated positively both with the progression of the disease and with the success with which virus could be isolated from the same patients by cocultivation methods. Of seven asymptomatic (Centers for Disease Control stage II) patients, all but one contained one provirus molecule per 6,000 to 80,000 cells; of five Centers for Disease Control stage IV patients, all but one contained one provirus molecule per 700 to 3,300 cells. When considered in conjunction with estimates of the frequency of PBMCs that express viral RNA, our results suggest that either (i) the majority of provirus-containing cells are monocytes or (ii) most provirus-containing lymphocytes are transcriptionally inactive. We also present nucleotide sequence data derived directly from provirus present in vivo which we show is not marred by the in vitro selection of potential virus variants or by errors introduced by Taq polymerase. We argue from these data that, of the provirus present in infected individuals, the proportion which is defective is not high in the regions sequenced.