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1.
Int J Mol Sci ; 25(5)2024 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-38474138

RESUMO

Antithrombin (AT) is the major plasma inhibitor of thrombin (FIIa) and activated factor X (FXa), and antithrombin deficiency (ATD) is one of the most severe thrombophilic disorders. In this study, we identified nine novel AT mutations and investigated their genotype-phenotype correlations. Clinical and laboratory data from patients were collected, and the nine mutant AT proteins (p.Arg14Lys, p.Cys32Tyr, p.Arg78Gly, p.Met121Arg, p.Leu245Pro, p.Leu270Argfs*14, p.Asn450Ile, p.Gly456delins_Ala_Thr and p.Pro461Thr) were expressed in HEK293 cells; then, Western blotting, N-Glycosidase F digestion, and ELISA were used to detect wild-type and mutant AT. RT-qPCR was performed to determine the expression of AT mRNA from the transfected cells. Functional studies (AT activity in the presence and in the absence of heparin and heparin-binding studies with the surface plasmon resonance method) were carried out. Mutations were also investigated by in silico methods. Type I ATD caused by altered protein synthesis (p.Cys32Tyr, p.Leu270Argfs*14, p.Asn450Ile) or secretion disorder (p.Met121Arg, p.Leu245Pro, p.Gly456delins_Ala_Thr) was proved in six mutants, while type II heparin-binding-site ATD (p.Arg78Gly) and pleiotropic-effect ATD (p.Pro461Thr) were suggested in two mutants. Finally, the pathogenic role of p.Arg14Lys was equivocal. We provided evidence to understand the pathogenic nature of novel SERPINC1 mutations through in vitro expression studies.


Assuntos
Deficiência de Antitrombina III , Antitrombinas , Humanos , Antitrombinas/química , Células HEK293 , Anticoagulantes , Heparina/metabolismo , Mutação , Deficiência de Antitrombina III/genética
2.
Int J Mol Sci ; 20(23)2019 Nov 27.
Artigo em Inglês | MEDLINE | ID: mdl-31783511

RESUMO

Cellular factor XIII (cFXIII, FXIII-A2), a transglutaminase, has been demonstrated in a few cell types. Its main function is to cross-link proteins by isopeptide bonds. Here, we investigated the presence of cFXIII in cells of human cornea. Tissue sections of the cornea were immunostained for FXIII-A in combination with staining for CD34 antigen or isopeptide cross-links. Isolated corneal keratocytes were also evaluated by immunofluorescent microscopy and flow cytometry. FXIII-A in the corneal stroma was quantified by Western blotting. FXIII-A mRNA was detected by RT-qPCR. The cornea of FXIII-A-deficient patients was evaluated by cornea topography. FXIII-A was detected in 68 ± 13% of CD34+ keratocytes. Their distribution in the corneal stroma was unequal; they were most abundant in the subepithelial tertile. cFXIII was of cytoplasmic localization. In the stroma, 3.64 ng cFXIII/mg protein was measured. The synthesis of cFXIII by keratocytes was confirmed by RT-qPCR. Isopeptide cross-links were detected above, but not within the corneal stroma. Slight abnormality of the cornea was detected in six out of nine FXIII-A-deficient patients. The presence of cFXIII in human keratocytes was established for the first time. cFXIII might be involved in maintaining the stability of the cornea and in the corneal wound healing process.


Assuntos
Ceratócitos da Córnea/metabolismo , Substância Própria/metabolismo , Fator XIII/metabolismo , Transglutaminases/metabolismo , Testes de Coagulação Sanguínea/métodos , Lesões da Córnea/metabolismo , Humanos , RNA Mensageiro/metabolismo , Cicatrização/fisiologia
3.
Ann Hematol ; 95(7): 1077-87, 2016 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-27130310

RESUMO

The effects of romiplostim on bone marrow morphology were evaluated in adults with immune thrombocytopenia (ITP). Patients with platelet counts <50 × 10(9)/L, ≥1 prior ITP therapies, and no collagen at baseline received weekly subcutaneous romiplostim starting at 1 µg/kg, adjusted to maintain platelet counts between 50 and 200 × 10(9)/L. Biopsies were scheduled after 1, 2, or 3 years of romiplostim (cohorts 1, 2, and 3, respectively). Irrespective of scheduled time, biopsies were performed earlier if patients discontinued or failed to achieve/maintain a response to romiplostim. Reticulin (silver stain) and collagen (trichrome stain) were graded by two hematopathologists using the modified Bauermeister scale (0-4). Of 169 patients, 131 had evaluable biopsies; 9/131 (6.9 %) had increases of ≥2 grades on the modified Bauermeister scale (cohort 1: 0/34; cohort 2: 2/39; cohort 3: 7/58), including two with collagen. Three of the nine patients had follow-up biopsies, including one patient with collagen; changes were reversible after romiplostim discontinuation. Of the nine patients, one had neutropenia detected by laboratory test and two had adverse events of anemia, both non-serious and not treatment-related. By actual exposure (as some biopsies did not occur as scheduled), the number of patients with grade increases ≥2 were year 1: 3/41, year 2: 1/38, year 3: 5/52. Twenty-four patients sustained platelet counts ≥50 × 10(9)/L for ≥6 months with no ITP medications after discontinuing romiplostim, i.e., they entered clinical remission of their ITP. In conclusion, in patients with ITP receiving romiplostim, bone marrow changes were observed in a small proportion of patients.ClinicalTrials.gov identifier: NCT#00907478.


Assuntos
Medula Óssea/efeitos dos fármacos , Medula Óssea/patologia , Púrpura Trombocitopênica Idiopática/tratamento farmacológico , Púrpura Trombocitopênica Idiopática/patologia , Receptores Fc/uso terapêutico , Proteínas Recombinantes de Fusão/uso terapêutico , Trombopoetina/uso terapêutico , Adulto , Idoso , Medula Óssea/metabolismo , Estudos de Coortes , Colágeno/metabolismo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Contagem de Plaquetas/métodos , Estudos Prospectivos , Púrpura Trombocitopênica Idiopática/metabolismo , Proteínas Recombinantes de Fusão/efeitos adversos , Reticulina/metabolismo , Trombopoetina/efeitos adversos , Resultado do Tratamento
4.
Orv Hetil ; 157(12): 443-50, 2016 Mar 20.
Artigo em Húngaro | MEDLINE | ID: mdl-26971644

RESUMO

Only vitamin K antagonists could be applied as oral anticoagulants over the past six decades. Coumarols have narrow therapeutic range, and unpredictable anticoagulant effects are resulted by multiple drug interactions. Therefore, regular routine monitoring of the international normalized ratio is necessary. There are two groups of factor-specific anticoagulants: molecules with anti-FIIa (dabigatran) and anti-FXa (rivaroxaban, apixaban and edoxaban) effect. Author summarizes the most important clinical features of the new oral anticoagulants, their indications and the possibilities of laboratory controls. Bleedings are the most important side effects of anticoagulants. This review summarizes the current published evidences for new oral anticoagulants reversal (non-specific and specific) agents, especially in cases with severe acute bleedings or urgent surgery procedures. It reports on how to use inhibitors, the recommended doses and the most important clinical results. The review focuses on idarucizumab - already approved by the U.S. Food and Drug Administration and the European Medicines Agency - which has a key role as the first specific inhibitor of dabigatran.


Assuntos
Anticorpos Monoclonais Humanizados/uso terapêutico , Anticoagulantes/administração & dosagem , Anticoagulantes/efeitos adversos , Antídotos/uso terapêutico , Dabigatrana/antagonistas & inibidores , Hemorragia/tratamento farmacológico , Procedimentos Cirúrgicos Operatórios , Doença Aguda , Administração Oral , Assistência Ambulatorial , Antitrombinas/administração & dosagem , Antitrombinas/efeitos adversos , Dabigatrana/administração & dosagem , Dabigatrana/efeitos adversos , Fator Xa , Inibidores do Fator Xa/administração & dosagem , Inibidores do Fator Xa/efeitos adversos , Hemorragia/induzido quimicamente , Humanos , Pirazóis/antagonistas & inibidores , Piridinas/antagonistas & inibidores , Piridonas/antagonistas & inibidores , Proteínas Recombinantes , Rivaroxabana/antagonistas & inibidores , Índice de Gravidade de Doença , Tiazóis/antagonistas & inibidores
5.
Clin Chem Lab Med ; 52(12): 1797-806, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24968404

RESUMO

BACKGROUND: Antithrombin (AT) is a slow-acting progressive inhibitor of activated clotting factors, particularly thrombin and activated factor X (FXa). However, the presence of heparin or heparan sulfate accelerates its effect by several magnitudes. AT deficiency, a severe thrombophilia, is classified as type I (quantitative) and type II (qualitative) deficiency. In the latter case mutations may influence the reactive site, the heparin binding-site (HBS) and exert pleiotropic effect. Heterozygous type II-HBS deficiency is a less severe thrombophilia than other heterozygous subtypes. However, as opposed to other subtypes, it also exists in homozygous form which represents a very high risk of venous thromboembolism. METHODS: A modified anti-FXa chromogenic AT assay was developed which determines both the progressive (p) and the heparin cofactor (hc) activities, in parallel. The method was evaluated and reference intervals were established. The usefulness of the assay in detecting type II-HBS AT deficiency was tested on 78 AT deficient patients including 51 type II-HBS heterozygotes and 18 homozygotes. RESULTS: Both p-anti-FXa and hc-anti-FXa assays showed excellent reproducibility and were not influenced by high concentrations of triglyceride, bilirubin and hemoglobin. Reference intervals for p-anti-FXa and hc-anti-FXa AT activities were 84%-117% and 81%-117%, respectively. Type II-HBS deficient patients demonstrated low (heterozygotes) or very low (homozygotes) hc-anti-FXa activity with normal or slightly decreased p-anti-FXa activity. The p/hc ratio clearly distinguished wild type controls, type II-HBS heterozygotes and homozygotes. CONCLUSIONS: Concomitant determination of p-anti-FXa and hc-anti-FXa activities provides a reliable, clinically important diagnosis of type II-HBS AT deficiency and distinguishes between homozygotes and heterozygotes.


Assuntos
Deficiência de Antitrombina III/diagnóstico , Antitrombina III/análise , Fator Xa/metabolismo , Antitrombina III/genética , Antitrombina III/normas , Deficiência de Antitrombina III/classificação , Bilirrubina/química , Testes de Coagulação Sanguínea/normas , Fator Xa/química , Hemoglobinas/química , Heparina/química , Heterozigoto , Homozigoto , Humanos , Masculino , Polimorfismo de Nucleotídeo Único , Valores de Referência , Triglicerídeos/química
6.
Blood Adv ; 8(6): 1405-1414, 2024 Mar 26.
Artigo em Inglês | MEDLINE | ID: mdl-38237075

RESUMO

ABSTRACT: Long-term prophylaxis with a von Willebrand factor (VWF) concentrate is recommended in patients with von Willebrand disease (VWD) who have a history of severe and frequent bleeds. However, data from prospective studies are scarce. WIL-31, a prospective, noncontrolled, international phase 3 trial, investigated the efficacy and safety of Wilate prophylaxis in severe patients with VWD. Male and female patients 6 years or older with VWD types 1, 2 (except 2N), or 3 who had completed a prospective, 6-month, on-demand, run-in study (WIL-29) were eligible to receive Wilate prophylaxis for 12 months. At baseline, patients (n = 33) had a median age of 18 years. Six (18%) patients had severe type 1, 5 (15%) had type 2, and 22 (67%) had type 3 VWD. The primary end point of a >50% reduction in mean total annualized bleeding rate (TABR) with Wilate prophylaxis vs prior on-demand treatment was met; mean TABR during prophylaxis was 5.2, representing an 84.4% reduction. The bleeding reduction was consistent across age, sex, and VWD types. The mean spontaneous ABR was 3.2, representing an 86.9% reduction vs on-demand treatment. During prophylaxis, 10 (30.3%) patients had 0 bleeding events and 15 (45.5%) patients had 0 spontaneous bleeding events. Of 173 BEs, 84.4% were minor and 69.9% treated. No serious adverse events related to study treatment and no thrombotic events were recorded. Overall, WIL-31 showed that Wilate prophylaxis was efficacious and well-tolerated in pediatric and adult patients with VWD of all types. The WIL-29 and WIL-31 trials were registered at www.ClinicalTrials.gov as #NCT04053699 and #NCT04052698, respectively.


Assuntos
Doenças de von Willebrand , Fator de von Willebrand , Adulto , Humanos , Masculino , Feminino , Criança , Adolescente , Fator de von Willebrand/efeitos adversos , Fator VIII/efeitos adversos , Doenças de von Willebrand/tratamento farmacológico , Estudos Prospectivos , Hemorragia/prevenção & controle , Hemorragia/induzido quimicamente
7.
N Engl J Med ; 363(13): 1222-32, 2010 Sep 23.
Artigo em Inglês | MEDLINE | ID: mdl-20860504

RESUMO

BACKGROUND: The efficacy and safety of anticoagulant treatment for patients with acute, symptomatic superficial-vein thrombosis in the legs, but without concomitant deep-vein thrombosis or symptomatic pulmonary embolism at presentation, have not been established. METHODS: In a randomized, double-blind trial, we assigned 3002 patients to receive either fondaparinux, administered subcutaneously at a dose of 2.5 mg once daily, or placebo for 45 days. The primary efficacy outcome was a composite of death from any cause or symptomatic pulmonary embolism, symptomatic deep-vein thrombosis, or symptomatic extension to the saphenofemoral junction or symptomatic recurrence of superficial-vein thrombosis at day 47. The main safety outcome was major bleeding. The patients were followed until day 77. RESULTS: The primary efficacy outcome occurred in 13 of 1502 patients (0.9%) in the fondaparinux group and 88 of 1500 patients (5.9%) in the placebo group (relative risk reduction with fondaparinux, 85%; 95% confidence interval [CI], 74 to 92; P<0.001). The incidence of each component of the primary efficacy outcome was significantly reduced in the fondaparinux group as compared with the placebo group, except for the outcome of death (0.1% in both groups). The rate of pulmonary embolism or deep-vein thrombosis was 85% lower in the fondaparinux group than in the placebo group (0.2% vs. 1.3%; 95% CI, 50 to 95; P<0.001). Similar risk reductions were observed at day 77. A total of 88 patients would need to be treated to prevent one instance of pulmonary embolism or deep-vein thrombosis. Major bleeding occurred in one patient in each group. The incidence of serious adverse events was 0.7% with fondaparinux and 1.1% with placebo. CONCLUSIONS: Fondaparinux at a dose of 2.5 mg once a day for 45 days was effective in the treatment of patients with acute, symptomatic superficial-vein thrombosis of the legs and did not have serious side effects. (Funded by GlaxoSmithKline; ClinicalTrials.gov number, NCT00443053.)


Assuntos
Anticoagulantes/uso terapêutico , Polissacarídeos/uso terapêutico , Trombose Venosa/tratamento farmacológico , Doença Aguda , Anticoagulantes/administração & dosagem , Anticoagulantes/efeitos adversos , Método Duplo-Cego , Feminino , Fondaparinux , Hemorragia/induzido quimicamente , Humanos , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Polissacarídeos/administração & dosagem , Polissacarídeos/efeitos adversos , Embolia Pulmonar/epidemiologia , Embolia Pulmonar/prevenção & controle , Recidiva , Risco , Resultado do Tratamento , Trombose Venosa/complicações , Trombose Venosa/mortalidade , Trombose Venosa/cirurgia
8.
Platelets ; 23(6): 495-8, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22309045

RESUMO

Heparin-induced thrombocytopenia (HIT) is one of the most common immune-mediated drug reactions. Immunoglobulin G-type antibodies against platelet factor 4(PF4)/heparin complexes are known to play a key role in the pathogenesis of HIT. Rapid-onset HIT is caused by the presence of circulating HIT antibodies at the time of heparin readministration. These antibodies are generally resulted from a recent immunizing exposure to heparin. Here we report a case of rapid-onset HIT developed after a septicemia without previous heparin exposure. The diagnosis of HIT as well as the presence of platelet activating and heparin-dependent antibodies was confirmed by ELISA and flow cytometric functional assays. Our case report reinforces that rapid-onset HIT cannot be excluded only based on the absence of previous heparin exposure. In addition, it may support the new theory of pre-immunization by PF4-coated bacteria in the pathomechanism of HIT. We also call the attention that venous limb gangrene can be rarely associated with HIT and thrombosis even in the absence of coumarin therapy. Furthermore, transient presence of anti-phospholipid antibodies can cause a differential diagnostic problem in the cases of HIT.


Assuntos
Anticoagulantes/efeitos adversos , Enoxaparina/efeitos adversos , Extremidades/patologia , Gangrena/induzido quimicamente , Trombocitopenia/induzido quimicamente , Trombose Venosa/induzido quimicamente , Anticorpos Antifosfolipídeos/sangue , Anticorpos Antifosfolipídeos/imunologia , Anticoagulantes/administração & dosagem , Complexo Antígeno-Anticorpo/sangue , Complexo Antígeno-Anticorpo/imunologia , Enoxaparina/administração & dosagem , Feminino , Gangrena/imunologia , Humanos , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Pessoa de Meia-Idade , Ativação Plaquetária , Fator Plaquetário 4/sangue , Fator Plaquetário 4/imunologia , Sepse/imunologia , Sepse/microbiologia , Staphylococcus epidermidis/crescimento & desenvolvimento , Trombocitopenia/imunologia , Trombose Venosa/imunologia
9.
Orv Hetil ; 153(13): 483-98, 2012 Apr 01.
Artigo em Húngaro | MEDLINE | ID: mdl-22430004

RESUMO

Atherosclerosis is a systemic disease affecting the coronary, carotid, intracerebral, renal and peripherial arteries. The early morphological and functional impairments could be detected in the second or third decades of life and their progression depend on the number and severity of risk factors and individual susceptility. Although the vascular risk factors (smoking, overweight, age, unhealthy diet, lack of physical exercise, hypertension, diabetes mellitus, chronic kidney disease and dyslipidemia) are the same and common in the different vascular diseases, the present clinical routine artificially classifies the diagnosis and therapy of different vascular diseases into different subfields of medicine with the negative impact of possible polypragmasia. Recently, worldwide health surveys (e.g. REACH registry) have proven the usefulness of a holistic approach in the diagnosis and therapy of multiorgan-affected vascular patients. This review summarizes the multidisciplinary advances and future perspective of vascular diseases.


Assuntos
Doenças Autoimunes/complicações , Diabetes Mellitus Tipo 2/complicações , Dislipidemias/complicações , Nefropatias/complicações , Doenças Vasculares/diagnóstico , Doenças Vasculares/terapia , Síndrome Coronariana Aguda/diagnóstico , Síndrome Coronariana Aguda/terapia , Fibrilação Atrial/diagnóstico , Fibrilação Atrial/terapia , Doenças Autoimunes/diagnóstico , Doenças Autoimunes/terapia , Estenose das Carótidas/diagnóstico , Estenose das Carótidas/terapia , Doença Crônica , Complicações do Diabetes/diagnóstico , Complicações do Diabetes/terapia , Dislipidemias/diagnóstico , Dislipidemias/terapia , Humanos , Hipertensão/diagnóstico , Hipertensão/terapia , Comunicação Interdisciplinar , Nefropatias/diagnóstico , Nefropatias/fisiopatologia , Nefropatias/terapia , Fatores de Risco , Doenças Vasculares/complicações , Doenças Vasculares/etiologia , Doenças Vasculares/imunologia , Doenças Vasculares/patologia , Doenças Vasculares/prevenção & controle
10.
Blood ; 113(3): 723-5, 2009 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-18955560

RESUMO

Acquired factor XIII (FXIII) deficiency due to autoantibody against FXIII is a very rare severe hemorrhagic diathesis. Antibodies directed against the A subunit of FXIII, which interfere with different functions of FXIII, have been described. Here, for the first time, we report an autoantibody against the B subunit of FXIII (FXIII-B) that caused life-threatening bleeding in a patient with systemic lupus erythematosus. FXIII activity, FXIII-A(2)B(2) complex, and individual FXIII subunits were undetectable in the plasma, whereas platelet FXIII activity and antigen were normal. Neither FXIII activation nor its activity was inhibited by the antibody, which bound to structural epitope(s) on both free and complexed FXIII-B. The autoantibody highly accelerated the elimination of FXIII from the circulation. FXIII supplementation combined with immunosuppressive therapy, plasmapheresis, immunoglobulin, and anti-CD20 treatment resulted in the patient's recovery. FXIII levels returned to around 20% at discharge and after gradual increase the levels stabilized above 50%.


Assuntos
Autoanticorpos/imunologia , Autoantígenos/imunologia , Deficiência do Fator XIII/imunologia , Fator XIII/imunologia , Hemorragia/imunologia , Adulto , Anticorpos Monoclonais/uso terapêutico , Anticorpos Monoclonais Murinos , Western Blotting , Ensaio de Imunoadsorção Enzimática , Fator XIII/análise , Deficiência do Fator XIII/terapia , Feminino , Humanos , Imunoglobulinas Intravenosas , Fatores Imunológicos/uso terapêutico , Imunossupressores/uso terapêutico , Falência Renal Crônica/etiologia , Lúpus Eritematoso Sistêmico/complicações , Plasmaferese , Diálise Renal , Rituximab
12.
Orv Hetil ; 151(34): 1365-74, 2010 Aug 22.
Artigo em Húngaro | MEDLINE | ID: mdl-20705551

RESUMO

A large proportion of hospitalized surgical and medical patients are at risk for venous thromboembolism. Depending on the type of surgical intervention, venous thrombosis develops in 15-60% of surgical patients without prophylaxis. Although venous thromboembolism is most often considered to be associated with recent surgery or trauma, 50 to 70% of symptomatic thromboembolic events and 70 to 80% of fatal pulmonary embolisms occur in nonsurgical patients. International and national registries show that the majority of at-risk surgical patients actually received the appropriate thromboembolic prophylaxis. However, despite of international and national recommendations, prophylaxis was not provided for a large proportion of at-risk medical patients. The rate of medical patients receiving prophylaxis should be increased, and appropriate thrombosis prophylaxis should be offered to at-risk medical patients. The thrombosis risk assessment is an important tool to identify patients at increased risk for venous thromboembolism, to simplify decision making on prophylaxis administration, and to improve the adherence to guidelines. When the risk is recognized, if there is no contraindication, prophylaxis should be ordered. The 4th Hungarian Antithrombotic Guideline entitled "Risk reduction and treatment of thromboembolism" calls attention to the importance of risk assessment and for the first time it includes and recommends risk assessment models for hospitalized surgical and medical patients. The risk assessment models are presented and the evidence based data for the different risk factors included in these models are reviewed.


Assuntos
Hospitalização , Procedimentos Cirúrgicos Operatórios/efeitos adversos , Inquéritos e Questionários/normas , Tromboembolia Venosa/etiologia , Tromboembolia Venosa/prevenção & controle , Fatores Etários , Anticoagulantes/uso terapêutico , Doenças Autoimunes/complicações , Índice de Massa Corporal , Anticoncepcionais Orais Hormonais/efeitos adversos , Predisposição Genética para Doença , Humanos , Hungria , Infecções/complicações , Doenças Inflamatórias Intestinais/complicações , Locomoção , Mutação , Neoplasias/complicações , Nefrose/complicações , Obesidade/complicações , Guias de Prática Clínica como Assunto , Prevenção Primária/métodos , Doença Pulmonar Obstrutiva Crônica/complicações , Sistema de Registros , Insuficiência Respiratória/complicações , Medição de Risco , Fatores de Risco , Varizes/complicações , Insuficiência Venosa/complicações , Tromboembolia Venosa/tratamento farmacológico , Tromboembolia Venosa/genética
13.
Stem Cells Int ; 2020: 8847038, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33144864

RESUMO

PURPOSE: This study is aimed at investigating the phenotype, differentiation potential, immunomodulatory properties, and responsiveness of saphenous vein vessel wall-derived mesenchymal stromal cells (SV-MSCs) to various TLR ligands and proinflammatory cytokines, as well as comparing their features to those of their bone marrow-derived counterparts (BM-MSCs). METHODS: SV-MSCs were isolated by enzymatic digestion of the saphenous vein vessel wall. Phenotype analysis was carried out by flow cytometry and microscopy, whereas adipogenic, chondrogenic, and osteogenic differentiation potentials were tested in in vitro assays. For comparative analysis, the expression of different stemness, proliferation, and differentiation-related genes was determined by Affymetrix gene array. To compare the immunomodulatory properties of SV-MSCs and BM-MSCs, mixed lymphocyte reaction was applied. To investigate their responses to various activating stimuli, MSCs were treated with TLR ligands (LPS, PolyI:C) or proinflammatory cytokines (TNFα, IL-1ß, IFNγ), and the expression of various early innate immune response-related genes was assessed by qPCR, while secretion of selected cytokines and chemokines was measured by ELISA. RESULTS: The isolated SV-MSCs were able to differentiate into bone, fat, and cartilage cells/direction in vitro. SV-MSCs expressed the most important MSC markers (CD29, CD44, CD73, CD90, and CD105) and shared almost identical phenotypic characteristics with BM-MSCs. Their gene expression pattern and activation pathways were close to those of BM-MSCs. SV-MSCs showed better immunosuppressive activity inhibiting phytohemagglutinin-induced T lymphocyte proliferation in vitro than BM-MSCs. Cellular responses to treatments mimicking inflammatory conditions were comparable in the bone marrow- and saphenous vein-derived MSCs. Namely, similar to BM-MSCs, SV-MSCs secreted increased amount of IL-6 and IL-8 after 12- or 24-hour treatment with LPS, PolyI:C, TNFα, or IL-1ß, compared to untreated controls. Interestingly, a different CXCL-10/IP-10 secretion pattern could be observed under inflammatory conditions in the two types of MSCs. CONCLUSION: Based on our results, cells isolated from saphenous vein vessel wall fulfilled the ISCT's (International Society for Cellular Therapy) criteria for multipotent mesenchymal stromal cells, and no significant differences in the phenotype, gene expression pattern, and responsiveness to inflammatory stimuli could be observed between BM-MSCs and SV-MSCs, while the latter cells have more potent immunosuppressive activity in vitro. Further functional assays have to be performed to reveal whether SV-MSCs could be useful for certain regenerative therapeutic applications or tissue engineering purposes.

14.
Orv Hetil ; 149(12): 531-40, 2008 Mar 23.
Artigo em Húngaro | MEDLINE | ID: mdl-18343769

RESUMO

BACKGROUND AND AIMS: Amputation is the only current option for relief of rest pain or gangrene in patients with severe peripheral arterial disease. Up to now, no effective blood-flow enhancement therapies are available. Autologous bone marrow-derived stem cell transplantation is an arising therapy modality with an option of building new blood vessels through endothelial stem and/or progenitor cells. PATIENTS AND METHODS: Five patients with severe peripheral arterial disorder were treated by autologous bone marrow-derived stem cell therapy. CD34+, CD133+ and CD45+/- cell number and ratio were determined. CD34+ cells were isolated by magnetic separation and collected into a 10 ml sample. The cell suspension was administered by local intramuscular injections (0.5-1.0 ml injections in the musculus gastrocnemius). The follow-up (before; 1, 3, 6, 9 and 12 months after the autologous bone marrow-derived stem cell therapy) based on clinical (rest pain, walking distance without pain, changes of non-healing ischaemic ulcers, ankle-brachial index) and laboratory (angiography, Color- and Laser-Doppler scan, measurement of transcutaneous oxygen tension and endothelial function test) parameters was documented and analyzed. RESULTS: Improvement of pain and walking distance was observed in all five cases. In three cases the non-healing ischaemic ulcers disappeared, in one other case they became smaller and thinner, and in one case no change was realized. The average of ankle-brachial index improved significantly (before: 0.41, twelve months after: 0.83). New collaterals were detected by angiography in three patients, but duplex ultrasonography detected improvement in one patient only. Before and 1, 6 and 12 months after stem cell therapy the transcutaneous oxygen tension changed on the foot from 18.80/16.78/23.83/37.50 mmHg, and on the calf from 36.66/31.25/45.00/37.30 mmHg. The macro- and microcirculation parameters did not show improvement after 1 month, however, after 3, 6, 9 and 12 months improved parameters were recorded. Severe adverse events were not observed. In one case elevated level of serum creatinine phosphokinase, and in another case a mild form of vasculitis were detected. CONCLUSION: autologous bone marrow-derived stem cell therapy with isolated CD34+ cells is effective, safe and results in sustained clinical benefit for patients with severe peripheral arterial disease.


Assuntos
Transplante de Medula Óssea , Perna (Membro)/irrigação sanguínea , Doenças Vasculares Periféricas/cirurgia , Transplante de Células-Tronco , Adulto , Angiografia , Antígenos CD34/análise , Biomarcadores/sangue , Pressão Sanguínea , Endotélio Vascular/fisiopatologia , Feminino , Humanos , Perna (Membro)/cirurgia , Úlcera da Perna/etiologia , Úlcera da Perna/cirurgia , Antígenos Comuns de Leucócito/análise , Masculino , Pessoa de Meia-Idade , Dor/etiologia , Doenças Vasculares Periféricas/sangue , Doenças Vasculares Periféricas/complicações , Doenças Vasculares Periféricas/diagnóstico por imagem , Doenças Vasculares Periféricas/fisiopatologia , Descanso , Índice de Gravidade de Doença , Fatores de Tempo , Resultado do Tratamento , Ultrassonografia Doppler , Caminhada
15.
Blood Coagul Fibrinolysis ; 29(3): 282-287, 2018 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-29474202

RESUMO

: In acquired haemophilia A (AHA), risk for recurrent bleeding exists until the inhibitor is detectable. Thus, patients with persisting inhibitor may benefit from prophylaxis with activated prothrombin complex concentrate (aPCC). Potential thromboembolic complications and cost are also factors to consider. Today, no high level evidence or clear recommendations are available on aPCC prophylaxis in AHA. Recently, a small prospective study demonstrated a favourable outcome with short-term, daily administered aPCC infusion. Here we report a retrospective case series of 19 patients with AHA to demonstrate our practice on aPCC prophylaxis. In our practice, clinical bleeding tendency guided our decision on the initiation of aPCC prophylaxis. In patients with serious bleeding tendency, aPCC infusion was prolonged beyond bleeding resolution in a twice-weekly or thrice-weekly regimen. Serious bleeding phenotype included a single episode of life-threatening bleeding or recurrent, severe haemorrhages. Patients who did not present such events were treated on-demand. The preventive dose of aPCC was equal with the lowest effective therapeutic dose. Prophylaxis was continued until the inhibitor disappeared. Eleven patients received aPCC prophylaxis. In nine cases, prophylaxis lasted beyond two months. No severe bleeding developed spontaneously and no thromboembolic complication occurred in the median 16 weeks (interquartile range 9-34) duration of prophylaxis. Eight patients of the nonprophylaxis group did not present any severe haemorrhage. According to our experience, we consider prophylaxis with aPCC effective and well tolerated for patients with AHA and serious bleeding tendency, until the acquired inhibitor persists.


Assuntos
Fatores de Coagulação Sanguínea/uso terapêutico , Hemofilia A/tratamento farmacológico , Pré-Medicação/métodos , Idoso , Inibidores dos Fatores de Coagulação Sanguínea/sangue , Fatores de Coagulação Sanguínea/efeitos adversos , Análise Custo-Benefício , Hemofilia A/imunologia , Hemorragia/prevenção & controle , Humanos , Pessoa de Meia-Idade , Estudos Retrospectivos , Centros de Atenção Terciária , Tromboembolia/induzido quimicamente
17.
Haematologica ; 92(5): 698-701, 2007 May.
Artigo em Inglês | MEDLINE | ID: mdl-17488698

RESUMO

In the platelets of a type II Glanzmann thrombasthenia patient, the amount of glycoprotein (GP) IIb and IIIa was significantly reduced. Three novel mutations were identified in the GPIIb gene (c.440C->G/p.Leu116Val, c.1772_1773insG/p.Asp560GlyfsX16 and c.2438C->A/p.His782Asn). p.Leu116Val did not represent a causative mutation. The c.1772_1773insG mutation resulted in an early stop codon and non-sense mediated decay of mRNA. When expressed in transfected BHK cells, the truncated protein was unable to form complex with GPIIIa. The p.His782Asn mutation compromised transport of the pro-GPIIb/IIIa complex from the endoplasmic reticulum to the Golgi, hindering its maturation and surface expression.


Assuntos
Integrina alfa2/genética , Trombastenia/genética , Animais , Linhagem Celular , Códon sem Sentido , Cricetinae , Análise Mutacional de DNA , Retículo Endoplasmático/metabolismo , Fibrinogênio/metabolismo , Genótipo , Complexo de Golgi/metabolismo , Humanos , Integrina alfa2/química , Integrina beta3/metabolismo , Masculino , Mesocricetus , Pessoa de Meia-Idade , Ligação Proteica , Mapeamento de Interação de Proteínas , Processamento de Proteína Pós-Traducional/genética , Estrutura Terciária de Proteína , Transporte Proteico/genética , Proteínas Recombinantes de Fusão/metabolismo , Relação Estrutura-Atividade , Transfecção
18.
J Antibiot (Tokyo) ; 60(8): 529-33, 2007 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-17827665

RESUMO

The water-soluble N-methoxy-PEG-yl-, N-beta-D-glucopyranosyl- and N-beta-D-maltosylthioureido aglyco-ristocetin were prepared which, in contrast to ristocetin A, did not induce thrombocyte aggregation. The antibacterial activity of N-beta-D-maltosylthioureido aglyco-ristocetin A against MRSA was comparable to that of ristocetin A, while its activity against Enterococcus faecalis (VRE, TSE) is somewhat stronger when compared to those of vancomycin and ristocetin A.


Assuntos
Agregação Plaquetária/efeitos dos fármacos , Ristocetina/farmacologia , Bactérias/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Ristocetina/síntese química , Ristocetina/química
19.
Thromb Res ; 160: 119-128, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-29153735

RESUMO

INTRODUCTION: Inherited antithrombin (AT) deficiency is a heterogeneous disease. Due to low prevalence, only a few studies are available concerning genotype-phenotype associations. The aim was to describe the clinical, laboratory and genetic characteristics of AT deficiency in a large cohort including children and to add further laboratory data on the different sensitivity of functional AT assays. PATIENTS AND METHODS: Non-related AT deficient patients (n=156) and their family members (total n=246) were recruited. Clinical and laboratory data were collected, the mutation spectrum of SERPINC1 was described. Three different AT functional assays were explored. RESULTS: Thirty-one SERPINC1 mutations including 11 novel ones and high mutation detection rate (98%) were detected. Heparin binding site deficiency (type IIHBS) was the most frequent (75.6%) including AT Budapest3 (ATBp3), AT Padua I and AT Basel (86%, 9% and 4% of type IIHBS, respectively). Clinical and laboratory phenotypes of IIHBS were heterogeneous and dependent on the specific mutation. Arterial thrombosis and pregnancy complications were the most frequent in AT Basel and AT Padua I, respectively. Median age at the time of thrombosis was the lowest in ATBp3 homozygotes. The functional assay with high heparin concentration and pH7.4 as assay conditions had low (44%) sensitivity for ATBp3 and it was insensitive for AT Basel and Padua I. CONCLUSION: Type IIHBS deficiencies behave differently in clinical and laboratory phenotypes from each other and from other AT deficiencies. Heparin concentration and pH seem to be the key factors influencing the sensitivity of AT functional assays to IIHBS.


Assuntos
Deficiência de Antitrombina III/diagnóstico , Trombose/diagnóstico , Adulto , Estudos de Coortes , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Adulto Jovem
20.
Thromb Res ; 158: 1-7, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28783511

RESUMO

INTRODUCTION: Hereditary antithrombin (AT) deficiency is a rare thrombophilic disorder with heterogeneous genetic background and various clinical presentations. In this study we identified a novel AT mutation. Genotype-phenotype correlations, molecular characteristics and thrombotic manifestations of the mutation were investigated. MATERIALS AND METHODS: Thirty-one members of a single family were included. Clinical data was collected regarding thrombotic history. The mutation was identified by direct sequencing of the SERPINC1 gene. HEK293 cells were transfected with wild type and mutant SERPINC1 plasmids. Western blotting, ELISA and functional amidolytic assay were used to detect wild type and mutant AT. After double immunostaining, confocal laser scanning microscopy was used to localize mutant AT in the cells. Molecular modeling was carried out to study the structural-functional consequences of the mutation. RESULTS: Unprovoked venous thrombotic events at early age, fatal first episodes and recurrences were observed in the affected individuals. The median AT activity was 59%. Genetic analysis revealed heterozygous form of the novel mutation p.Leu205Pro (AT Debrecen). The mutant AT was expressed and synthesized in HEK293 cells but only a small amount was secreted. The majority was trapped intracellularly in the trans­Golgi and 26S proteasome. The mutation is suspected to cause considerable structural distortion of the protein. The low specific activity of the mutant AT suggested functional abnormality. CONCLUSIONS: AT Debrecen was associated with very severe thrombotic tendency. The mutation led to misfolded AT, impaired secretion and altered function. Detailed clinical and molecular characterization of a pathogenic mutation might provide valuable information for individualized management.


Assuntos
Deficiência de Antitrombina III/genética , Antitrombina III/genética , Mutação , Adolescente , Adulto , Idoso , Deficiência de Antitrombina III/sangue , Deficiência de Antitrombina III/patologia , Feminino , Células HEK293 , Humanos , Masculino , Simulação de Dinâmica Molecular , Linhagem , Trombose/sangue , Trombose/genética , Trombose/patologia
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