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1.
J Thromb Haemost ; 22(10): 2910-2921, 2024 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-38925490

RESUMO

BACKGROUND: Scientific and clinical interest in extracellular vesicles (EVs) is growing. EVs that expose tissue factor (TF) bind factor VII/VIIa and can trigger coagulation. Highly procoagulant TF-exposing EVs are detectable in the circulation in various diseases, such as sepsis, COVID-19, or cancer. Many in-house and commercially available assays have been developed to measure EV-TF activity and antigen, but only a few studies have compared some of these assays. OBJECTIVES: The International Society on Thrombosis and Haemostasis Scientific and Standardization Committee Subcommittee on Vascular Biology initiated a multicenter study to compare the sensitivity, specificity, and reproducibility of these assays. METHODS: Platelet-depleted plasma samples were prepared from blood of healthy donors. The plasma samples were spiked either with EVs from human milk or EVs from TF-positive and TF-negative cell lines. Plasma was also prepared from whole human blood with or without lipopolysaccharide stimulation. Twenty-one laboratories measured EV-TF activity and antigen in the prepared samples using their own assays representing 18 functional and 9 antigenic assays. RESULTS: There was a large variability in the absolute values for the different EV-TF activity and antigen assays. Activity assays had higher specificity and sensitivity compared with antigen assays. In addition, there was a large intra-assay and interassay variability. Functional assays that used a blocking anti-TF antibody or immunocapture were the most specific and sensitive. Activity assays that used immunocapture had a lower coefficient of variation compared with assays that isolated EVs by high-speed centrifugation. CONCLUSION: Based on this multicenter study, we recommend measuring EV-TF using a functional assay in the presence of an anti-TF antibody.


Assuntos
Vesículas Extracelulares , Tromboplastina , Humanos , Tromboplastina/metabolismo , Vesículas Extracelulares/metabolismo , Reprodutibilidade dos Testes , Coagulação Sanguínea , COVID-19/sangue , COVID-19/diagnóstico , COVID-19/imunologia , Valor Preditivo dos Testes
2.
ACS Appl Mater Interfaces ; 16(10): 12986-12995, 2024 Mar 13.
Artigo em Inglês | MEDLINE | ID: mdl-38426266

RESUMO

This paper describes the synthesis and use of multifunctional methacrylic monomers, which contain basic (amine) functional groups, including an example in which an acid-labile tert-butylcarbamate-protected glycine is used to form a novel methacrylic monomer. The "protected" amino acid-derived functional monomer (BOC-Gly-MA) is copolymerized with an epoxide functional methacrylic monomer (GMA), to deliver novel multifunctional polymers, which are processed into powder coatings and used to study filiform corrosion at the surface of an aluminum substrate. The BOC-Gly-MA-containing copolymers were shown to improve a coating's anticorrosion performance, presenting the lowest average filiform corrosion (FFC) track length, total FFC number, and total corroded surface area (CSA) of the coatings investigated. Further to this, a mode of action for the role of BOC-Gly functional polymers in corrosion protection is proposed, supported by both solution and polymer-aluminum interface studies, delivering new insights into the mode of action of pH-responsive polymer coatings.

3.
J Med Case Rep ; 17(1): 464, 2023 Nov 08.
Artigo em Inglês | MEDLINE | ID: mdl-37936226

RESUMO

BACKGROUND: Tamoxifen is used in low dose concentrations (20-40 mg per day) as a therapy for breast cancer but is known to have ocular side effects. In this case report, the foveal cone integrity in a tamoxifen-treated patient who complained of a small central scotoma in the left eye while reading was examined using high resolution adaptive optics imaging. CASE PRESENTATION: Both eyes of a 54-year-old Caucasian, non-hispanic female who had been treated with tamoxifen for 1.5 years were examined using various imaging modalities including fundus photography, fundus autofluorescence, fluorescein angiography, spectral-domain optical coherence tomography, and adaptive optics scanning laser ophthalmoscopy. Clinical spectral-domain optical coherence tomography showed a very small disruption to the photoreceptor layer at the fovea in the left eye only. However, adaptive optics scanning laser ophthalmoscopy imaging revealed foveal cone loss in both eyes, but to a lesser extent in the right eye. Inner retinal changes were not observed in either eye. CONCLUSION: The area of cone loss was similar in size to a single newsprint letter when projected onto the retina, matching the patient's description of a scotoma in the left eye. Given the isolated loss of foveal cone photoreceptors with the absence of previously reported inner retinal and vascular changes, our results may indicate the earliest retinal changes associated with tamoxifen retinopathy.


Assuntos
Degeneração Macular , Doenças Retinianas , Humanos , Feminino , Pessoa de Meia-Idade , Células Fotorreceptoras Retinianas Cones , Tamoxifeno/efeitos adversos , Doenças Retinianas/induzido quimicamente , Doenças Retinianas/diagnóstico por imagem , Escotoma , Tomografia de Coerência Óptica/métodos , Angiofluoresceinografia/métodos
4.
Blood Adv ; 7(3): 458-467, 2023 02 14.
Artigo em Inglês | MEDLINE | ID: mdl-35839077

RESUMO

Adeno-associated virus (AAV) gene therapy has the potential to functionally cure hemophilia B by restoring factor (F)IX concentrations into the normal range. Next-generation AAV therapies express a naturally occurring gain-of-function FIX variant, FIX-Padua (R338L-FIX), that increases FIX activity (FIX:C) by approximately eightfold compared with wild-type FIX (FIX-WT). Previous studies have shown that R338L-FIX activity varies dramatically across different clinical FIX:C assays, which complicates the monitoring and management of patients. To better understand mechanisms that contribute to R338L-FIX assay discrepancies, we characterized the performance of R338L-FIX in 13 1-stage clotting assays (OSAs) and 2 chromogenic substrate assays (CSAs) in a global field study. This study produced the largest R338L-FIX assay dataset to date and confirmed that clinical FIX:C assay results vary over threefold. Both phospholipid and activating reagents play a role in OSA discrepancies. CSA generated the most divergent FIX:C results. Manipulation of FIX:C CSA kits demonstrated that specific activity gains for R338L-FIX were most profound at lower FIX:C concentrations and that these effects were enhanced during the early phases of FXa generation. Supplementing FX into CSA had the effect of dampening FIX-WT activity relative to R338L-FIX activity, suggesting that FX impairs WT tenase formation to a greater extent than R338L-FIX tenase. Our data describe the scale of R338L-FIX assay discrepancies and provide insights into the causative mechanisms that will help establish best practices for the measurement of R338L-FIX activity in patients after gene therapy.


Assuntos
Fator IX , Hemofilia B , Humanos , Fator IX/genética , Hemofilia B/diagnóstico , Hemofilia B/genética , Hemofilia B/terapia , Testes de Coagulação Sanguínea , Cisteína Endopeptidases
5.
Metab Eng ; 70: 155-165, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-35038554

RESUMO

Heparin is an essential anticoagulant used for treating and preventing thrombosis. However, the complexity of heparin has hindered the development of a recombinant source, making its supply dependent on a vulnerable animal population. In nature, heparin is produced exclusively in mast cells, which are not suitable for commercial production, but mastocytoma cells are readily grown in culture and make heparan sulfate, a closely related glycosaminoglycan that lacks anticoagulant activity. Using gene expression profiling of mast cells as a guide, a multiplex genome engineering strategy was devised to produce heparan sulfate with high anticoagulant potency and to eliminate contaminating chondroitin sulfate from mastocytoma cells. The heparan sulfate purified from engineered cells grown in chemically defined medium has anticoagulant potency that exceeds porcine-derived heparin and confers anticoagulant activity to the blood of healthy mice. This work demonstrates the feasibility of producing recombinant heparin from mammalian cell culture as an alternative to animal sources.


Assuntos
Edição de Genes , Heparina , Animais , Anticoagulantes , Heparitina Sulfato/metabolismo , Camundongos , Suínos
6.
PLoS One ; 15(5): e0233644, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32469940

RESUMO

Cytotoxic and pro-inflammatory histones are present in neutrophil extracellular traps (NETs) and are elevated in blood in several inflammatory conditions, sepsis being a major example. Compounds which can attenuate activities of histones are therefore of interest, with heparin being one such material that has previously been shown to bind to histones. Heparin, a successful anticoagulant for nearly a century, has been shown experimentally to bind to histones and exhibit a protective effect in inflammatory conditions. In the present study carried out in whole blood, heparin and selectively desulfated heparin reduced histone induced inflammatory markers such as interleukin 6 (IL 6), interleukin 8 (IL 8) and tissue factor and C3a, a complement component. The selectively desulfated heparins, with reduced anticoagulant activities, retained a high degree of effectiveness as an anti-histone agent, whereas fully desulfated heparin was found to be ineffective. The results from this study indicate that the presence of sulfate and other specific structural features are required for heparin to attenuate the inflammatory action of histones in whole blood.


Assuntos
Anti-Inflamatórios/farmacologia , Anticoagulantes/farmacologia , Heparina/farmacologia , Histonas/imunologia , Inflamação/tratamento farmacológico , Anti-Inflamatórios/química , Anticoagulantes/química , Complemento C3a/análise , Complemento C3a/imunologia , Heparina/análogos & derivados , Histonas/sangue , Humanos , Inflamação/sangue , Inflamação/imunologia , Interleucina-6/sangue , Interleucina-6/imunologia , Interleucina-8/sangue , Interleucina-8/imunologia
7.
Ophthalmic Surg Lasers Imaging Retina ; 49(12): 946-954, 2018 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-30566702

RESUMO

BACKGROUND AND OBJECTIVE: Subthreshold micropulse laser (SML) treatment at 577 nm has been proposed as a safe and efficacious therapy for diabetic macular edema (DME). The study objective was to evaluate the integrity of individual cone photoreceptors after SML treatment using high-resolution retinal imaging. PATIENTS AND METHODS: An observational cohort study of four subjects with DME treated using SML was followed over time. Cone inner and outer segment lengths and total retinal thicknesses (TRT) were measured as the edema resolved. The primary outcome was the detection of any laser-induced photoreceptor damage / change following the SML treatment using adaptive optics imaging. RESULTS: Individual cones observed pre-treatment remained visible, whereas cones that were initially obscured by the DME became more discernable after the treatment. TRT showed statistically significant thinning in half of the subjects. One subject showed no significant change, whereas another showed a statistically significant increase in TRT despite the treatment. No subject was found to have photoreceptor damage following treatment. CONCLUSIONS: SML at 577 nm did not result in measurable structural damage to the underlying photoreceptor layer, supporting previous work that SML is a safe alternative for treating DME. [Ophthalmic Surg Lasers Imaging Retina. 2018;49:946-954.].


Assuntos
Retinopatia Diabética/cirurgia , Fotocoagulação a Laser/métodos , Lasers Semicondutores/uso terapêutico , Edema Macular/cirurgia , Células Fotorreceptoras Retinianas Cones/patologia , Acuidade Visual , Adulto , Retinopatia Diabética/complicações , Retinopatia Diabética/diagnóstico , Angiofluoresceinografia , Fundo de Olho , Humanos , Edema Macular/diagnóstico , Edema Macular/etiologia , Masculino , Pessoa de Meia-Idade , Período Pós-Operatório , Tomografia de Coerência Óptica/métodos
8.
Front Med (Lausanne) ; 5: 46, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29541637

RESUMO

Until recently, the role of factor XII (FXII) in hemostasis was not considered to be important since patients with FXII deficiency do not present with bleeding. The activation of FXII by agents including mast cells and platelet polyphosphates suggests that it may have a role in thrombogenesis. The inhibition of FXII therefore presents an option for antithrombotic therapy, and antibodies and inhibitors are already in development. Assays for FXII will be required to support these technologies, and an international standard (IS) for FXII would be useful for the development of these methods and for the clinical monitoring of patients. The purpose of this study was to develop an IS for FXII, with values for functional activity (FXII:C) and antigen (FXII:Ag). Double-spun normal plasma was pooled, filled into siliconized glass ampoules, and freeze-dried to prepare the candidate material. Data from 20 laboratories using the one-stage clotting assay were used to assign the functional activity value in units (u). The antigen value was calculated using data from eight laboratories that carried out antigen assays. Each laboratory was requested to collect two local normal plasma pools. Units of activity and antigen were calculated relative to these pools, as is usual for new coagulation factor analytes. The amount of activity or antigen in 1 ml of normal plasma from each pool was taken to be 1 unit. A total of 566 donors were used across the pools for the FXII:C study and 216 donors for the FXII:Ag study. The overall geometric mean per ampoule for FXII:C was 0.86 u and for FXII:Ag was 0.80 u. The inter-laboratory variation was 10 and 11%, respectively (expressed as the geometric coefficient of variation). Based on these data, the candidate was deemed suitable for use as an IS for FXII. In 2017, the candidate was established by the World Health Organization (WHO) Expert Committee on Biological Standardization as the WHO first IS for blood coagulation FXII, Plasma (National Institute for Biological Standards and Control code 15/180). The values assigned were 0.86 international units (IU) of functional activity (FXII:C) per ampoule and 0.80 IU/ampoule of antigen (FXII:Ag).

9.
Pharmacol Rev ; 68(1): 76-141, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26672027

RESUMO

Heparin has been recognized as a valuable anticoagulant and antithrombotic for several decades and is still widely used in clinical practice for a variety of indications. The anticoagulant activity of heparin is mainly attributable to the action of a specific pentasaccharide sequence that acts in concert with antithrombin, a plasma coagulation factor inhibitor. This observation has led to the development of synthetic heparin mimetics for clinical use. However, it is increasingly recognized that heparin has many other pharmacological properties, including but not limited to antiviral, anti-inflammatory, and antimetastatic actions. Many of these activities are independent of its anticoagulant activity, although the mechanisms of these other activities are currently less well defined. Nonetheless, heparin is being exploited for clinical uses beyond anticoagulation and developed for a wide range of clinical disorders. This article provides a "state of the art" review of our current understanding of the pharmacology of heparin and related drugs and an overview of the status of development of such drugs.


Assuntos
Anticoagulantes/farmacologia , Heparina/farmacologia , Anti-Inflamatórios/farmacologia , Anticoagulantes/efeitos adversos , Antineoplásicos/farmacologia , Antivirais/farmacologia , Moléculas de Adesão Celular/metabolismo , Quimiocinas/metabolismo , Proteínas do Sistema Complemento/metabolismo , Citocinas/metabolismo , Heparina/efeitos adversos , Heparina de Baixo Peso Molecular/farmacologia , Heparinoides/farmacologia , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Agregados Proteicos/fisiologia , Selectinas/metabolismo , Venenos de Serpentes/metabolismo , Relação Estrutura-Atividade
10.
PLoS One ; 9(12): e114524, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25474412

RESUMO

RATIONALE: The chemokine interleukin-8 is implicated in the development of bronchopulmonary dysplasia in preterm infants. The 77-amino acid isoform of interleukin-8 (interleukin-877) is a less potent chemoattractant than other shorter isoforms. Although interleukin-877 is abundant in the preterm circulation, its regulation in the preterm lung is unknown. OBJECTIVES: To study expression and processing of pulmonary interleukin-877 in preterm infants who did and did not develop bronchopulmonary dysplasia. METHODS: Total interleukin-8 and interleukin-877 were measured in bronchoalveolar lavage fluid from preterm infants by immunoassay. Neutrophil serine proteases were used to assess processing. Neutrophil chemotaxis assays and degranulation of neutrophil matrix metalloproteinase-9 were used to assess interleukin-8 function. MAIN RESULTS: Peak total interleukin-8 and interleukin-877 concentrations were increased in infants who developed bronchopulmonary dysplasia compared to those who did not. Shorter forms of interleukin-8 predominated in the preterm lung (96.3% No-bronchopulmonary dysplasia vs 97.1% bronchopulmonary dysplasia, p>0.05). Preterm bronchoalveolar lavage fluid significantly converted exogenously added interleukin-877 to shorter isoforms (p<0.001). Conversion was greater in bronchopulmonary dysplasia infants (p<0.05). This conversion was inhibited by α-1 antitrypsin and antithrombin III (p<0.01). Purified neutrophil serine proteases efficiently converted interleukin-877 to shorter isoforms in a time- and dose-dependent fashion; shorter interleukin-8 isoforms were primarily responsible for neutrophil chemotaxis (p<0.001). Conversion by proteinase-3 resulted in significantly increased interleukin-8 activity in vitro (p<0.01). CONCLUSIONS: Shorter, potent, isoforms interleukin-8 predominate in the preterm lung, and are increased in infants developing bronchopulmonary dysplasia, due to conversion of interleukin-877 by neutrophil serine proteases and thrombin. Processing of interleukin-8 provides an attractive therapeutic target to prevent development of bronchopulmonary dysplasia.


Assuntos
Displasia Broncopulmonar/metabolismo , Interleucina-8/sangue , Interleucina-8/metabolismo , Respiração Artificial/efeitos adversos , Serina Proteases/metabolismo , Displasia Broncopulmonar/enzimologia , Displasia Broncopulmonar/etiologia , Células Cultivadas , Quimiotaxia , Humanos , Lactente , Recém-Nascido , Recém-Nascido Prematuro , Neutrófilos/fisiologia
11.
Eur J Hum Genet ; 19(1): 10-7, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-20736975

RESUMO

Fragile X syndrome is the most common inherited form of mental retardation. It is caused by expansion of a trinucleotide (CGG)n repeat sequence in the 5' untranslated region of the FMR1 gene, resulting in promoter hypermethylation and suppression of FMR1 transcription. Additionally, pre-mutation alleles in carrier males and females may result in Fragile X tremor ataxia syndrome and primary ovarian insufficiency, respectively. Fragile X is one of the most commonly requested molecular genetic tests worldwide. Quality assessment schemes have identified a wide disparity in allele sizing between laboratories. It is therefore important that clinical laboratories have access to characterized reference materials (RMs) to aid accurate allele sizing and diagnosis. With this in mind, a panel of genotyping RMs for Fragile X syndrome has been developed, which should be stable over many years and available to all diagnostic laboratories. Immortalized cell lines were produced by Epstein-Barr virus transformation of lymphocytes from consenting patients. Genomic DNA was extracted in bulk and RM aliquots were freeze-dried in glass ampoules. Twenty-one laboratories from seventeen countries participated in a collaborative study to assess their suitability. Participants evaluated the samples (blinded, in triplicate) in their routine methods alongside in-house and commercial controls. The panel of five genomic DNA samples was endorsed by the European Society of Human Genetics and approved as an International Standard by the Expert Committee on Biological Standardization at the World Health Organization.


Assuntos
Proteína do X Frágil da Deficiência Intelectual/normas , Síndrome do Cromossomo X Frágil/genética , Testes Genéticos/normas , Linhagem Celular Transformada , DNA/genética , DNA/isolamento & purificação , DNA/metabolismo , Feminino , Proteína do X Frágil da Deficiência Intelectual/genética , Síndrome do Cromossomo X Frágil/diagnóstico , Genótipo , Herpesvirus Humano 4 , Humanos , Linfócitos/virologia , Masculino , Mutação , Padrões de Referência , Organização Mundial da Saúde
13.
Melanoma Res ; 19(5): 301-8, 2009 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-19550359

RESUMO

A correlation between cancer and hypercoagulability has been described for more than a century. Patients with cancer are at increased risk for thrombotic complications, and the clotting initiator protein, tissue factor (TF), is possibly involved in this process. In addition to TF, the presence of negatively charged phospholipids, particularly phosphatidylserine (PS), is necessary to support some of the blood-clotting reactions. There are few reports describing PS exposure by tumor cells. In this study, we characterized the procoagulant properties of the murine B16F10 and the human WM-266-4 melanoma cell lines. Flow cytometry analyses showed constitutive TF expression by both cell lines, in contrast to negative staining observed for the nontumorigenic melanocyte lineage, melan-A. In addition, tumor cells accelerate plasma clotting in a number-dependent manner. For WM-266-4, this ability was partially reversed by an anti-TF antibody but not by aprotinin, a nonspecific serine-protease inhibitor. Furthermore, flow-cytometric analyses showed the presence of PS at the outer leaflet of both cell lines. This phenomenon was determinant for the assembly of the intrinsic tenase (FIXa/FVIIIa) and prothrombinase (FXa/FVa) complexes, resulting in the activation of FX to FXa and prothrombin to thrombin, respectively. As a result, incubation of WM-266-4 with human plasma produces robust thrombin generation. In conclusion, simultaneous TF expression and PS exposure are responsible for the highly procoagulant pattern of the aggressive melanoma cell lines B16F10 and WM-266-4. Therefore, these cell lines might be regarded as useful models for studying the role of blood coagulation proteins in tumor biology.


Assuntos
Melanoma/sangue , Fosfatidilserinas/farmacologia , Tromboplastina/biossíntese , Animais , Coagulação Sanguínea/efeitos dos fármacos , Coagulação Sanguínea/fisiologia , Linhagem Celular Tumoral , Citometria de Fluxo , Humanos , Melanoma/metabolismo , Melanoma Experimental/sangue , Melanoma Experimental/química , Camundongos , Trombina/biossíntese , Trombina/metabolismo , Tromboplastina/metabolismo
14.
Thromb Haemost ; 99(5): 807-18, 2008 May.
Artigo em Inglês | MEDLINE | ID: mdl-18449410

RESUMO

Heparin is one of the oldest biological medicines, and has an established place in the prevention and treatment of venous thrombosis. Low-molecular-weight heparins (LMWH) have been developed by several manufacturers and have advantages in terms of pharmacokinetics and convenience of administration. They have been shown to be at least as effective and safe as unfractionated heparin and have replaced the latter in many indications. In this article the chemistry, mechanisms of action, measurement of anticoagulant activities, and clinical status of heparin and LMWH are reviewed.


Assuntos
Anticoagulantes/uso terapêutico , Coagulação Sanguínea/efeitos dos fármacos , Heparina de Baixo Peso Molecular/uso terapêutico , Heparina/uso terapêutico , Trombose/tratamento farmacológico , Animais , Anticoagulantes/efeitos adversos , Anticoagulantes/química , Testes de Coagulação Sanguínea , Doença da Artéria Coronariana/sangue , Doença da Artéria Coronariana/tratamento farmacológico , Monitoramento de Medicamentos , Inibidores do Fator Xa , Heparina/efeitos adversos , Heparina/química , Heparina de Baixo Peso Molecular/efeitos adversos , Heparina de Baixo Peso Molecular/química , Humanos , Estrutura Molecular , Peso Molecular , Neoplasias/sangue , Neoplasias/tratamento farmacológico , Seleção de Pacientes , Relação Estrutura-Atividade , Trombina/metabolismo , Trombose/sangue , Resultado do Tratamento , Tromboembolia Venosa/sangue , Tromboembolia Venosa/tratamento farmacológico , Trombose Venosa/sangue , Trombose Venosa/tratamento farmacológico
15.
J Pharm Biomed Anal ; 47(3): 460-8, 2008 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-18374535

RESUMO

We have investigated the use of quantitative reverse transcription-polymerase chain reaction (qRT-PCR) as an alternative to a selection of late-stage functional bioassays for determination of the potency of preparations of vascular endothelial growth factor (VEGF). Responses were measured in cultures of human umbilical vein endothelial cells (HUVECs). Late-stage responses measured were cell survival and proliferation, and production of interleukin-8 (IL-8), interleukin-6 (IL-6), and tissue factor. The dose-response range was similar across the assays, increasing from 2 ng/mL VEGF and reaching a maximum between 30 ng/mL and 125 ng/mL VEGF. A number of VEGF-induced mRNA species demonstrated dose-response curves suitable for VEGF potency determination. IL-8 mRNA induction after 45 min incubation with VEGF, which showed maximal responses between 15.6 ng/mL and 62.5 ng/mL VEGF, was selected for further characterization. This gene-expression bioassay was robust across a range of cell seeding densities and could be used for samples processed immediately following incubation with VEGF and for cell lysates stored at -80 degrees C for 3 months. We also compared this gene-expression bioassay and the assays of late-stage responses in the potency measurement of the inhibitors of VEGF activity, anti-VEGF monoclonal antibody MAB293, and a VEGF soluble receptor VEGFsR1 preparation. We present a critical evaluation of the use of qRT-PCR in assaying the potency of VEGF and its inhibitors, and of the potential of this platform for measuring the potency of other biological therapeutics.


Assuntos
Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Fator A de Crescimento do Endotélio Vascular/farmacologia , Anticorpos Monoclonais/farmacologia , Bioensaio , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Células Endoteliais/efeitos dos fármacos , Humanos , Interleucina-6/biossíntese , Interleucina-8/biossíntese , Interleucina-8/genética , RNA Mensageiro/análise , Tromboplastina/biossíntese , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores
16.
Blood ; 109(4): 1414-21, 2007 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-17090654

RESUMO

The safety and efficacy of peripheral venous administration of a self-complementary adeno-associated viral vector encoding the human FIX gene (scAAV-LP1-hFIXco) was evaluated in nonhuman primates for gene therapy of hemophilia B. Peripheral vein infusion of 1x10(12) vg/kg scAAV-LP1-hFIXco pseudotyped with serotype 8 capsid, in 3 macaques, resulted in stable therapeutic expression (more than 9 months) of human FIX (hFIX) at levels (1.1+/-0.5 microg/mL, or 22% of normal) that were comparable to those achieved after direct delivery of the same vector dose into the portal circulation (1.3+/-0.3 microg/mL, or 26% of normal). Importantly, the pattern of vector biodistribution after systemic and portal vein administration of scAAV-LP1-hFIXco was almost identical. Additionally, comparable levels of gene transfer were achieved in macaques with preexisting immunity to AAV8 following peripheral vein administration of 1x10(12) vg/kg AAV5-pseudotyped scAAV-LP1-hFIXco. This confirms that alternative serotypes can circumvent preexisting naturally acquired immunity to AAV. Thus, peripheral venous administration of AAV5 and AAV8 vectors is safe and as effective at transducing the liver in nonhuman primates as direct vector administration into the portal circulation. These results should make vector administration to patients, especially those with a severe bleeding diathesis, significantly easier and safer.


Assuntos
Fator IX/administração & dosagem , Terapia Genética/métodos , Vetores Genéticos/administração & dosagem , Hemofilia B/terapia , Fígado/metabolismo , Transdução Genética/métodos , Animais , Formação de Anticorpos , Fator IX/farmacocinética , Vetores Genéticos/farmacocinética , Humanos , Macaca , Distribuição Tecidual , Resultado do Tratamento
17.
Blood ; 101(11): 4372-9, 2003 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-12560230

RESUMO

Interaction of platelets with collagen under conditions of blood flow is a multi-step process with tethering via glycoprotein IbIXV (GPIbIXV) over von Willebrand factor, adhesion by direct interaction with the integrin GPIaIIa, and signaling via GPVI. GPVI can be specifically agonized by cross-linked collagen-related peptide (CRP-XL), which results in a signaling cascade very similar to that evoked by native collagen. The GPVI gene has 2 common alleles that differ by 3 replacements in the glycosylated stem and 2 in the cytoplasmic domain. We used CRP-XL to elucidate the variation in responses observed in platelet function in different individuals. We observed a 3-fold difference in the response to CRP-XL in platelet aggregation when comparing platelets from 10 high-frequency allele homozygotes with 8 low-frequency ones (2-way analysis of variance [ANOVA], P <.0001). The difference in functional responses was reflected in fibrinogen binding and in downstream signaling events as measured by tyrosine phosphorylation, the expression of P-selectin, and the binding of annexin V and the generation of thrombin on the platelet surface (2-way ANOVA, P <.001). Platelets homozygous for the low-frequency allele tended to be less able to form a thrombus on a collagen surface in flowing whole blood or in the platelet function analyzer-100 (t test, P =.065 and P =.061, respectively). The functional difference was correlated to a difference in total and membrane-expressed GPVI measured by monoclonal and polyclonal antibodies. This study demonstrates for the first time that platelet function may be altered by allelic differences in GPVI.


Assuntos
Peptídeos , Glicoproteínas da Membrana de Plaquetas/genética , Glicoproteínas da Membrana de Plaquetas/fisiologia , Polimorfismo de Nucleotídeo Único , Adulto , Proteínas de Transporte/farmacologia , Colágeno/farmacologia , Feminino , Frequência do Gene , Homozigoto , Humanos , Masculino , Pessoa de Meia-Idade , Fosforilação/efeitos dos fármacos , Ativação Plaquetária/efeitos dos fármacos , Adesividade Plaquetária/efeitos dos fármacos , Agregação Plaquetária/efeitos dos fármacos , Receptores de Colágeno , Trombina/biossíntese , Trombina/efeitos dos fármacos
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