RESUMO
BAX 855 is a PEGylated recombinant factor VIII preparation that showed prolonged circulatory half-life in nonclinical and clinical studies. This paper describes the development, validation, and application of a novel ligand-binding assay (LBA) to selectively measure BAX 855 in plasma. The LBA is based on PEG-specific capture of BAX 855, followed by immunological factor VIII (FVIII)-specific detection of the antibody-bound BAX 855. This assay principle enabled sensitive measurement of BAX 855 down to the low nanomolar range without interference from non-PEGylated FVIII as demonstrated by validation data for plasma from animals typically used for nonclinical characterization of FVIII. The selectivity of an in-house-developed anti-PEG and a commercially available preparation, shown by competition studies to primarily target the terminating methoxy group of PEG, also allowed assessment of the intactness of the attached PEG chains. Altogether, this new LBA adds to the group of methods to selectively, accurately, and precisely measure a PEGylated drug in complex biological matrices. The feasibility and convenience of using this method was demonstrated during extensive nonclinical characterization of BAX 855.
Assuntos
Bioensaio/métodos , Fator VIII/análise , Proteínas Recombinantes/análise , Animais , Anticorpos/química , Fator VIII/genética , Fator VIII/imunologia , Fator VIII/metabolismo , Meia-Vida , Humanos , Imunoglobulina G , Ligantes , Macaca , Camundongos , Polietilenoglicóis/química , Coelhos , Ratos , Proteínas Recombinantes/sangue , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sensibilidade e EspecificidadeRESUMO
Neutralizing antibodies against factor VIII (FVIII) remain the major complication in the replacement therapy of hemophilia A patients. To better understand the evolution of these antibodies it is important to generate comprehensive datasets which include both neutralizing and nonneutralizing antibodies, their isotypes, and IgG subclasses. We developed sensitive ELISAs to analyze FVIII-binding antibodies in different cohorts of hemophilia A patients and in healthy individuals. Our data reveal the prevalence of FVIII-binding antibodies among healthy individuals (n = 600) to be as high as 19%, with a prevalence of antibody titers > or =1:80 of 2%. The prevalence of FVIII-binding antibodies was 34% (5% for titers > or =1:80) in patients without FVIII inhibitors (n = 77), 39% (4% for titers > 1:80) in patients after successful immune tolerance induction therapy (n = 23), and 100% (n = 20, all titers > or =1:80) in patients with FVIII inhibitors. We found significant differences for IgG subclasses of FVIII-binding antibodies between the different study cohorts. IgG4 and IgG1 were the most abundant IgG subclasses in patients with FVIII inhibitors. Strikingly, IgG4 was completely absent in patients without FVIII inhibitors and in healthy subjects. These findings point toward a distinct immune regulatory pathway responsible for the development of FVIII-specific IgG4 associated with FVIII inhibitors.
Assuntos
Formação de Anticorpos/imunologia , Fator VIII/imunologia , Hemofilia A/imunologia , Imunoglobulina G/imunologia , Adolescente , Adulto , Idoso , Anticorpos Neutralizantes/sangue , Anticorpos Neutralizantes/imunologia , Inibidores dos Fatores de Coagulação Sanguínea/uso terapêutico , Estudos de Coortes , Ensaio de Imunoadsorção Enzimática , Fator VIII/antagonistas & inibidores , Feminino , Hemofilia A/sangue , Hemofilia A/tratamento farmacológico , Humanos , Tolerância Imunológica/efeitos dos fármacos , Tolerância Imunológica/imunologia , Imunoglobulina G/sangue , Imunoglobulina G/classificação , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Adulto JovemRESUMO
Deficiency of a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS13), a VWF-cleaving protease, is the key factor in the pathogenesis of thrombotic thrombocytopenic purpura (TTP), a life-threatening thrombotic microangiopathy. It is well established that ADAMTS13 deficiency results in elevated plasma levels of ultra-large VWF multimers (ULVWF), which are prone to induce platelet aggregation; however, the actual trigger of TTP development remains uncertain. Here we describe a new animal model in which some TTP-like symptoms can be triggered in ADAMTS13 knockout mice by challenge with 2000 units/kg body weight of recombinant human VWF containing ULVWF multimers. Animals rapidly showed clinical symptoms and developed severe thrombocytopenia. Schistocytosis, a decrease in hematocrit, and elevated serum lactate dehydrogenase levels were observed. The heart was identified as the most sensitive target organ with rapid onset of extensive platelet aggregation in the ventricles and myocardial necrosis. Prophylactic administration of 200 units/kg recombinant human ADAMTS13 protected ADAMTS13 knockout mice from developing TTP. Therapeutic administration of 320 units/kg rhADAMTS13 reduced the incidence and severity of TTP findings in a treatment interval-dependent manner. We therefore consider this newly established mouse model of thrombotic microangiopathy highly predictive for investigating the efficacy of new treatments for TTP.
Assuntos
Proteínas ADAM/uso terapêutico , Modelos Animais de Doenças , Metaloendopeptidases/genética , Camundongos Knockout , Púrpura Trombocitopênica Trombótica/tratamento farmacológico , Púrpura Trombocitopênica Trombótica/genética , Proteínas ADAM/administração & dosagem , Proteína ADAMTS13 , Animais , Peso Corporal/efeitos dos fármacos , Relação Dose-Resposta a Droga , Feminino , Hematócrito , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Contagem de Plaquetas , Púrpura Trombocitopênica Trombótica/sangue , Púrpura Trombocitopênica Trombótica/patologia , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/uso terapêutico , Resultado do TratamentoRESUMO
Today it is generally accepted that B cells require cognate interactions with CD4(+) T cells to develop high-affinity antibodies against proteins. CD4(+) T cells recognize peptides (epitopes) presented by MHC class II molecules that are expressed on antigen-presenting cells. Structural features of both the MHC class II molecule and the peptide determine the specificity of CD4(+) T cells that can bind to the MHC class II-peptide complex. We used a new humanized hemophilic mouse model to identify FVIII peptides presented by HLA-DRB1*1501. This model carries a knockout of all murine MHC class II molecules and expresses a chimeric murine-human MHC class II complex that contains the peptide-binding sites of the human HLA-DRB1*1501. When mice were treated with human FVIII, the proportion of mice that developed antibodies depended on the application route of FVIII and the activation state of the innate immune system. We identified 8 FVIII peptide regions that contained CD4(+) T-cell epitopes presented by HLA-DRB1*1501 to CD4(+) T cells during immune responses against FVIII. CD4(+) T-cell responses after intravenous and subcutaneous application of FVIII involved the same immunodominant FVIII epitopes. Interestingly, most of the 8 peptide regions contained promiscuous epitopes that bound to several different HLA-DR proteins in in vitro binding assays.
Assuntos
Formação de Anticorpos/imunologia , Linfócitos T CD4-Positivos/imunologia , Modelos Animais de Doenças , Epitopos de Linfócito T/imunologia , Fator VIII/administração & dosagem , Fator VIII/imunologia , Cadeias HLA-DRB1/imunologia , Hemofilia A/imunologia , Animais , Apresentação de Antígeno , Linfócitos T CD4-Positivos/metabolismo , Células Dendríticas/citologia , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Cadeias HLA-DRB1/metabolismo , Haplótipos/genética , Hemofilia A/metabolismo , Hemofilia A/patologia , Humanos , Injeções Intravenosas , Injeções Subcutâneas , Masculino , Camundongos , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
INTRODUCTION: Infants with congenital adrenal hyperplasia (CAH) require higher doses of fludrocortisone (FC) due to physiological mineralocorticoid resistance. The adequacy of mineralocorticoid replacement should be closely monitored to avoid hypertension. OBJECTIVE: To evaluate blood pressure (BP) in infants with CAH due to 21-hydroxylase deficiency. PATIENTS AND METHODS: Thirty-three patients (18f/15 m) diagnosed by newborn screening were followed until the age of 4 years. Mean start of HC and FC treatment was day 9·8 ± 9·2 postnatally. Mean daily HC dose ranged from 8·6 to 12·3 mg/m(2) /day. RESULTS: During the first year of life prevalence of systolic hypertension was up to 45·5%. At 12 and at 18 months, BP was highest. Prevalence of systolic hypertension was up to 57·6% at 18 months of age. After 24 months BP levels were lower and at 48 months prevalence of hypertension decreased to 15·2%. Systolic and diastolic BP correlated significantly with the administered fludrocortisone dose (r = 0·3, P = 0·005), but not with body mass index. Hypertensive children received significantly higher FC doses and had significantly lower plasma renin activity during the study period. CONCLUSION: High prevalence of transient, most likely FC induced hypertension was found in young children with classic CAH diagnosed by newborn screening. The changing mineralocorticoid sensitivity in infants is a risk factor for the development of hypertension in patients with CAH, who are treated with FC. Therefore suppressed plasma renin activity should be avoided to prevent arterial hypertension.
Assuntos
Hiperplasia Suprarrenal Congênita/tratamento farmacológico , Fludrocortisona/efeitos adversos , Glucocorticoides/efeitos adversos , Terapia de Reposição Hormonal/efeitos adversos , Hipertensão/induzido quimicamente , Renina/metabolismo , Hiperplasia Suprarrenal Congênita/metabolismo , Pressão Sanguínea , Pré-Escolar , Ensaios Enzimáticos , Feminino , Fludrocortisona/administração & dosagem , Glucocorticoides/administração & dosagem , Humanos , Lactente , Recém-Nascido , Masculino , Radioimunoensaio , Renina/efeitos dos fármacosRESUMO
PURPOSE: This study reports the identification of a novel heterozygous IKBA missense mutation (p.M37K) in a boy presenting with ectodermal dysplasia with immunodeficiency (EDA-ID) who had wild type IKBKG gene encoding NEMO. Our aim was to characterize the clinical course of this IκB-α gain-of-function mutant and to investigate if the p.M37K substitution affects NF-κB activation by interfering with IκB-α degradation, thus impairing NF-κB signaling and causing the EDA-ID phenotype. METHODS: NF-κB signaling was evaluated by measuring IκB-α degradation in patient fibroblasts. In addition, transiently transfected HeLa cells expressing either the M37K-mutant IκB-α allele, the previously characterized S36A-mutant IκB-α allele, or wild type IκB-α were evaluated for IκB-α degradation and NF-κB nuclear translocation following stimulation with TNF-α. RESULTS: Clinical findings revealed a classical ectodermal dysplasia phenotype complicated by recurrent mucocutaneous candidiasis, hypothyroidism, hypopituitarism, and profound combined immunodeficiency with decreased numbers of IL-17 T cells. IκB-α degradation after TNF-α and TLR agonist stimulation was abolished in patient fibroblasts as well as in HeLa cells expressing M37K-IκB-α similar to cells expressing S36A-IκB-α resulting in impaired nuclear translocation of NF-κB and reduced NF-κB dependent luciferase activity compared to cells expressing wild type IκB-α. Patient whole blood cells failed to secrete IL-6 in response to IL-1ß, Pam2CSK4, showed reduced responses to LPS and PMA/Ionomycin, and lacked IL-10 production in response to TNF-α. CONCLUSION: The novel heterozygous mutation p.M37K in IκB-α impairs NF-κB activation causing autosomal dominant EDA-ID with an expanded clinical phenotype.
Assuntos
Núcleo Celular/metabolismo , Displasia Ectodérmica/imunologia , Fibroblastos/imunologia , Quinase I-kappa B/metabolismo , Síndromes de Imunodeficiência/imunologia , Poliendocrinopatias Autoimunes/imunologia , Transporte Ativo do Núcleo Celular/genética , Pré-Escolar , Citocinas/imunologia , Células HeLa , Humanos , Quinase I-kappa B/genética , Lactente , Ativação Linfocitária/genética , Masculino , Mutação de Sentido Incorreto/genética , Proteólise , Células Th17/imunologia , Transgenes/genéticaRESUMO
Factor VIII (FVIII)-specific memory B cells are essential components for regulating anamnestic antibody responses against FVIII in hemophilia A with FVIII inhibitors. We asked how stimulation and inhibition of FVIII-specific memory B cells by low and high concentrations of FVIII, respectively, are affected by concurrent activation of the innate immune system. Using CD138(-) spleen cells from hemophilic mice treated with FVIII to study restimulation and differentiation of memory B cells in vitro, we tested modulating activities of agonists for Toll-like receptors (TLRs) 2, 3, 4, 5, 7, and 9. Ligands for TLR7 and 9 were most effective. They not only amplified FVIII-specific memory responses in the presence of stimulating concentrations of FVIII, but also countered inhibition in the presence of inhibitory concentrations of FVIII. Notably, CpG oligodeoxynucleotide (CpG-ODN), a ligand for TLR9, expressed biphasic effects. It amplified memory responses at low concentrations and inhibited memory responses at high concentrations, both in vitro and in vivo. Both stimulatory and inhibitory activities of CpG-ODN resulted from specific interactions with TLR9. Despite their strong immunomodulatory effects in the presence of FVIII, ligands for TLR induced negligible restimulation in the absence of FVIII in vitro and no restimulation in the absence of FVIII in vivo.
Assuntos
Linfócitos B/imunologia , Fator VIII/imunologia , Hemofilia A/imunologia , Memória Imunológica/imunologia , Oligodesoxirribonucleotídeos/farmacologia , Receptor Toll-Like 9/imunologia , Animais , Linfócitos B/metabolismo , Diferenciação Celular , Células Cultivadas , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Fator VIII/administração & dosagem , Fator VIII/metabolismo , Hemofilia A/metabolismo , Humanos , Ligantes , Ativação Linfocitária , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Baço/citologia , Baço/imunologia , Baço/metabolismo , Receptor Toll-Like 9/agonistas , Receptor Toll-Like 9/metabolismoRESUMO
Memory B cells are involved in long-term maintenance of antibody-dependent immunologic disorders. Therefore, it is essential to understand how the restimulation of FVIII-specific memory B cells in hemophilia A with FVIII inhibitors is regulated. We asked whether concurrent activation of the innate immune system by an agonist for toll-like receptor (TLR) 7 is able to facilitate the differentiation of FVIII-specific memory B cells in the absence of T-cell help. TLR7 recognizes single-stranded RNA as contained in RNA viruses such as influenza, Sendai, and Coxsackie B viruses. Our results indicate that highly purified murine memory B cells do not differentiate into FVIII-specific antibody-secreting cells in the presence of FVIII and the TLR7 agonist when cultured in the absence of CD4(+) T cells. However, CD11c(+) dendritic cells facilitate the T cell-independent differentiation of FVIII-specific memory B cells but only in the presence of FVIII and the TLR7 agonist. In contrast to T cell-dependent restimulation, the antibody response after T cell-independent restimulation of FVIII-specific memory B cells is skewed toward IgG2a, an antibody subclass that is efficient in activating the complement system and in inducing Fc-receptor-mediated effector functions, both are required for effective immune responses against pathogens.
Assuntos
Linfócitos B/imunologia , Células Dendríticas/fisiologia , Fator VIII/imunologia , Memória Imunológica/imunologia , Receptor 7 Toll-Like/agonistas , Animais , Linfócitos B/citologia , Linfócitos B/efeitos dos fármacos , Células Cultivadas , Células Dendríticas/imunologia , Epitopos/efeitos dos fármacos , Epitopos/imunologia , Memória Imunológica/efeitos dos fármacos , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Especificidade por Substrato/efeitos dos fármacos , Especificidade por Substrato/imunologia , Linfócitos T/imunologia , Linfócitos T/fisiologiaRESUMO
Replacement of the missing factor VIII (FVIII) is the current standard of care for patients with hemophilia A. However, the short half-life of FVIII makes frequent treatment necessary. Current efforts focus on the development of longer-acting FVIII concentrates by introducing chemical and genetic modifications to the protein. Any modification of the FVIII protein, however, risks increasing its immunogenic potential to induce neutralizing antibodies (FVIII inhibitors), and this is one of the major complications in current therapy. It would be highly desirable to identify candidates with a high risk for increased immunogenicity before entering clinical development to minimize the risk of exposing patients to such altered FVIII proteins. In the present study, we describe a transgenic mouse line that expresses a human F8 cDNA. This mouse is immunologically tolerant to therapeutic doses of native human FVIII but is able to mount an antibody response when challenged with a modified FVIII protein that possesses altered immunogenic properties. In this situation, immunologic tolerance breaks down and antibodies develop that recognize both the modified and the native human FVIII. The applicability of this new model for preclinical immunogenicity assessment of new FVIII molecules and its potential use for basic research are discussed.
Assuntos
Fator VIII/genética , Fator VIII/imunologia , Hemofilia A/genética , Tolerância Imunológica/genética , Memória Imunológica/genética , Animais , Formação de Anticorpos/genética , Formação de Anticorpos/fisiologia , Modelos Animais de Doenças , Fator VIII/antagonistas & inibidores , Feminino , Hemofilia A/imunologia , Hemofilia A/patologia , Humanos , Tolerância Imunológica/fisiologia , Memória Imunológica/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Modelos Biológicos , Especificidade da EspécieRESUMO
PURPOSE: Human factor VIIa (FVIIa) is commonly used as bypassing therapy to treat bleeding episodes in hemophilia patients with neutralizing antibodies to factors VIII (FVIII) or IX (FIX). There is a need for a suitable animal model to assess the immunogenicity of new FVIIa products during preclinical development. The aim of this study was the design of a novel transgenic mouse model with immune tolerance to human FVIIa. METHODS: The model was generated by transgenic expression of human F7 cDNA. FVIIa-specific immune responses after treatment with human FVIIa were assessed by analyzing circulating antibodies, antibody producing plasma cells and CD4(+) T cells. RESULTS: In contrast to wild-type mice, human FVII transgenic mice did not develop antibodies when treated with human FVIIa. The immune tolerance was specific and could be broken by application of human FVIIa together with a strong stimulus of the innate immune system. Break of tolerance was associated with increased numbers of pro-inflammatory FVIIa-specific CD4(+) T cells. CONCLUSIONS: The new mouse model is suitable to study the influence of the innate immune system on maintenance and break of immune tolerance against FVIIa and could be used to assess the immunogenicity of new FVIIa products during pre-clinical development.
Assuntos
Fator VIIa/genética , Fator VIIa/imunologia , Tolerância Imunológica , Transgenes , Animais , Linfócitos T CD8-Positivos/imunologia , DNA Complementar/genética , Fator VIIa/uso terapêutico , Feminino , Humanos , Imunidade Inata , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Modelos AnimaisRESUMO
OBJECTIVE: Poor growth early in life is associated with numerous adverse conditions including decreased bone mass. The aim is to investigate bone and body composition in young adults born small for gestational age (SGA). DESIGN: Observational study. PARTICIPANTS: A total of 76 young adults born SGA (34f) at a mean age of 19·68 ± 0·5 years were enrolled. METHOD: Bone mineral density (BMD), bone geometry and body composition were analysed using peripheral quantitative computed tomography. RESULTS: Adults born SGA had significantly lower z-score for height (-0.86 ± 0·87), weight (-0·61 ± 0·78) and BMI (-0·38 ± 1·04) as well as fat cross-sectional area (CSA) (-0·62 ± 0·80) compared with a healthy reference population (P < 0·05). Z-scores for trabecular and cortical BMD were normal. After correction for reduced height, z-scores for total CSA (-0·14 ± 1·11) and muscle CSA (-0·21 ± 0·99) were normal and medullary cavity (-0·71 ± 0·80) was reduced. Those with a birthweight of ≤ 1500 g had even lower height-corrected z-scores for medullary cavity (-1·12 ± 0·69) and total bone CSA (-0·58 ± 0·93) (P < 0·05). After adjustment for sex and weight, significant partial correlations were detectable between BMI at the age of 48 months and height-corrected z-scores for medullary cavity (r = 0·33, P = 0·020) and total CSA (r = 0·29, P = 0·04). CONCLUSION: Environmental factors early in life seem to influence bone geometry in adulthood. Young adults born SGA have normal total bone CSA but smaller medullary cavity. Those with very low birthweight, however, show compromised bone size development that may alter bone stability later in life.
Assuntos
Peso ao Nascer/fisiologia , Densidade Óssea/fisiologia , Adulto , Antropometria , Composição Corporal/fisiologia , Feminino , Humanos , Recém-Nascido , Recém-Nascido Pequeno para a Idade Gestacional/fisiologia , Masculino , Tomografia Computadorizada por Raios X , Adulto JovemRESUMO
BACKGROUND: A typical growth pattern with decreased pubertal growth spurt has been identified in patients with classical congenital adrenal hyperplasia (CAH). OBJECTIVE: To evaluate the accuracy of final height predictions in patients with CAH using the Bayley and Pinneau (B&P) method. PATIENTS AND METHODS: Using growth and final height data of 92 patients (57 F/35 M) with CAH due to 21-hydroxylase deficiency (38 SV/54 SW), final height predictions with the B&P method were compared to actual final heights. RESULTS: In females, mean final height was 159.9 +/- 5.3 cm (-1.0 +/- 0.7 SDS) compared to predicted mean final height of 167.9 +/- 10.7 cm (+0.5 +/- 1.7 SDS), p < 0.001, overestimation 7.3 +/- 9.5 cm. In males, mean final height was 170.1 +/- 6 cm (-1.2 +/- 0.8 SDS) compared to predicted mean final height of 185.6 +/- 13.4 cm (+1.2 +/- 1.9 SDS), p < 0.001, overestimation 13.9 +/- 10.8 cm. CONCLUSION: In classical CAH, final height prediction using the B&P method results in significant overestimation of final height.
Assuntos
Hiperplasia Suprarrenal Congênita/diagnóstico , Hiperplasia Suprarrenal Congênita/fisiopatologia , Estatura , Pesos e Medidas Corporais/métodos , Estatística como Assunto/métodos , Determinação da Idade pelo Esqueleto/métodos , Estatura/fisiologia , Criança , Pré-Escolar , Feminino , Seguimentos , Previsões , Humanos , Lactente , Recém-Nascido , Masculino , Prognóstico , Reprodutibilidade dos TestesRESUMO
At final height, somatic maturity has not been reached yet. We investigated bone and body composition in patients, who completed pediatric growth hormone (GH) treatment at final height. After a mean period of 0.55 ± 0.17 yr off GH treatment 90 (66 m/24 f) childhood-onset growth hormone deficiency (GHD) patients were reinvestigated for GHD by insulin tolerance testing at a mean age of 17.52 ± 1.50 yr. Thirty-seven (25 m/12 f) patients remained GH deficient (persistent GHD). Bone and body composition were measured using peripheral quantitative computed tomography of the nondominant forearm. Bone mineral density (BMD) was within normal limits. Total cross-sectional bone area Z-score (0.64 ± 1.3) was significantly higher as a result of an enlarged medullary cavity Z-score (1.12 ± 1.2) leading to reduction of cortical thickness Z-score (-1.21 ± 1.0). Patients with persistent GHD had a significantly higher fat mass (13.3 ± 8.7 and 6.8 ± 4.6 cm(2), p<0.05), which was more pronounced in multiple pituitary hormone deficiency patients. Shortly after cessation of GH treatment in patients treated for childhood-onset GHD age adequate normal BMD and enlarged diaphysis was detectable. Patients with persistent GHD status had a significant higher fat mass.
Assuntos
Composição Corporal , Transtornos do Crescimento/tratamento farmacológico , Hormônio do Crescimento/deficiência , Adolescente , Criança , Feminino , Hormônio do Crescimento/uso terapêutico , Humanos , Lipídeos/sangue , Masculino , Rádio (Anatomia)/diagnóstico por imagem , Rádio (Anatomia)/fisiopatologia , Tomografia Computadorizada por Raios X/métodosRESUMO
The complex structure, large size, and multiple posttranslational modifications of von Willebrand factor (VWF) presented a technological challenge for the production of recombinant VWF (rVWF). Nonetheless, we developed an rVWF product for treating von Willebrand disease, whereupon rVWF is coexpressed with recombinant factor VIII (rFVIII) in Chinese hamster ovary cells used to produce rFVIII for the treatment of hemophilia A. Here we describe the characterization of the structure and function of the rVWF drug product, with a focus on its in vitro platelet aggregation and matrix protein binding functions. Electron microscopy and multimer analysis revealed a highly organized structure for the rVWF protein, with a homogeneous multimer distribution including ultrahigh molecular weight multimers. The specific activity for binding to collagen and platelets mediated by ristocetin is higher in rVWF than in commercial plasma-derived VWF-FVIII complex products. The affinity and binding capacity of rVWF to FVIII is comparable to VWF in plasma. rVWF effectively binds to platelets and promotes platelet adhesion under shear stress similar to VWF in human plasma.
Assuntos
Doenças de von Willebrand/tratamento farmacológico , Fator de von Willebrand/química , Fator de von Willebrand/farmacologia , Animais , Humanos , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacologiaRESUMO
Von Willebrand factor (VWF) is cleaved by the plasma metalloprotease ADAMTS13 ( A Disintegrin and Metalloproteinase with Thrombo Spondin repeats, number 13) that regulates the hemostatic activity of VWF by limiting its multimeric size in the human system. In vitro and ex vivo studies have shown that human recombinant VWF (rVWF) is virtually resistant to the proteolytic activity of murine ADAMTS13. In contrast, rabbit and cynomolgus ADAMTS13 is able to cleave human rVWF. These findings were consistent with in vivo results showing distinct pharmacological behavior of human rVWF depending on the cleaving capacity of ADAMTS13 present in the species tested. Studies were performed using three mouse strains (ADAMTS13 deficient, C57BL/6J [wild type], VWF deficient), rats, rabbits, and cynomolgus monkeys. All animals were infused once with different doses of human rVWF and, in addition, 14 daily doses were given to rats and cynomolgus monkeys. Exaggerated pharmacological effects were observed in mice, with the ADAMTS13 knockout mouse being the most sensitive strain. Similar findings with decreased incidence and severity were seen in normal C57BL/6J mice and also in VWF-deficient mice, where they were least pronounced. In rats, exaggerated pharmacological effects were observed only after 14 doses. Rabbits and cynomolgus monkeys showed no exaggerated pharmacological effects. These differences between species and between mouse strains suggest that the efficiency of ADAMTS13 to cleave rVWF determines the severity of clinical, laboratory and pathohistological findings. These observations highlight the importance of evaluating species' suitability for the generation of meaningful preclinical data for determining the therapeutic safety margins for human patients. Only animals with a sufficient rVWF cleavage capacity by endogenous ADAMTS13 (rabbits and cynomolgus monkeys) are considered appropriate animal models for preclinical evaluation of the rVWF product.
Assuntos
Proteínas ADAM/metabolismo , Fator de von Willebrand/farmacologia , Animais , Humanos , Proteínas Recombinantes/farmacologiaRESUMO
Von Willebrand factor (VWF), an adhesive glycoprotein with an approximate molecular weight (MW) of the monomer of 260 kDa, circulates in human blood plasma as a series of multimers ranging in size up to 20.000 kDa; thus the determination of the accurate MW of the monomer is of great importance and due to its high MW quite challenging. In this study accurate MW determination of intact recombinant VWF monomer (rVWF) was performed with GEMMA (gas-phase electrophoretic mobility macromolecular analysis) and MALDI TOF MS (matrix-assisted laser desorption/ionization linear time-of-flight mass spectrometry). Three rVWF preparations with differing buffer systems and glycoprotein concentrations were analyzed. First investigations directed towards heterogeneity determination by means of capillary gel electrophoresis (CGE)-on-the-chip with a laser-induced fluorescence detector revealed two compounds (MW of 277 kDa (migration time 44.3 s) and 341 kDa (migration time 49.5 s)) present in each sample to varying extents, namely mature and pro-rVWF. MALDI MS analysis in the linear positive ion mode allowed the detection of mature rVWF with an exact MW of 256.1 kDa (+/-0.8%) and pro-rVWF with a MW of 349.8 kDa (+/-0.8%). Two samples containing pro-rVWF in very minor concentration resulted in GEMMA detection of the mature rVWF with a MW of 227.4 kDa (+/-2.5%), derived from the measured globular size of 10.9 nm. For one sample containing both rVWF species in almost equal concentrations no differentiation of the two species was possible with GEMMA. Due to its lower resolution only a peak representing a mixture of both species at 11.8 nm could be observed, yielding a MW of 298.8 kDa (+/-1.6%).
Assuntos
Eletroforese em Microchip/métodos , Eletroforese em Gel de Poliacrilamida/métodos , Espectrometria de Massas por Ionização por Electrospray/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Fator de von Willebrand/química , Humanos , Peso Molecular , Proteínas Recombinantes/químicaRESUMO
CONTEXT: Cardiovascular disease caused by atherosclerosis is a major cause of morbidity and mortality in adult diabetic patients. In children, we detected signs of subclinical atherosclerosis in a large patient cohort. This study reports the results of a longitudinal observation in this patient group. PATIENTS AND METHODS: Of the 37/150 diabetic children in whom an increased intima-media thickness (IMT) of the carotid artery had been found, 27 (mean age 14.6 +/- 2.6 yrs) could be reevaluated 2 yrs after the initial study. Of the 27, 5 patients were on medication with angiotensin-converting enzyme (ACE) inhibitors, and all patients underwent detailed counselling of their lifestyle, sports activity, and nutritional habits. RESULTS: Mean IMT increased significantly (0.49 +/- 0.02 mm vs. 0.51 +/- 0.026 mm, p < 0.05) However, there was no significant change compared to normal values (mean IMT z-score 2.4 +/- 0.3 vs. 2.6 +/- 0.5). Of the 27, 13 patients (48%) showed a progression of the IMT whereas in 14/27 patients the IMT values remained stable. In these subgroups, patients with IMT progression showed a higher hemoglobin A1c (HbA1c) (7.5 +/- 0.8 vs. 7.1 +/- 0.7, p < 0.05) and a slightly higher systolic blood pressure (120 +/- 14.4 vs. 113.9 +/- 12.1, p = 0.08). CONCLUSIONS: In a well-selected group of diabetic children, mean IMT progression during a 2-yr period did not exceed the physiological increase. Children with a higher HbA1c and a higher systolic blood pressure showed a progression of the IMT. Control of atherogenic risk factors in diabetic children may help to avoid subclinical atherosclerosis progression.
Assuntos
Doenças das Artérias Carótidas/epidemiologia , Diabetes Mellitus Tipo 1/epidemiologia , Angiopatias Diabéticas/epidemiologia , Adolescente , Doenças das Artérias Carótidas/diagnóstico por imagem , Artéria Carótida Primitiva/diagnóstico por imagem , Artéria Carótida Primitiva/patologia , Criança , Progressão da Doença , Feminino , Humanos , Estudos Longitudinais , Masculino , Túnica Íntima/patologia , Túnica Média/patologia , UltrassonografiaRESUMO
Diamond-Blackfan anemia (DBA) is a rare disorder characterized by congenital pure red cell aplasia. Mutations in ribosomal protein S19 (RPS19) have been identified in 25% of DBA patients. More recently, mutations in other ribosomal protein genes, namely RPS7, RPS15, RPS24, RPS17, RPS27A, RPL35a, RPL36, RPL11, and RPL5, have also been found in patients with DBA. Approximately 30-40% of affected patients have various associated physical anomalies, mostly craniofacial and at the extremities, but also cardiac or urogenital malformations. Anomalies of the urogenital tract in DBA patients comprise changes in the kidney (dysplasia, agenesis, duplication, horseshoe kidney) and genitalia (hypospadias). To date, disorders of sex development (DSD) have only been described once in association with DBA. We report here four DBA patients who exhibited DSD.
Assuntos
Anemia de Diamond-Blackfan/complicações , Transtornos do Desenvolvimento Sexual/complicações , Proteínas Ribossômicas/genética , Anormalidades Múltiplas/genética , Anormalidades Múltiplas/patologia , Anemia de Diamond-Blackfan/genética , Anemia de Diamond-Blackfan/patologia , Transtornos do Desenvolvimento Sexual/genética , Transtornos do Desenvolvimento Sexual/patologia , Idade Gestacional , Humanos , Recém-Nascido , Masculino , MutaçãoAssuntos
Anti-Inflamatórios não Esteroides/imunologia , Doenças Autoimunes/terapia , Imunoglobulinas Intravenosas/imunologia , Imunoglobulinas/imunologia , Imunoterapia , Doenças Neurodegenerativas/imunologia , Receptores Fc/imunologia , Animais , Anti-Inflamatórios não Esteroides/uso terapêutico , Autoanticorpos/imunologia , Doenças Autoimunes/imunologia , Ensaios Clínicos como Assunto , Modelos Animais de Doenças , Humanos , Imunoglobulinas/uso terapêutico , Imunoglobulinas Intravenosas/uso terapêutico , Camundongos , Doenças Neurodegenerativas/terapia , Inflamação Neurogênica , Estados UnidosRESUMO
OBJECTIVES: To evaluate insulin sensitivity in short children born small for gestational age (SGA) treated with growth hormone (GH), and to study the relationship between growth response and insulin levels. STUDY DESIGN: In 29 children (16 female, 13 male) who were short and SGA, an oral glucose tolerance test was performed before (mean age, 8.8 years; range, 4.5-14.3 years) and after 1 year of GH treatment (33 microg/kg/day). Insulin sensitivity was calculated with the homeostasis model assessment (HOMA) and the insulin sensitivity index (ISI) of Matsuda. RESULTS: The mean height increased from -3.1 to -2.4 SD. Insulin resistance (ISI<5) was seen in 17.2% of children before and in 48.3% (mainly pubertal) children after GH treatment. Insulin sensitivity decreased significantly: ISI fell from 12.2 to 6.1 (P= .02) and HOMA increased from 1.2 to 2.2 (P= .001). Glucose and HbA1c levels did not change significantly. ISI after 1 year did not correlate with height gain, but it did correlate with age (r= -0.469; P= .01) and body mass index (r= -0.52; P= .004). CONCLUSIONS: Insulin sensitivity is impaired in some children who are SGA already at baseline and decreases with GH treatment in most of them. Children close to puberty and children who are less underweight have the highest risk to become insulin resistant.