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1.
Int J Lab Hematol ; 40(4): 442-447, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-29573340

RESUMO

INTRODUCTION: The direct thrombin inhibitor dabigatran interferes with thrombophilia screening and with the diagnosis of hemostasis disorders that develop during treatment with the anticoagulant. In vitro addition of idarucizumab, a humanized antibody fragment that binds dabigatran, to plasma samples containing dabigatran fully neutralizes the drug. This study was carried out to determine whether binding of dabigatran on selected insoluble commercial adsorbent material, DOAC-STOPR , was as efficient as idarucizumab to neutralize the anticoagulant activity of the drug in vitro. METHODS: Coagulation assays sensitive to dabigatran were carried out with patient and control plasma samples spiked with dabigatran and supplemented with idarucizumab or incubated with adsorbent material. RESULTS: In samples containing upto 10 000 ng/mL dabigatran, the adsorption procedure was at least as efficient as the addition of idarucizumab to neutralize the activity of the anticoagulant drug. Neither the adsorption procedure nor the addition of idarucizumab did impair routine coagulation assays carried out with plasma devoid of dabigatran, such as the activated partial thromboplastin time, prothrombin time, fibrinogen Clauss, and the thrombophilia screening assays used to detect antiphospholipid antibodies or activated protein C resistance. In addition, the adsorption procedure did not interfere with the detection of lupus anticoagulant samples. CONCLUSIONS: Adsorption of dabigatran in plasma samples containing the drug neutralizes its activity as efficiently as the addition of idarucizumab. This method allows the evaluation of thrombophilia markers without interruption of anticoagulation therapy or the detection of hemostasis disorders in patients treated with the drug.


Assuntos
Anticorpos Monoclonais Humanizados/administração & dosagem , Testes de Coagulação Sanguínea/normas , Dabigatrana/isolamento & purificação , Trombofilia/diagnóstico , Adsorção , Anticoagulantes/uso terapêutico , Interações Medicamentosas , Humanos
2.
Haemophilia ; 24(4): 675-682, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-28124445

RESUMO

INTRODUCTION: Postinfusion ReFacto AF levels can be difficult to measure accurately due to discrepancies between one-stage and chromogenic FVIII assays. To overcome this, the use of the ReFacto AF laboratory standard (RAFLS) is recommended, but there are discordant reports regarding its usefulness. AIM: We investigated whether calibration with RAFLS and measurement of ReFacto AF levels are influenced by the choice of reagents and patient-specific factors in one-stage FVIII assays. METHODS: Calibration curves were generated with both the RAFLS and a plasma standard using different F8DPs and one-stage FVIII assay reagents. This selection of reagents was then used to determine FVIII levels in the plasma of patients repeatedly treated with ReFacto AF. Results were compared with those obtained with a chromogenic assay. RESULTS: F8DP devoid of von Willebrand factor (VWF) falsely increased the values of RAFLS pro-coagulant activity generated using the APTT reagent. The resulting RAFLS calibration curve underestimated ReFacto AF levels to be half of their true concentration. The use of RAFLS with F8DP containing VWF reduced the discrepancy observed between the one-stage and chromogenic FVIII assays. However, the mean difference between the two assays still varied up to 50% depending on the patient. CONCLUSIONS: The RAFLS is a suitable calibrator for one-stage FVIII assays carried out with F8DP containing VWF. However, calibration with the RAFLS does not avoid the effect of patient-specific variables that contribute to discrepancies between the measurements of ReFacto AF levels with one-stage and chromogenic FVIII assays.


Assuntos
Testes de Coagulação Sanguínea/normas , Fator VIII/genética , Fator VIII/farmacologia , Deleção de Sequência , Calibragem , Fator VIII/química , Humanos , Indicadores e Reagentes/química , Domínios Proteicos , Padrões de Referência
3.
Hernia ; 21(6): 973-982, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-28752425

RESUMO

PURPOSE: Surgical restoration of soft tissue defects often requires implantable devices. The clinical outcome of the surgery is determined by the properties inherent to the used matrix. Mesenchymal stem cells (MSC) modulate the immune processes after in vivo transplantation and their addition to matrices is associated with constructive remodeling. Herein we evaluate the potential of MSC derived from the amniotic fluid (AF-MSC), an interesting MSC source for cell therapeutic applications in the perinatal period, for immune modulation when added to a biomaterial. METHODS: We implant cell free small intestinal submucosa (SIS) or SIS seeded with AF-MSC at a density of 1 × 105/cm2 subcutaneously at the abdominal wall in immune competent rats. The host immune response is evaluated at 3, 7 and 14 days postoperatively. RESULTS: The matrix-specific or cellular characteristics are not altered after 24 h of in vitro co-culture of SIS with AF-MSC. The host immune response was not different between animals implanted with cell free or AF-MSC-seeded SIS in terms of cellular infiltration, vascularity, macrophage polarization or scaffold replacement. Profiling the mRNA expression level of inflammatory cytokines at the matrix interface shows a significant reduction in the expression of the pro-inflammatory marker Tnf-α and a trend towards lower iNos expression upon AF-MSC-seeding of the SIS matrix. Anti-inflammatory marker expression does not alter upon cell seeding of matrix implants. CONCLUSION: We conclude that SIS is a suitable substrate for in vitro culture of AF-MSC and fibroblasts. AF-MSC addition to SIS does not significantly modulate the host immune response after subcutaneous implantation in rats.


Assuntos
Líquido Amniótico/citologia , Imunidade nas Mucosas/fisiologia , Mucosa Intestinal/fisiologia , Células-Tronco Mesenquimais/fisiologia , Animais , Técnicas de Cultura de Células , Citocinas/metabolismo , Feminino , Fibroblastos/fisiologia , Intestino Delgado , Masculino , Ratos
4.
Int J Lab Hematol ; 39(3): 301-307, 2017 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-28318107

RESUMO

INTRODUCTION: Thrombin time (TT) tests are useful for diagnosing coagulation disorders involving abnormal fibrinogen but do not allow us to distinguish between qualitative and quantitative defects. However, with the widening availability of optical coagulation automates, more information about the coagulation process is becoming increasingly accessible. METHODS: In this study, we compared the coagulation curves of TT tests carried out with plasma from healthy donors with those from patients with acquired low Clauss fibrinogen levels or with dysfibrinogenemia caused by a heterozygous point mutation in the fibrinogen γ-chain that results in a p.Arg301(275)Cys substitution. The functional fibrinogen levels of these three groups of samples were also measured with the Clauss method, and their fibrinogen protein levels were determined by ELISA. RESULTS: Our data indicate that the amplitude and maximal velocity of coagulation curves from plasma samples from FGG p.Arg301(275)Cys dysfibrinogenemic patients were comparable to those from plasma samples with fibrinogen in the normal range, whereas the amplitude of coagulation curves from patients with acquired low fibrinogen levels was lower. CONCLUSIONS: Examination of the amplitude of coagulation curves generated during TT tests may provide additional information to enable the differential diagnoses of diseases following a low fibrinogen measurement by the Clauss method.


Assuntos
Afibrinogenemia/sangue , Afibrinogenemia/genética , Fibrinogênios Anormais/genética , Fibrinogênios Anormais/metabolismo , Mutação de Sentido Incorreto , Substituição de Aminoácidos , Feminino , Humanos , Masculino , Tempo de Trombina/métodos
5.
Ultrasound Obstet Gynecol ; 49(6): 704-713, 2017 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-27312047

RESUMO

OBJECTIVES: To identify antenatal predictors of persistent pulmonary hypertension (PPH) and the need for extracorporeal membrane oxygenation (ECMO) in fetuses with congenital diaphragmatic hernia (CDH). METHODS: We performed a systematic literature review on antenatal diagnostic tests in fetuses with isolated CDH. The primary outcomes assessed were PPH within 28 days of age and the need for ECMO. Quality of studies was assessed with the QUADAS-2 tool. Meta-analysis was performed when at least three studies reported on the same test. Sensitivity analysis was performed according to prenatal management of CDH (tracheal occlusion vs expectant management). RESULTS: Thirty-eight studies met the inclusion criteria. Fifteen reported on the incidence of PPH only, 19 on the need for ECMO only and four reported on both outcomes. The general quality of the studies was moderate; most studies were retrospective (61%) and single-center series (92%). One study included only fetuses undergoing tracheal occlusion, 22 included only fetuses managed expectantly in utero and 15 included both populations. We could not identify antenatal predictors of PPH. The need for ECMO was predicted by parameters indicative of lung size: lung-to-head ratio (LHR) (relative risk (RR) for LHR < 1, 1.65 (95% CI, 1.27-2.14)) and observed/expected LHR (standardized mean difference (SMD), -0.70 (95% CI, -0.98 to -0.42)) measured by ultrasound and observed/expected total lung volume (SMD, -1.00 (95% CI, -1.52 to -0.48)) measured by magnetic resonance imaging. Liver herniation was also associated with an increased risk of need for ECMO (RR, 3.04 (95% CI, 2.23-4.14)). These results were confirmed by a sensitivity analysis of studies that included only expectantly managed cases. Data on vascular assessment for the prediction of PPH could not be pooled as most of the parameters were evaluated in a single series or in different series by the same principal investigator. CONCLUSIONS: In fetuses with CDH, lung size and liver herniation predict the need for ECMO, however a predictor for PPH is still lacking. Further studies aimed at diagnosing impaired vascular development in utero should therefore be undertaken. Copyright © 2016 ISUOG. Published by John Wiley & Sons Ltd.


Assuntos
Hérnias Diafragmáticas Congênitas/fisiopatologia , Hepatopatias/complicações , Pulmão/patologia , Oxigenação por Membrana Extracorpórea , Feminino , Hérnias Diafragmáticas Congênitas/complicações , Hérnias Diafragmáticas Congênitas/diagnóstico por imagem , Hérnias Diafragmáticas Congênitas/terapia , Humanos , Hepatopatias/congênito , Valor Preditivo dos Testes , Gravidez , Índice de Gravidade de Doença , Ultrassonografia Pré-Natal
6.
J Thromb Haemost ; 13(11): 2087-92, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26347330

RESUMO

BACKGROUND: The anticoagulant effect of dabigatran can be approximated by its prolongation of routine coagulation assays. Consequently, dabigatran also interferes with thrombophilia screening or with diagnosing hemostasis disorders that have developed after the initiation of anticoagulant treatment, such as vitamin K deficiency or acquired hemophilia A. OBJECTIVES: This study was carried out to determine whether idarucizumab, a humanized antibody fragment that binds dabigatran, could fully neutralize dabigatran in routine diagnostic coagulation assays conducted in vitro, thereby preventing false-positive or false-negative diagnostic readouts. METHODS: Preliminary experiments identified coagulation assays that were sensitive to dabigatran, and identified a concentration of idarucizumab that neutralized the effects of dabigatran. These assays were then carried out with patient and control plasma samples spiked with dabigatran, with or without a molar excess of idarucizumab. RESULTS: Dabigatran altered the prothrombin time, activated partial thromboplastin time and thrombin time, and the measurement of intrinsic and extrinsic factor levels. Screening and confirmation tests used for lupus anticoagulant detection were prolonged by dabigatran, falsely suggesting the presence of lupus anticoagulant. Conversely, the addition of dabigatran falsely corrected an abnormal activated protein C resistance ratio. Addition of idarucizumab completely normalized these measurements, and allowed the correct identification of normal and abnormal samples with these assays. CONCLUSIONS: In vitro addition of idarucizumab to plasma samples containing dabigatran fully neutralizes the drug, and facilitates the use of routine coagulation assays to allow the diagnosis of hemostasis disorders that may be concurrently present in patients taking dabigatran.


Assuntos
Anticorpos Monoclonais Humanizados/farmacologia , Anticorpos Neutralizantes/farmacologia , Antitrombinas/sangue , Transtornos da Coagulação Sanguínea/sangue , Testes de Coagulação Sanguínea , Dabigatrana/sangue , Resistência à Proteína C Ativada/sangue , Anticorpos Monoclonais Humanizados/imunologia , Anticorpos Neutralizantes/imunologia , Especificidade de Anticorpos , Antitrombinas/imunologia , Antitrombinas/farmacologia , Transtornos da Coagulação Sanguínea/tratamento farmacológico , Dabigatrana/antagonistas & inibidores , Dabigatrana/imunologia , Dabigatrana/farmacologia , Relação Dose-Resposta Imunológica , Reações Falso-Negativas , Reações Falso-Positivas , Hemofilia A/sangue , Humanos , Inibidor de Coagulação do Lúpus/sangue
7.
Eur J Pediatr ; 172(6): 763-7, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23371429

RESUMO

Phalangeal microgeodic syndrome is a rare but benign disorder that affects the fingers of children. This condition was originally described by Maroteaux in 1970. We present two patients who consulted a pediatrician with swelling of the digits of one or both hands. Both lacked additional clinical or biochemical signs. Radiological examination showed multiple small osteolytic areas with sclerotic lining and periostal reactions in the phalanges of the affected hands. These cases were treated with a conservative approach and spontaneous resolution occurred within weeks to months. As it is a rare disease, the clinical presentation can be misinterpreted as an infectious, inflammatory, or even malignant condition and prompts clinicians to expand the diagnostic process with radiological or nuclear imaging and even biopsy. In these patients, a timely clinical diagnosis by a physician who is aware of the disease prevented further investigations.


Assuntos
Acro-Osteólise/diagnóstico , Falanges dos Dedos da Mão , Pré-Escolar , Falanges dos Dedos da Mão/diagnóstico por imagem , Falanges dos Dedos da Mão/patologia , Humanos , Lactente , Masculino , Radiografia , Remissão Espontânea , Esclerose/diagnóstico , Síndrome
8.
Haemophilia ; 19(3): e133-8, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23374099

RESUMO

The assessment of recombinant FVIII (rFVIII) activity (FVIII:C) in plasma of patients is dependent on the assay. Notably, a calibration with a product-specific laboratory standard is recommended when measuring Refacto-AF(R) activity in plasma with a one-stage assay. The objective of this study was to facilitate the measurement of rFVIII, taking into account the recent demonstration that a calibration curve does not have to be included in each run. FVIII:C was measured in patients' samples after infusion of different types of rFVIII with a one-stage and a chromogenic assay calibrated either with pooled normal plasma or a product-specific laboratory standard. Results obtained with the one-stage coagulation assay were compared with these provided by a chromogenic assay. We confirmed that a calibration curve can be used for a prolonged period of time without loss of precision and accuracy. In such conditions, a stable relation between the calibration curves generated with a product-specific laboratory standard and plasma can be established. In patients' plasma, Refacto-AF levels measured with a one-stage FVIII assay calibrated with plasma or a product-specific laboratory standard diverged from -58% to -17% and from -25% to +18%, respectively, from the activity determined with a chromogenic substrate assay. By comparison, FVIII:C levels of full-length rFVIII measured with the one-stage assay calibrated with plasma were 6-49% lower than with the chromogenic assay. In a monocentric setting, the long-term stability of the calibration curves allows the implementation of a practical and cost-effective approach to determine rFVIII:C levels.


Assuntos
Ensaios Enzimáticos , Fator VIII/análise , Proteínas Recombinantes/análise , Calibragem , Compostos Cromogênicos/química , Compostos Cromogênicos/metabolismo , Ensaios Enzimáticos/normas , Fator VIII/biossíntese , Fator VIII/normas , Hemofilia A/sangue , Humanos , Laboratórios , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/normas , Padrões de Referência
9.
Cell Transplant ; 21(9): 1945-67, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22513051

RESUMO

Among the animal models of Duchenne muscular dystrophy (DMD), the Golden Retriever muscular dystrophy (GRMD) dog is considered the best model in terms of size and pathological onset of the disease. As in human patients presenting with DMD or Becker muscular dystrophies (BMD), the GRMD is related to a spontaneous X-linked mutation of dystrophin and is characterized by myocardial lesions. In this respect, GRMD is a useful model to explore cardiac pathogenesis and for the development of therapeutic protocols. To investigate whether cardiac progenitor cells (CPCs) isolated from healthy and GRMD dogs may differentiate into myocardial cell types and to test the feasibility of cell therapy for cardiomyopathies in a preclinical model of DMD, CPCs were isolated from cardiac biopsies of healthy and GRMD dogs. Gene profile analysis revealed an active cardiac transcription network in both healthy and GRMD CPCs. However, GRMD CPCs showed impaired self-renewal and cardiac differentiation. Population doubling and telomerase analyses highlighted earlier senescence and proliferation impairment in progenitors isolated from GRMD cardiac biopsies. Immunofluorescence analysis revealed that only wt CPCs showed efficient although not terminal cardiac differentiation, consistent with the upregulation of cardiac-specific proteins and microRNAs. Thus, the pathological condition adversely influences the cardiomyogenic differentiation potential of cardiac progenitors. Using PiggyBac transposon technology we marked CPCs for nuclear dsRed expression, providing a stable nonviral gene marking method for in vivo tracing of CPCs. Xenotransplantation experiments in neonatal immunodeficient mice revealed a valuable contribution of CPCs to cardiomyogenesis with homing differences between wt and dystrophic progenitors. These results suggest that cardiac degeneration in dystrophinopathies may account for the progressive exhaustion of local cardiac progenitors and shed light on cardiac stemness in physiological and pathological conditions. Furthermore, we provide essential information that canine CPCs may be used to alleviate cardiac involvement in a large preclinical model of DMD.


Assuntos
Distrofia Muscular Animal/patologia , Miocárdio/citologia , Miocárdio/patologia , Células-Tronco/patologia , Animais , Diferenciação Celular/fisiologia , Linhagem da Célula , Modelos Animais de Doenças , Cães , Feminino , Citometria de Fluxo , Humanos , Masculino , Camundongos , Camundongos SCID , Distrofia Muscular Animal/genética , Distrofia Muscular Animal/metabolismo , Miocárdio/metabolismo , Ratos , Células-Tronco/metabolismo , Transcriptoma
10.
Gene Ther ; 18(5): 517-27, 2011 May.
Artigo em Inglês | MEDLINE | ID: mdl-21326331

RESUMO

Dysfunction of the nigrostriatal system is the major cause of Parkinson's disease (PD). This brain region is therefore an important target for gene delivery aiming at disease modeling and gene therapy. Recombinant adeno-associated viral (rAAV) vectors have been developed as efficient vehicles for gene transfer into the central nervous system. Recently, several serotypes have been described, with varying tropism for brain transduction. In light of the further development of a viral vector-mediated rat model for PD, we performed a comprehensive comparison of the transduction and tropism for dopaminergic neurons (DNs) in the adult Wistar rat substantia nigra (SN) of seven rAAV vector serotypes (rAAV 2/1, 2/2, 2/5, 2/6.2, 2/7, 2/8 and 2/9). All vectors were normalized by titer and volume, and stereotactically injected into the SN. Gene expression was assessed non-invasively and quantitatively in vivo by bioluminescence imaging at 2 and 5 weeks after injection, and was found to be stable over time. Immunohistochemistry at 6 weeks following injection revealed the most widespread enhanced green fluorescence protein expression and the highest number of positive nigral cells using rAAV 2/7, 2/9 and 2/1. The area transduced by rAAV 2/8 was smaller, but nevertheless almost equal numbers of nigral cells were targeted. Detailed confocal analysis revealed that serotype 2/7, 2/9, 2/1 and 2/8 transduced at least 70% of the DNs. In conclusion, these results show that various rAAV serotypes efficiently transduce nigral DNs, but significant differences in transgene expression pattern and level were observed.


Assuntos
Dependovirus/genética , Dopamina/metabolismo , Proteínas de Fluorescência Verde/genética , Substância Negra/metabolismo , Transdução Genética , Animais , Vetores Genéticos , Proteínas de Fluorescência Verde/metabolismo , Ratos , Ratos Wistar , Sorotipagem , Substância Negra/citologia
11.
Gene Ther ; 18(6): 594-605, 2011 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-21346786

RESUMO

The development of in vivo imaging protocols to reliably track transplanted cells or to report on gene expression is critical for treatment monitoring in (pre)clinical cell and gene therapy protocols. Therefore, we evaluated the potential of lentiviral vectors (LVs) and adeno-associated viral vectors (AAVs) to express the magnetic resonance imaging (MRI) reporter gene ferritin in the rodent brain. First, we compared the induction of background MRI contrast for both vector systems in immune-deficient and immune-competent mice. LV injection resulted in hypointense (that is, dark) changes of T(2)/T(2)(*) (spin-spin relaxation time)-weighted MRI contrast at the injection site, which can be partially explained by an inflammatory response against the vector injection. In contrast to LVs, AAV injection resulted in reduced background contrast. Moreover, AAV-mediated ferritin overexpression resulted in significantly enhanced contrast to background on T(2)(*)-weighted MRI. Although sensitivity associated with the ferritin reporter remains modest, AAVs seem to be the most promising vector system for in vivo MRI reporter gene imaging.


Assuntos
Encéfalo/metabolismo , Dependovirus/genética , Ferritinas/genética , Genes Reporter , Vetores Genéticos , Lentivirus/genética , Imageamento por Ressonância Magnética , Animais , Linhagem Celular , Ferritinas/metabolismo , Técnicas de Transferência de Genes , Humanos , Camundongos , Imagem Molecular , Sensibilidade e Especificidade
12.
Facts Views Vis Obgyn ; 3(1): 22-35, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-24753844

RESUMO

The widespread use of prenatal ultrasound has made the fetus a patient. A number of conditions diagnosed as such may require therapy prior to birth. Herein we describe past, current and potential future procedures designed to treat pulmonary conditions in the antenatal period. When congenital cystic adenomatoid malformation (CCAM) is -associated with fetal hydrops, treatment is required. Prior to viability this may be in utero resection of the pathologic lung lobe or shunting of cystic lesions. More recently, fetuses with isolated congenital diaphragmatic hernia (CDH) with lethal lung hypoplasia have been offered percutaneous fetal tracheal occlusion to provoke lung growth. A very rare condition is laryngeal atresia, which requires peripartum re-establishment of the airways. As we get more -experience with access to the fetal airways, this may open the doors for novel therapies. One of these is gene delivery to treat fetuses with serious monogenic disorders or to induce transient overexpression of certain proteins. We review the individual hurdles that are being met by researchers when designing fetal gene therapeutic strategies, in particular for the fetal lung. Also the use of stem cells for pulmonary disorders is currently explored.

13.
Facts Views Vis Obgyn ; 3(3): 221-7, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-24753868

RESUMO

The pregnant patient is a vulnerable subject, and even more so when a serious fetal condition is diagnosed. (Invasive) fetal therapy should only be offered when there is a good chance that the life of the fetus will be saved, or irreversible damage by the disease or disability is prevented. Following diagnosis of a potentially treatable condition, the patient needs to be referred to a center with sufficient expertise in diagnosis and all therapeutic options. Preferences of the physician towards one or another antenatal intervention is not at stake prior to that moment. When fetal therapy is justified--, it should be offered with full respect for maternal choice and individual assessment and perception of potential-- risks, and should be at the location where there is sufficient expertise. For therapies of unproven benefit, the absence of evidence must be disclosed, and therapy should only be undertaken with full voluntary consent of the mother. These ought to be undertaken within well designed and approved trials and only by experts in the treatment modality. Potential risks and eventual morbidities in case of therapeutic failure should be part of the counselling, neither-- should fetal therapy be presented as an alternative to termination of pregnancy.

14.
Nucleic Acids Res ; 27(10): 2202-10, 1999 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-10219094

RESUMO

Integration of the human immunodeficiency virus type 1 (HIV-1) cDNA into the genome of a human cell is an essential step in the viral replication cycle. Understanding of the integration process has been facilitated by the development of in vitro assays using specific oligonucleotides and recombinant integrase. However, understanding of the biology of retroviral integration will require in vitro and in vivo model systems using long DNA substrates that mimic the HIV cDNA. We have now studied the activity of recombinant HIV-1 integrase on a linear 4.7 kb double-stranded DNA, containing flanking regions of approximately 200 bp that represent the intact ends of the HIV-1 long terminal repeat (LTR) sequences (mini-HIV). The strand transfer products of the integration reaction can be directly visualized after separation in agarose gels by ethidium bromide staining. The most prominent reaction product resulted from integration of one LTR end into another LTR end (U5 into U5 and U5 into U3). Sequence analysis of the reaction products showed them to be products of legitimate integration preceded by correct processing of the viral LTR ends. Hotspots for integration were detected. Electron microscopy revealed the presence of a range of reaction products resulting from single or multiple integration events. The binding of HIV-1 integrase to mini-HIV DNA was visualized. Oligomers of integrase seem to induce DNA looping whereby the enzyme often appears to be bound to the DNA substrate that adopts the structure of a three-site synapsis that is reminiscent of the Mu phage transposase complex.


Assuntos
DNA Viral/metabolismo , Integrase de HIV/metabolismo , HIV-1/enzimologia , Sequência de Bases , Primers do DNA/genética , DNA Viral/genética , DNA Viral/ultraestrutura , Integrase de HIV/genética , Integrase de HIV/ultraestrutura , Repetição Terminal Longa de HIV , HIV-1/genética , Humanos , Substâncias Macromoleculares , Microscopia Eletrônica , Dados de Sequência Molecular , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Integração Viral
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