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Importance: Several antifungal drugs are available for antifungal prophylaxis in patients with hematological disease or who are undergoing hematopoietic stem cell transplantation (HSCT). Objective: To summarize the evidence on the efficacy and adverse effects of antifungal agents using an integrated comparison. Data Sources: Medline, EMBASE, and the Cochrane Central Register of Controlled Clinical Trials were searched to collect all relevant evidence published in randomized clinical trials that assessed antifungal prophylaxis in patients with hematological disease. Sources were search from inception up to October 2019. Study Selection: Studies that compared any antifungal agent with a placebo, no antifungal agent, or another antifungal agent among patients with hematological disease or undergoing HSCT were included. Of 39â¯709 studies identified, 69 met the criteria for inclusion. Data Extraction and Synthesis: The outcome from each study was estimated using the relative risk (RR) with 95% CIs. The Mantel-Haenszel random-effects model was used. The reliability and validity of the networks were estimated by addressing inconsistencies in the evidence from comparative studies of different treatments. Data were analyzed from December 2019 to February 2020. Reporting followed the Preferred Reporting Items for Systematic Reviews and Meta-analyses for Network Meta-analysis (PRISMA-NMA) guideline. Main Outcomes and Measures: The primary outcomes were invasive fungal infections (IFIs) and mortality. The secondary outcomes were fungal infections, proven IFIs, invasive candidiasis, invasive aspergillosis, fungi-related death, and withdrawal owing to adverse effects of the drug. Results: We identified 69 randomized clinical trials that reported comparisons of 12 treatments with at total of 14â¯789 patients. Posaconazole was the treatment associated with the best probability of success against IFIs (surface under the cumulative ranking curve, 86.7%; mean rank, 2.5). Posaconazole treatment was associated with a significant reduction in IFIs (RR, 0.57; 95% CI, 0.42-0.79) and invasive aspergillosis (RR, 0.36; 95% CI, 0.15-0.85) compared with placebo. Voriconazole was associated with a significant reduction in invasive candidiasis (RR, 0.15; 95% CI, 0.09-0.26) compared with placebo. However, posaconazole was associated with a higher incidence of withdrawal because of the adverse effects of the drug (surface under the cumulative ranking curve, 17.5%; mean rank, 9.2). In subgroup analyses considering efficacy and tolerance, voriconazole might be the best choice for patients undergoing HSCT, especially allogenic HSCT; however, posaconazole was ranked as the best choice for patients with acute myeloid leukemia or myelodysplastic syndrome. Conclusions and Relevance: These findings suggest that voriconazole may be the best prophylaxis option for patients undergoing HSCT, and posaconazole may be the best prophylaxis option for patients with acute myeloid leukemia or myelodysplastic syndrome.
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Antifúngicos/administração & dosagem , Doenças Hematológicas/tratamento farmacológico , Transplante de Células-Tronco Hematopoéticas/métodos , Micoses/prevenção & controle , Profilaxia Pré-Exposição/métodos , Triazóis/administração & dosagem , Voriconazol/administração & dosagem , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
INTRODUCTION AND AIMS: Age-related severity and distribution of haemophilic arthropathy (HA) among Chinese patients with haemophilia using the Haemophilia Early Detection with Ultrasound (HEAD-US) system have not been extensively studied. METHODS: In our study, 89 patients with moderate and severe haemophilia were recruited. A total of 534 joints (knees, ankles and elbows on both sides included) were evaluated using musculoskeletal ultrasound (MSKUS) and scored using the HEAD-US system. RESULTS: Prevalence and average number of HA were 39.1% and 0.7, 90.6% and 3.2, 94.1% and 4.5, and 100% and 4.3 for ages ≤10, 11-20, 21-30 and 31-40 years, respectively. Prevalence and mean number of knee, ankle and elbow arthropathies also increased with age, although joint damages progressed in unparallel patterns. A significant difference in synovium subscores was observed between patients aged <10 and >10 years. An increasing tendency was observed in cartilage and subchondral bone subscores along with age before 30 years. No significant difference in mean joint scores was found between patients receiving on-demand therapy and those receiving on-demand to low-dose prophylactic therapy. CONCLUSIONS: Haemophilic arthropathy developed in early childhood and progressed mainly before 30 years of age among Chinese patients with haemophilia, although in different ways among the knee, ankle and elbow.
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Tornozelo/patologia , Povo Asiático , Cotovelo/patologia , Hemofilia A/complicações , Artropatias/complicações , Joelho/patologia , Adolescente , Adulto , Distribuição por Idade , Fatores Etários , Tornozelo/diagnóstico por imagem , Criança , Pré-Escolar , Cotovelo/diagnóstico por imagem , Hemofilia A/tratamento farmacológico , Humanos , Artropatias/diagnóstico por imagem , Joelho/diagnóstico por imagem , Prevalência , Índice de Gravidade de Doença , Membrana Sinovial/diagnóstico por imagem , Membrana Sinovial/patologia , Adulto JovemRESUMO
Primary immune thrombocytopenia (ITP) is a bleeding disorder commonly encountered in clinical practice. The International Working Group (IWG) on ITP has published several landmark papers on terminology, definitions, outcome criteria, bleeding assessment, diagnosis, and management of ITP. The Chinese consensus reports for diagnosis and management of adult ITP have been updated to the 4th edition. Based on current consensus positions and new emerging clinical evidence, the thrombosis and hemostasis group of the Chinese Society of Hematology issued Chinese guidelines for management of adult ITP, which aim to provide evidence-based recommendations for clinical decision making.
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Medicina Baseada em Evidências , Hematologia/organização & administração , Guias de Prática Clínica como Assunto , Púrpura Trombocitopênica Idiopática/diagnóstico , Púrpura Trombocitopênica Idiopática/tratamento farmacológico , Sociedades Médicas/organização & administração , Idoso , China , Feminino , Humanos , Masculino , Índice de Gravidade de DoençaRESUMO
PURPOSE: We estimated the expression of nuclear factor kappa B/p65 in non-germinal center B-cell-like subtype diffuse large B-cell lymphoma, to investigate its relationship to clinicopathological features, and to further evaluate its prognostic value and clarify its impact on survival. RESULTS: Among the 49 patients enrolled in this study, 14 (28.6%) had positive p65 expression. The negative p65 group had significantly better survival compared to the positive p65 group in terms of both the 3-year estimated OS (91.2% vs. 39.3%, p = 0.003) and PFS (75.6% vs. 26.5%, p = 0.002). In patients with 4 or more risk factors, p65 was an independent prognostic factor of OS (HR 5.99, 95%CI=1.39-25.75, p=0.016) and PFS (HR 4.01, 95%CI=1.15-14.00, p=0.029). MATERIALS AND METHODS: The expression of the NF-κB/p65 protein was deteremined by immunohistochemistry in 49 non-GCB DLBCL. Survival was assessed by the Kaplan-Meier method and Cox multivariate analysis. The median patient follow-up period was 24 months. CONCLUSIONS: The expression of NF-κB/p65 has prognostic value in high risk non-GCB DLBCL, and it is a suitable target for the development of new therapies.
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Biomarcadores Tumorais/análise , Linfoma Difuso de Grandes Células B/química , Fator de Transcrição RelA/análise , Adolescente , Adulto , Idoso , Intervalo Livre de Doença , Feminino , Humanos , Imuno-Histoquímica , Estimativa de Kaplan-Meier , Linfoma Difuso de Grandes Células B/mortalidade , Linfoma Difuso de Grandes Células B/patologia , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Valor Preditivo dos Testes , Modelos de Riscos Proporcionais , Estudos Retrospectivos , Fatores de Risco , Fatores de Tempo , Adulto JovemRESUMO
BACKGROUND: Mutations in Janus kinase 2 (JAK2), myeloproliferative leukemia (MPL), and CALR are highly relevant to Philadelphia chromosome (Ph)-negative myeloproliferative neoplasms. METHODS: Assessing the prevalence of molecular mutations in Chinese Han patients with essential thrombocythemia (ET), and correlating their mutational profile with disease characteristics/phenotype. RESULTS: Of the 110 subjects studied, 62 carried the JAK2 V617F mutation, 21 had CALR mutations, one carried an MPL (W515) mutation, and 28 had non-mutated JAK2, CALR, and MPL (so-called triple-negative ET). Mutations in JAK2 exon 12 were not detected in any patient. Two ET patients had both CALR and JAK2 V617F mutations. Comparing the hematological parameters of the patients with JAK2 mutations with those of the patients with CALR mutations showed that the ET patients with CALR mutations were younger (p = 0.045) and had higher platelet counts (p = 0.043). CONCLUSION: Genotyping for CALR could be a useful diagnostic tool for JAK2/MPL-negative ET, since the data suggest that CALR is much more prevalent than MPL, therefore testing for CALR should be considered in patients who are JAK2 negative as its frequency is almost 20 times that of MPL mutation.
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Povo Asiático/genética , Calreticulina/genética , Janus Quinase 2/genética , Mutação , Receptores de Trombopoetina/genética , Trombocitemia Essencial/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Alelos , China , Análise Mutacional de DNA , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Fenótipo , Estudos Retrospectivos , Trombocitemia Essencial/diagnóstico , Trombocitemia Essencial/tratamento farmacológico , Adulto JovemRESUMO
This study aims to investigate whether neutrophil to lymphocyte ratio (NLR) is an independent predictor in newly diagnosed diffuse large B-cell lymphoma (DLBCL) patients in the rituximab era. Data from newly diagnosed DLBCL patients at Nanjing Drum Tower Hospital from 2006 to 2015 were retrospectively reviewed. We used the receiver operating characteristic (ROC) curve analysis to generate the optimal cutoff value for NLR. Among those 156 patients enrolled, the NLR was < 3.0 in 46.8% (73/156) of the patients, and the remaining 53.2% (83/156) had an NLR ≥ 3.0. Patients with higher pretreatment NLR were found to correlate with poorer OS and PFS than these with lower NLR (hazard ratio [HR] = 2.66, 95% confidence interval [CI] = 1.43-4.97, P = 0.002 and HR = 1.79, 95% CI = 1.05-3.07, P = 0.034, respectively). The multivariate Cox proportional hazard model analysis further showed that high NLR was found independently predictive of poor OS (HR = 0.40; CI = 0.19-0.84, P = 0.015) and PFS (HR = 0.57; CI = 0.33-0.98, P = 0.042). Consequently, pretreatment NLR was an independent prognostic predictor in patients with DLBCL in the rituximab era.
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Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Linfócitos/patologia , Linfoma Difuso de Grandes Células B/diagnóstico , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Neutrófilos/patologia , Idoso , Anticorpos Monoclonais Murinos/uso terapêutico , Ciclofosfamida/uso terapêutico , Doxorrubicina/uso terapêutico , Feminino , Humanos , Contagem de Linfócitos , Linfoma Difuso de Grandes Células B/mortalidade , Masculino , Pessoa de Meia-Idade , Prednisona/uso terapêutico , Prognóstico , Modelos de Riscos Proporcionais , Curva ROC , Estudos Retrospectivos , Rituximab/administração & dosagem , Análise de Sobrevida , Vincristina/uso terapêuticoRESUMO
The International Prognostic Index (IPI) has been the basis for determining prognosis in patients with diffuse large B-cell lymphoma (DLBCL) for the past 20 years. The utility of the IPI must be reassessed in the era of immunochemotherapy. Seven risk factors at diagnosis were identified, and a maximum of 7 points were assigned to each patient. Four risk groups were created: low (0-1), low-intermediate (2-3), high-intermediate (4), and high (5-7). Using MYC and BCL-2 clinical data from the Drum Tower Hospital collected during the rituximab era, we performed a retrospective analysis of patients with DLBCL treated with R-CHOP and built an biological markers adjusted IPI with the goal of improving risk stratification.Clinical features from 60 adults with de novo DLBCL diagnosed from 2008-2013 were assessed for their prognostic significance. The IPI remains predictive, but it cannot identify the high-risk subgroup. Compared with the IPI, the MYC and BCL-2 adjusted-IPI (A-IPI) better discriminated patients in the high-risk subgroup (4-year overall survival [OS]: 33.3%) than did the IPI (4 year OS: 48.0%). In the era of R-CHOP treatment, MYC and BCL-2 adjusted-IPI is more powerful than the IPI for helping guide treatment planning and interpretation of clinical trials.
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Biomarcadores Tumorais/análise , Linfoma Difuso de Grandes Células B/patologia , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Proteínas Proto-Oncogênicas c-myc/biossíntese , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Anticorpos Monoclonais Murinos , Protocolos de Quimioterapia Combinada Antineoplásica , Ciclofosfamida , Intervalo Livre de Doença , Doxorrubicina , Feminino , Humanos , Imuno-Histoquímica , Estimativa de Kaplan-Meier , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Linfoma Difuso de Grandes Células B/mortalidade , Masculino , Pessoa de Meia-Idade , Prednisona , Prognóstico , Proteínas Proto-Oncogênicas c-bcl-2/análise , Proteínas Proto-Oncogênicas c-myc/análise , Estudos Retrospectivos , Fatores de Risco , Rituximab , Vincristina , Adulto JovemRESUMO
This study aims to investigate the prognostic significance of the MYC protein expression in diffuse large B cell lymphoma (DLBCL) patients treated with RCHOP (rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone). A total of 60 patients with DLBCL from 2008 to 2013 were included. Formalin-fixed, paraffin-embedded DLBCL samples were analyzed for MYC protein expression and divided into high or low MYC group. The MYC protein expression and the international prognostic variables were evaluated. The high MYC protein expression predicted a shorter 3-year estimated overall survival (OS) and progression-free survival (PFS) versus the low MYC protein expression (57 % vs. 96 %, P < 0.001 and 50 % vs. 96 %, P = 0.001, respectively). Multivariate analysis confirmed the prognostic significance of the MYC protein expression for both OS (HR, 11.862; 95 % CI, 1.462-96.218; P = 0.021) and PFS (HR, 6.073; 95 % CI, 1.082-34.085; P = 0.040). MYC protein expression with International Prognostic Index (IPI) score distinguished patients into three risk groups with different 3-year OS rates (χ (2) 23.079; P < 0.001) and distinct 3-year PFS rates (χ (2) 15.862; P < 0.001). This study suggests that the MYC protein expression is an important inferior prognostic factor for survival in patients with DLBCL treated with RCHOP. The combinative model with IPI score and MYC protein expression could stratify DLBCL patients into prognostically relevant subgroups more effectively than either the IPI or the MYC alone.
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Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Linfoma Difuso de Grandes Células B/genética , Proteínas Proto-Oncogênicas c-myc/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Anticorpos Monoclonais Murinos/administração & dosagem , Ciclofosfamida/administração & dosagem , Intervalo Livre de Doença , Doxorrubicina/administração & dosagem , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Linfoma Difuso de Grandes Células B/patologia , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Prednisona/administração & dosagem , Prognóstico , Proteínas Proto-Oncogênicas c-myc/biossíntese , Rituximab , Vincristina/administração & dosagemRESUMO
BACKGROUND: To determine whether the use of idarubicin+cytarabine (IA) is more effective than the use of daunorubicin+cytarabine (DA) as induction chemotherapy for patients with newly diagnosed acute myeloid leukaemia. METHODS: A computer-based search was performed. Randomised trials comparing IA with DA as induction therapy for newly diagnosed AML were included in this meta-analysis. The primary outcome of interest for our analysis was survival (disease-free survival, event-free survival and overall survival); the secondary endpoint was complete remission. RESULTS: Ten trials with 4,060 patients were eligible for this meta-analysis. Our pooled results suggest that IA is associated with a significant advantage in CR (RRâ=â1·23; 95% CIâ=â1·07-1·41, pâ=â0.004), EFS (HRâ=â0·64; 95% CIâ=â0·45-0·91, pâ=â0.013), and OS (HRâ=â0·88; 95% CIâ=â0·81-0·95, pâ=â0.02) but not in DFS (HRâ=â0·90; 95% CIâ=â0·80-1·00, pâ=â0.06). In the subgroup analysis, age had a significant interaction with OS and CR benefits. CONCLUSION: Our analysis indicated that IA could improve the duration of overall survival compared to DA as induction therapy for young patients with newly diagnosed AML. Further study is needed to determine whether IA can produce clinical benefits in selected genetic or molecular subgroups of young AML patients.
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Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Leucemia Mieloide Aguda/tratamento farmacológico , Adulto , Idoso , Citarabina/administração & dosagem , Daunorrubicina/administração & dosagem , Humanos , Idarubicina/administração & dosagem , Quimioterapia de Indução , Pessoa de Meia-Idade , Viés de Publicação , Ensaios Clínicos Controlados Aleatórios como Assunto , Resultado do TratamentoRESUMO
OBJECTIVE: To study the relationship between polymorphism of genes XPA, XPC, XPD, XRCC1 and susceptibility to acute lymphoblastic leukemia (ALL) in a Chinese population. METHODS: Polymorphism were determined by a case-control study through matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry method of Mass-ASSAY platform in 114 confirmed ALL cases and 169 age- and sex-matched controls, so as to compare the relationship between different genotypes and ALL risk. RESULTS: Multivariate logistic regression analysis revealed that individuals carrying at least one 23G variant allele (AG/GG genotypes) had a significantly increased risk for ALL (adjusted OR 2.02; 95%CI 1.08 - 3.78) compared with the wild-type genotype (23AA), and evidence that positive interactions between the polymorphisms in XPC C499T and XPA A23G might occur. Furthermore, individuals with both putative risk genotypes had a significantly higher risk (adjusted OR 5.60; 95%CI 1.57 - 19.90), compared with those with both wild-genotypes. By contrast, no significant association was observed between the XPD T751G, XRCC1 G399A, C194T polymorphism and ALL risk. CONCLUSIONS: XPA A23G and XPC C499T polymorphism may contribute to the risk of developing ALL. There are significant combinations between XPC C499T and XPA A23G.
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Predisposição Genética para Doença , Polimorfismo de Nucleotídeo Único , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Adolescente , Adulto , Idoso , Alelos , Povo Asiático/genética , Estudos de Casos e Controles , Criança , Reparo do DNA , Proteínas de Ligação a DNA/genética , Feminino , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Proteína 1 Complementadora Cruzada de Reparo de Raio-X , Proteína de Xeroderma Pigmentoso Grupo A/genética , Proteína Grupo D do Xeroderma Pigmentoso/genética , Adulto JovemRESUMO
OBJECTIVE: To explore the profiles of Th1, Th2, Th17 and Regulatory T (Treg) cells in patients with chronic idiopathic thrombocytopenic purpura (ITP). METHODS: Samples of peripheral blood were collected from 35 chronic ITP patients (21 in an active stage group, 5 in a non-remission stage group, 9 in a remission stage group) and also from 18 healthy subjects. Flow cytometry was used to measure the intracellular cytokines interferon (IFN)gamma, interleukin (IL)-4 and IL-17 so as to identify the Th1, Th2 and IL-17 cells. Treg cells were identified with CD(4)(+) CD(25)(+) Foxp3(+) cells and uncultured peripheral blood was used to measure the CD(4)(+) CD(+)(25) Foxp3(+) cells with flow cytometry. The concentrations of IFNgamma, IL-4, IL-17 and IL-10 in plasma specimens were detected with ELISA method and its correlation with peripheral platelets counts and megakaryocytes number was analyzed, respectively. RESULTS: There were no statistically significant differences between any two of the three groups for the percentage of Th1 cells, Th17 cells and Th1/Th17 ratio. The percentage of Th2 cells was (1.01 +/- 0.88)% in active stage and (1.22 +/- 1.04)% in non-remission stage, being significantly decreased than those in remission stage (1.93 +/- 1.04)% (P < 0.05) and the controls (1.86 +/- 0.59)% (P < 0.05). Th1/Th2 ratio was 15.04 +/- 9.67 in active stage, 11.65 +/- 9.32 in non-remission stage, which were significantly higher than those in remission stage (7.17 +/- 5.38, P < 0.05) and the controls (7.02 +/- 3.01, P < 0.05). The percentage of Treg cells was (0.89 +/- 0.58)% in active stage and (1.46 +/- 1.27)% in non-remission stage, being significantly decreased than those in remission stage (6.41 +/- 1.86)% (P < 0.01) and the control (5.73 +/- 0.71)% (P < 0.01). There was no statistic difference between any two of the three groups for plasma IFNgamma and IL-17 level. The plasma IL-4 level was (2.25 +/- 2.05) ng/L in active stage and (2.33 +/- 2.14) ng/L in non-remission stage, being significantly decreased than those in remission stage (6.00 +/- 4.57) ng/L (P < 0.05) and the controls (5.54 +/- 4.00) ng/L (P < 0.05). The plasma IL-10 level was (5.07 +/- 4.10) ng/L in active stage and (5.66 +/- 4.35) ng/L in non-remission stage, being significantly decreased than those in remission stage (10.92 +/- 6.17) ng/L (P < 0.01) and the controls (14.21 +/- 7.31) ng/L (P < 0.01). The plasma level of IL-10 in patients in active stage was positively related to the platelet counts (r = 0.16, P = 0.03). CONCLUSION: Deficiency of Treg cells might be one of mechanisms that cause immune regulation dysfunction in chronic ITP. Th17 cells might not play a role in the development of chronic ITP.
Assuntos
Púrpura Trombocitopênica Idiopática/metabolismo , Linfócitos T Reguladores/metabolismo , Adolescente , Adulto , Idoso , Estudos de Casos e Controles , Feminino , Humanos , Interferon gama/metabolismo , Interleucina-10/metabolismo , Interleucina-17/metabolismo , Interleucina-4/metabolismo , Masculino , Pessoa de Meia-Idade , Púrpura Trombocitopênica Idiopática/imunologia , Células Th1/metabolismo , Células Th2/metabolismo , Adulto JovemRESUMO
OBJECTIVE: To study the molecular mechanisms of antithrombin (AT) deficiency caused by AT gene mutations T98I and A404T. METHODS: Wild-type and mutant AT cDNA expression plasmids (ATwt, AT T98I and AT A404T) were constructed and transfected into the monkey fibroblast of the line COS-7 or Chinese hamster ovary (CHO) cells. NH4Cl. ALLN and brefeldin A were added. ELISA was used to detect the AT: Ag. Pulse-chase experiment and immunofluorescence assay were used to detect the radioactivity of the 35S-labeled AT. Fluorescence real-time PCR was used to detect the expression of AT mRNA, protein degradation inhibition was used to elucidate the mutant T98I degradation pathway inside the cells. RESULTS: AT T98I was not secreted from the cells and was gradually degraded inside the cells. There was partial secretion of AT A404T, but most of the molecule was not secreted but was degraded intracellularly. Fluorescence real-time PCR indicated that the mutant AT mRNA was transcribed at a similar or even higher level as that of wild-type (wt). Pulse-chase labeling studies suggested both AT variants did not accumulate, but degraded intracellularly. Protein degradation inhibition experiment showed that mutant AT T98I was degraded intracellularly through the proteasome pathway. Immunohistochemical staining of the transfected cells revealed that CHO cells expressing the AT T98I mutant were stained diffusely without perinuclear enhancement and cells expressing AT A404T mutant mainly in the whole cytoplasm with weaker perinuclear enhancement. CONCLUSION: Impaired secretion of the mutant AT molecules, due to intracellular degradation, is the molecular mechanism of AT deficiency caused by T98I and A404T mutation.
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Deficiência de Antitrombina III/genética , Antitrombinas/genética , Mutação , Animais , Antitrombinas/metabolismo , Células CHO , Células COS , Chlorocebus aethiops , Cricetinae , Cricetulus , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , TransfecçãoRESUMO
OBJECTIVE: To identify the phenotype and gene mutation in two Chinese pedigrees with hereditary protein C deficiency. METHODS: The plasma level of protein C activity (PC: A) , protein C antigen (PC: Ag), protein S activity (PS: A), and antithrombin activity (AT: A) of the probands and their family members were detected using chromogenic assay and ELISA, respectively. All of the nine exons and intron-exon boundaries of protein C gene were amplified by PCR and analyzed by direct sequencing of the probands. Restriction enzyme site analysis was used to confirm the mutation. RESULTS: The plasma PC: A and PC: Ag for proband 1 was 1.2% and 0, respectively. Compound heterozygous mutations, C(TGC)64W (TGG) and F(TTC) 139V(GTC) , were identified in her, the former being inherited from the maternal side and the later the paternal side. Further genetic analysis showed that her husband ( II 8) had the heterozygous deletion mutation (K150 or 151 Del) in exon 7, her daughter had the same heterozygous deletion mutation and a F139V. The plasma PC: A and PC: Ag for proband 2 was 50. 3% and 1.9 mg/L, respectively. He had the heterozygous Lys150 or Lys151 deletion mutation, which was inherited from his father. Polymorphisms of C/T at position - 1654, A/G at - 1641 , and A/T at - 1476A/T in the promoter region of protein C were confirmed in all members of the two pedigrees, of which, proband 2 had homozygous CC/GG/TT. The F139V mutation was confirmed by restriction enzyme site analysis and polymorphism for this mutation was excluded. PS: A and AT: A were in normal range for all members. CONCLUSION: Compound heterozygous mutation C64W and F139V of protein C gene lead to type I hereditary protein C deficiency for proband 1. K150 or 151 deletion mutation and polymorphism of CC/GG/TT might lead to type I hereditary protein C deficiency for proband 2. C64W is a novel mutation for protein C gene. F139V and K150 or 151 deletion mutation are reported for the first time in China.
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Mutação , Polimorfismo Genético , Deficiência de Proteína C/genética , Adulto , Povo Asiático/genética , China , Feminino , Genótipo , Humanos , Masculino , Linhagem , FenótipoRESUMO
OBJECTIVE: To study the molecular mechanisms of protein C (PC) deficiency caused by PC gene mutations of C64W, F139V and K150 deletion (K150d). METHODS: Wild-type and mutant PC cDNA expression plasmids (PCwt, PC C64W, PC F139V, PC K150d) were constructed and transfected into COS-7 cells or CHO cells respectively for in vitro expression study and immunofluorescent assay. Fluorescent real-time PCR was used to detect the expression of PC mRNA, protein degradation inhibition and endo-beta-N-acetylglucosaminidase H (Endo H) digestion experiments to explain the mutant protein degradation pathway and its localizations inside the cells. RESULTS: PC C64W was not secreted from the cells and was gradually degraded inside the cells. There was partial secretion of PC F139V, most of the protein molecule was not secreted and degraded intracellularly. Mutant PC K150d was secreted normally from the cells. Fluorescent realtime PCR analysis of total mRNA from transfected cells showed no reduction of the mutant PC mRNA expression compared with that of wild-type PC mRNA. Protein degradation inhibition experiments showed that mutants PC C64W and PC F139V were degraded intracellularly through the proteasome pathway. Endo H digestion experiments and immunofluorescence results suggested that mutant PC molecules were located mainly in pre-Golgi apparatus. CONCLUSIONS: Impaired secretion and degradation intracellularly of the mutants might be the molecular mechanisms of PC deficiency caused by C64W and F139V mutations. K150 deletion mutation might not affect the secretion of the mutant.
Assuntos
Mutação , Deficiência de Proteína C/genética , Proteína C/genética , Animais , Células CHO , Células COS , Chlorocebus aethiops , Cricetinae , Cricetulus , Humanos , Plasmídeos/genética , TransfecçãoRESUMO
OBJECTIVE: To investigate the antithrombotic mechanisms of holothurian glycosaminoglycan (GAG) extracted from sea cucumber. METHODS: Human endothelial cell line EA. hy926 cells were treated with 10 mg/L GAG or 10U/mL unfractionated heparin (UFH) by short-term (15 min - 2 h) and longer-time incubation (6 h - 48 h). Different doses of GAG were used to stimulate EA. hy926. Released free tissue factor pathway inhibitor(TFPI) was determined by ELISA assay. TFPI expression was investigated by immunofluorescent method and TFPI mRNA level by real-time PCR. In a 96-wells microtitre plate, pooled normal plasma containing different concentrations of GAG was allowed to clot by addition of thrombin and calcium chloride, fibrinolysis was induced by addition of t-PA. TRR (TAFI-related retardation of clot lysis) was used to assess thrombin-activatable fibrinolysis inhibitor(TAFI) functional activity. RESULTS: GAG increased TFPI synthesis, expression and secretion in a dose- and time dependent manner. GAG at low concentrations could lengthen while at intermediate concentrations could shorten clot lysis times significantly as compared to control values. TRR was dose-dependently decreased on addition of GAG. CONCLUSIONS: GAG increases TFPI synthesis, expression and secretion of endothelial cells. GAG at intermediate concentrations significantly affects clot stability of a developing clot by means of diminishing TAFI activation.
Assuntos
Células Endoteliais/efeitos dos fármacos , Glicosaminoglicanos/farmacologia , Holothuria , Lipoproteínas/biossíntese , Extratos de Tecidos/farmacologia , Animais , Carboxipeptidase B2/antagonistas & inibidores , Linhagem Celular , Relação Dose-Resposta a Droga , Células Endoteliais/metabolismo , Heparina/farmacologia , Humanos , Lipoproteínas/genética , RNA Mensageiro/genéticaRESUMO
OBJECTIVE: To investigate the antithrombin (AT) activity (AT: A) and AT antigen (AT: Ag) level in a Chinese family with type I antithrombin (AT) deficiency, and to explore the molecular mechanism of AT deficiency. METHODS: Immuno-nephelometry and chromogenic assay were used to detect the plasma level of AT: A and AT: Ag, respectively. Genomic DNA was isolated from the peripheral blood, and all the seven exons and exon-intron boundaries of AT gene were amplified by PCR and direct sequencing. RESULTS: The plasma levels of AT: A and AT: Ag of the proband were 45% and 97 mg/L, respectively, which led to a type I AT deficiency. A heterozygous T to A mutation was found at nucleotide 9833 in exon 5 resulting in a Tyr363Stop nonsense mutation. The sequencing results from the pedigree indicated that four other members also had this mutation. CONCLUSION: This heterozygous nonsense mutation of T9833A in exon 5 resulting in venous thrombosis is a novel genetic defect of hereditary AT deficiency, which has not been described before.
Assuntos
Deficiência de Antitrombina III/genética , Antitrombinas/genética , Testes de Coagulação Sanguínea , Feminino , Humanos , Masculino , Mutação , Linhagem , Reação em Cadeia da Polimerase , Análise de Sequência de DNARESUMO
BACKGROUND AND OBJECTIVES: Most secreted proteins, including coagulation factor X (FX), are synthesized with a signal peptide, which is necessary for targeting the nascent polypeptide into the endoplasmic reticulum. Characterization of naturally occurring mutations may provide insights into the functional roles of the amino acids in the signal peptide. DESIGN AND METHODS: A 52-year old male patient with type I FX deficiency was studied. Mutations were searched for by FX gene (F10) sequencing. The wild-type and the mutant FX proteins were expressed in transfected cells and then immunological assays were performed. Pulse-chase experiments and cell-free expression studies were conducted to determine the cellular fate of the mutant FX molecules. RESULTS: The patient we studied was homozygous for a substitution of arginine for serine at codon -30 in the signal sequence of F10. Immunoassays detected low FX antigen levels in both the conditioned media and lysates of the cells expressing the mutant protein. Pulse-chase analysis showed that only trace amounts of the mutant FX protein were detectable in the conditioned media, and that the mutant molecules did not accumulate inside the cells either. The results of cell-free expression studies showed that although the transcription and translation of the mutant construct were normal, no post-translational processing, such as N-linked glycosylation, occurred in the presence of microsomes. INTERPRETATION AND CONCLUSIONS: These findings suggest that substitution of a neutral polar amino acid, serine by arginine, in the hydrophobic core of FX signal peptide severely impairs the ability of the protein to enter the endoplasmic reticulum and results in FX deficiency.