RESUMO
BACKGROUND: Plasma thymus and activation-regulated chemokine is a potential biomarker for classical Hodgkin's lymphoma. To define its value as a marker to monitor treatment response, we correlated serial plasma thymus and activation-regulated chemokine levels with clinical response in newly diagnosed and relapsed classical Hodgkin's lymphoma patients. DESIGN AND METHODS: Plasma was collected from 60 (39 early stage and 21 advanced stage) newly diagnosed classical Hodgkin's lymphoma patients before, during, and after treatment, and from 12 relapsed patients before and after treatment. Plasma thymus and activation-regulated chemokine levels were determined by enzyme-linked immunosorbent assay and were related to pre-treatment metabolic tumor volume, as measured by quantification of 2-[18F]fluoro-2-deoxyglucose positron emission tomography images, and to treatment response. RESULTS: Baseline plasma thymus and activation-regulated chemokine levels correlated with stage of disease and bulky disease, and more closely with metabolic tumor volume. Response to treatment was observed among 38 of 39 early stage and 19 of 21 advanced stage patients. Reduction in plasma thymus and activation-regulated chemokine to normal range levels could be observed as early as after one cycle of chemotherapy in all responsive patients, while plasma levels remained elevated during and after treatment in the 3 non-responsive patients. Plasma thymus and activation-regulated chemokine was elevated in all 12 relapsed patients at time of relapse and remained elevated after salvage treatment in the 4 non-responsive patients. CONCLUSIONS: Baseline plasma thymus and activation-regulated chemokine levels correlate with classical Hodgkin's lymphoma tumor burden and serial levels correlate with response to treatment in patients with classical Hodgkin's lymphoma.
Assuntos
Quimiocina CCL17/sangue , Doença de Hodgkin/diagnóstico , Doença de Hodgkin/tratamento farmacológico , Adolescente , Adulto , Idoso , Biomarcadores/sangue , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Prognóstico , Recidiva , Resultado do Tratamento , Carga Tumoral , Adulto JovemRESUMO
BACKGROUND: The c-Met signaling pathway regulates a variety of biological processes, including proliferation, survival and migration. Deregulated c-Met activation has been implicated in the pathogenesis and prognosis of many human malignancies. We studied the function and prognostic significance of c-Met and hepatocyte growth factor protein expression in patients with classical Hodgkin's lymphoma. DESIGN AND METHODS: Expression of c-Met and its ligand, hepatocyte growth factor, were determined by immunohistochemistry. Prognostic values were defined in cohorts of German and Dutch patients with classical Hodgkin's lymphoma. Functional studies were performed on Hodgkin's lymphoma cell lines. RESULTS: Expression of c-Met was detected in the tumor cells of 52% (80/153) of the patients and expression of its ligand, hepatocyte growth factor, in 8% (10/121) of the patients. c-Met expression correlated with a 5-year freedom from tumor progression of 94%, whereas lack of expression correlated with a 5-year freedom from tumor progression of 73% (P<0.001) in the combined cohort. In multivariate analysis both c-Met (hazard ratio 5.0, 95% confidence interval 1.9-13.3, P<0.001) and stage (hazard ratio 2.8, 95% confidence interval 1.2-6.4, P=0.014) were independent predictors for freedom from tumor progression. In functional studies activation with hepatocyte growth factor did not affect cell growth, while the c-Met inhibitor SU11274 suppressed cell growth by inducing G2/M cell cycle arrest. CONCLUSIONS: Although functional studies showed an oncogenic role of the hepatocyte growth factor/c-Met signaling pathway in cell cycle progression, expression of c-Met in tumor cells from patients with classical Hodgkin's lymphoma strongly correlated with a favorable prognosis in two independent cohorts.
Assuntos
Doença de Hodgkin/metabolismo , Proteínas Proto-Oncogênicas c-met/metabolismo , Adolescente , Adulto , Idoso , Ciclo Celular , Linhagem Celular , Criança , Feminino , Expressão Gênica , Fator de Crescimento de Hepatócito/genética , Fator de Crescimento de Hepatócito/metabolismo , Doença de Hodgkin/genética , Doença de Hodgkin/mortalidade , Humanos , Masculino , Pessoa de Meia-Idade , Prognóstico , Proteínas Proto-Oncogênicas c-met/genética , Transdução de Sinais , Análise de Sobrevida , Adulto JovemRESUMO
BACKGROUND: The incidence of classical Hodgkin lymphoma (cHL) and its association with Epstein-Barr virus (EBV) varies significantly with age, sex, ethnicity and geographic location. This is the first report on epidemiological features of cHL patients from Northern regions of China. These features are compared to data from a previously published Dutch cHL population. METHODOLOGY/PRINCIPAL FINDINGS: 157 cHL patients diagnosed between 1997 and 2008 in the North of China were included after histopathological re-evaluation. The Dutch population-based cohort consisted of 515 cHL patients diagnosed between 1987 and 2000. EBV status was determined by in situ hybridization of EBV- encoded small RNAs. In the Chinese population, tumor cells of 39% of the cHL patients were EBV+ and this was significantly associated with male sex, mixed cellularity subtype and young age (<20 y). The median age of the Chinese patients was 9 years younger than that of the Dutch patients (28 y vs. 37 y). In addition, the age distribution between the two populations was strikingly different in both the EBV+ subgroups (p<0.001) and the EBV- subgroups (p = 0.01). The mixed cellularity subtype was almost 3x more frequent amongst the Chinese (p<0.001). CONCLUSION/SIGNIFICANCE: CHL patients from Northern regions of China show a distinctive age distribution pattern with a striking incidence peak of EBV+ mixed cellularity cases among children and adolescents and another high incidence peak of EBV- nodular sclerosis cases in young adults. In comparison to Dutch cHL patients there are pronounced differences in age distribution, subtype and EBV status, presumably caused by complex gene-environmental interactions.
Assuntos
Herpesvirus Humano 4/fisiologia , Doença de Hodgkin/epidemiologia , Doença de Hodgkin/virologia , Adolescente , Adulto , Distribuição por Idade , Idoso , Criança , Pré-Escolar , China/epidemiologia , Feminino , Doença de Hodgkin/patologia , Humanos , Masculino , Pessoa de Meia-Idade , Distribuição por Sexo , Adulto JovemAssuntos
Coagulação Sanguínea/genética , Desogestrel/farmacologia , Antagonistas de Estrogênios/farmacologia , Levanogestrel/farmacologia , Fígado/efeitos dos fármacos , Útero/efeitos dos fármacos , Animais , Coagulação Sanguínea/efeitos dos fármacos , Anticoncepcionais Orais/farmacologia , Etinilestradiol/farmacologia , Feminino , Humanos , Fígado/metabolismo , Fígado/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Receptores de Estrogênio/metabolismo , Receptores de Progesterona/metabolismo , Transcrição Gênica/efeitos dos fármacosRESUMO
BACKGROUND: In Caucasian populations, the tumor cells of Epstein Barr virus (EBV)-positive classical Hodgkin Lymphomas (cHL) patients more frequently express HLA class I and HLA class II molecules compared to EBV-negative cHL patients. HLA expression (in relation to EBV) in Asian cHL patients has not been previously investigated. METHODOLOGY/PRINCIPAL FINDINGS: We randomly selected 145 cHL patients with formalin-fixed, paraffin embedded tissue blocks available from 5 hospitals from the Northern part of China. Hematoxylin & Eosin-stained slides were used to re-classify the histological subtypes according to the WHO classification. EBV status was determined by visualization of EBERs in tumor cells using in situ hybridization. Membranous expression of HLA molecules was detected by immunohistochemistry using antibodies HC-10 (class I heavy chain) and anti-beta2-microglobulin for HLA class I, and CR3/43 for HLA class II. EBV+ tumor cells were observed in 40% (58/145) of the cHL patients. As expected, the percentage of EBV+ cases was much higher in the mixed cellularity subtype (71%) than in the nodular sclerosis subtype (16%) (p<0.001). Expression of HLA class I was observed in 79% of the EBV+ cHL cases and in 30% of the EBV-cases (p<0.001). For HLA class II, 52% of EBV+ cHL cases were positive, compared to 43% in EBV- cases (p = 0.28). CONCLUSIONS: The results in the Northern China population were similar to those in the Caucasian population for HLA class I, but not for HLA class II.
Assuntos
Povo Asiático , Antígenos de Histocompatibilidade Classe II/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Doença de Hodgkin/imunologia , Adolescente , Adulto , Idoso , Criança , Pré-Escolar , China , Feminino , Herpesvirus Humano 4/imunologia , Doença de Hodgkin/patologia , Doença de Hodgkin/virologia , Humanos , Imuno-Histoquímica , Hibridização In Situ , Masculino , Pessoa de Meia-Idade , Países Baixos , RNA Viral/genética , Adulto JovemAssuntos
Aquaporina 2/genética , Fator VIII/biossíntese , Variação Genética , Trombose Venosa/genética , Fator de von Willebrand/biossíntese , Estudos de Casos e Controles , Éxons , Fator VIII/genética , Feminino , Humanos , Íntrons , Masculino , Peptídeos/química , Polimorfismo de Nucleotídeo Único , Risco , Trombose Venosa/diagnóstico , Fator de von Willebrand/genéticaRESUMO
Hodgkin and Reed-Sternberg (HRS) cells in Hodgkin lymphoma (HL) secrete factors that interact with inflammatory background cells and may serve as biomarkers for disease activity. To detect new proteins related to pathogenesis, we analyzed the secretome of HRS cells. Proteins in cell culture supernatant of 4 HL cell lines were identified using 1DGE followed by in-gel trypsin digestion and LC-MS/MS. In total, 1290 proteins, including 368 secreted proteins, were identified. Functional grouping of secreted proteins revealed 37 proteins involved in immune response. Sixteen of the 37 proteins (ie, ALCAM, Cathepsin C, Cathepsin S, CD100, CD150, CD26, CD44, CD63, CD71, Fractal-kine, IL1R2, IL25, IP-10, MIF, RANTES, and TARC) were validated in HL cell lines and patient material using immunohistochemistry and/or ELISA. Expression of all 16 proteins was confirmed in HL cell lines, and 15 were also confirmed in HL tissues. Seven proteins (ALCAM, cathepsin S, CD26, CD44, IL1R2, MIF, and TARC) revealed significantly elevated levels in patient plasma compared with healthy controls. Proteomics analyses of HL cell line supernatant allowed detection of new secreted proteins, which may add to our insights in the interaction between HRS cells and infiltrating lymphocytes and in some instances might serve as biomarkers.
Assuntos
Doença de Hodgkin/genética , Células de Reed-Sternberg/patologia , Adolescente , Adulto , Células Sanguíneas/citologia , Células Sanguíneas/efeitos dos fármacos , Células Sanguíneas/patologia , Comunicação Celular , Linhagem Celular Tumoral , Criança , Humanos , Pessoa de Meia-Idade , Proteômica , Valores de ReferênciaRESUMO
Von Willebrand factor (VWF) contains a large number of cysteine residues, which all form disulfide bonds. Mutations of cysteines located in the cystine-knot (CK) domain of VWF have been identified in both qualitative type 2A (IID) and quantitative type 3 von Willebrand disease (VWD). Our objective was to test the hypothesis that the difference in phenotype is related to whether the mutated cysteine residue is involved in either interchain- or intrachain-disulfide-bond formation. The effects of three cysteine mutations which are all located in the CK-domain of VWF, C2773S (type 2A(IID)), C2739Y (type 3), and C2754W (type 3), were studied by transient expression in 293T cells. Cotransfection of wild-type (wt) and C2773S VWF constructs reproduced the plasma phenotype of heterozygous type 2A(IID) patients, with normal to high levels of VWF antigen (VWF:Ag), absence of high-molecular-weight multimers, and the presence of intervening bands between the normal multimers. In contrast, single transfections of C2739Y or C2754W resulted in a quantitative VWF defect with low VWF:Ag levels, and co-transfections of wt and mutant constructs resulted in a 50% reduction of VWF:Ag and only a minor effect on VWF multimerization. We demonstrated N-terminal dimerization of VWF-C2773S and both N- and C-terminal dimerization of VWF-C2754W. Our data suggest that loss of a single disulfide bond in the CK-domain of VWF leads to a recessive, quantitative VWF deficiency if an intrachain-disulfide bond is involved, and to a dominant-negative, qualitative defect of VWF if an interchain-disulfide bond is involved.
Assuntos
Coagulação Sanguínea , Motivos Nó de Cisteína , Dissulfetos/química , Processamento de Proteína Pós-Traducional , Doenças de von Willebrand/metabolismo , Fator de von Willebrand/metabolismo , Proteínas ADAM/metabolismo , Proteína ADAMTS13 , Linhagem Celular , Cisteína , Análise Mutacional de DNA , Dimerização , Eletroforese em Gel de Poliacrilamida , Feminino , Humanos , Masculino , Mutação de Sentido Incorreto , Linhagem , Fenótipo , Conformação Proteica , Transfecção , Doenças de von Willebrand/sangue , Doenças de von Willebrand/genética , Fator de von Willebrand/química , Fator de von Willebrand/genéticaRESUMO
OBJECTIVE: The protein C anticoagulant pathway is an essential process for attenuating thrombin generation by the membrane-bound procoagulant complexes tenase and prothrombinase. In this pathway, protein S (PS) serves as a cofactor for activated protein C. PS circulates in plasma both in a free form and in complex with complement component 4b-binding protein (C4BP). C4BP is a known acute phase reactant, thereby suggesting a relation between PS and the acute phase response. Interleukin (IL)-6 has been shown to increase both PS and C4BP gene expression. Our objective was to study the regulation of PS gene expression by IL-6 in detail. METHODS AND RESULTS: IL-6 upregulates both PS mRNA and protein levels in liver-derived HepG2 cells. The promoter of the PS gene (PROS1) was cloned upstream from a luciferase reporter gene. After transfection in HepG2 cells, the luciferase activity was shown to be stimulated by the addition of IL-6. IL-6 exerts its effect through Signal Transducer and Activator of Transcription 3 (STAT3) that interacts with the PROS1 promoter at a binding site in between nucleotides 229 to 207 upstream from the translational start. CONCLUSIONS: IL-6 induces PS expression via STAT3. A possible function for IL-6-induced PS expression in cell survival is discussed.
Assuntos
Interleucina-6/fisiologia , Proteína S/biossíntese , Fator de Transcrição STAT3/fisiologia , Linhagem Celular Tumoral , Regulação da Expressão Gênica , Humanos , Interleucina-6/farmacologia , Fosforilação , Regiões Promotoras Genéticas , Proteína S/genética , Proteína S/metabolismo , RNA Mensageiro/biossíntese , Elementos de Resposta , Fator de Transcrição STAT3/metabolismoRESUMO
Protein S (PS) is a vitamin K-dependent plasma protein that inhibits blood coagulation by serving as a nonenzymatic cofactor for activated protein C in the protein C anticoagulant pathway. Low PS levels are a risk factor for the development of deep venous thrombosis. The regulation of PS levels through transcriptional regulation of the PS gene was investigated in this report. A minimal PS gene promoter 370 bp upstream from the translational initiation codon was sufficient for maximal promoter activity in transient transfections regardless of the cell type. A pivotal role for Sp1 in the constitutive expression of the PS gene was demonstrated through electrophoretic mobility shift assay experiments, transient expression of mutant PS promoter-reporter gene constructs, and chromatin immunoprecipitations in HepG2 cells. At least four Sp-binding sites were identified. The two sites most proximal to the translational start codon were found to be indispensable for PS promoter activity, whereas mutation of the two most distal Sp-binding sites had a negligible influence on basal promoter activity. In addition, all other major promoter-binding proteins that were found by electrophoretic mobility shift assay could be positively identified in supershift assays. We identified binding sites for the hepatocyte-specific forkhead transcription factor FOXA2, nuclear factor Y, and the cAMP-response element-binding protein/activating transcription factor family of transcription factors. Their relevance was investigated using site-directed mutagenesis.
Assuntos
Regiões Promotoras Genéticas/fisiologia , Proteína S/genética , Proteína S/metabolismo , Fator de Transcrição Sp1/metabolismo , Fator de Transcrição Sp3/metabolismo , Sequência de Bases , Sítios de Ligação/fisiologia , Fator de Ligação a CCAAT/metabolismo , Carcinoma Hepatocelular , Cromatina/fisiologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Endotélio Vascular/citologia , Regulação da Expressão Gênica/fisiologia , Células HeLa , Fator 3-beta Nuclear de Hepatócito/metabolismo , Humanos , Neoplasias Hepáticas , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Proteína S/química , Fatores de Transcrição/metabolismo , Transfecção , Veias Umbilicais/citologiaRESUMO
BACKGROUND: Elevated plasminogen activator inhibitor-1 (PAI-1) concentrations are associated with cardiovascular diseases. PAI-1 antigen levels are influenced by environmental factors such as body mass index (BMI), and by genetic factors. The PAI-1 promoter of the PAI-1 gene contains two common polymorphisms (-844A/G and -675(4G/5G)) and the 4G allele of the -675(4G/5G) variation has been associated with elevated PAI-1 concentrations and on some occasions with an increased risk of cardiovascular disease. OBJECTIVES: The aim of our study was to investigate the effect of the PAI-1 promoter haplotype on PAI-1 concentrations and to determine the role of BMI. METHODS: The association between the PAI-1 promoter haplotype and PAI-1 antigen levels was investigated in two independent populations, each including 600 healthy Caucasians. Furthermore, to assess the effect of the PAI-1 promoter haplotype on PAI-1 promoter activity, in vitro reporter gene assays were performed in HepG2 and BAEC cells. RESULTS: We observed significantly higher PAI-1 concentrations in A-4G homozygotes than in G-5G carriers in lean subjects (BMI in the lowest quartile). In these lean subjects, the PAI-1 concentrations in A-4G/G-5G heterozygotes were reduced to 60-75%, and the concentrations in G-5G homozygotes to 45-55%, compared to the PAI-1 concentrations of A-4G homozygotes (p < 0.01). PAI-1 concentrations increased approximately four-fold from the lowest to the highest BMI quartile (p < 0.01). The reporter gene assays did not support a direct effect of the PAI-1 promoter haplotype on promoter activity in HepG2 or BEAC cells. CONCLUSIONS: Our study suggests that the PAI-1 promoter haplotype and BMI affect PAI-1 concentrations and that BMI is a stronger determinant than PAI-1 promoter variation.
Assuntos
Índice de Massa Corporal , Peso Corporal/genética , Inibidor 1 de Ativador de Plasminogênio/sangue , Inibidor 1 de Ativador de Plasminogênio/genética , Regiões Promotoras Genéticas/genética , Adulto , Idoso , Carcinoma Hepatocelular , Linhagem Celular Tumoral , Feminino , Genes Reporter , Variação Genética , Haplótipos , Humanos , Neoplasias Hepáticas , Masculino , Pessoa de Meia-IdadeRESUMO
An elevated plasma fibrinogen level is an independent risk factor for cardiovascular disease. Therefore, an understanding of the regulation of fibrinogen expression is important. Inflammation and genetic variation of the fibrinogen beta gene regulate plasma fibrinogen levels, and there are indications that inflammation and genetic variation interact. The aim of our study was to gain more understanding of the regulation of the inflammatory response of the fibrinogen beta gene and to determine the effects of genetic variation. Luciferase reporter gene assays in hepatoma cells, mutation analysis, and electrophoretic mobility shift assays were used to investigate the transcriptional regulation of the fibrinogen beta promoter. We identified a hepatocyte nuclear factor-3 (HNF-3) site located just upstream of previously identified interleukin-6 (IL6)-responsive sequences. This HNF-3 site is essential for a full response of the promoter to IL6, which is a new function for HNF-3. The activity of the CCAAT box/enhancer-binding protein site (located 18 nucleotides downstream of the HNF-3 site and important to the IL6 response) depends on the integrity of the HNF-3 site and vice versa, explaining the necessity of HNF-3 in the IL6 response of the fibrinogen beta promoter. Furthermore, small interfering RNA to HNF-3 reduces the fibrinogen beta mRNA levels. The rare T allele of the -148C/T polymorphism, which is present between the binding sites of HNF-3 and CCAAT box/enhancer-binding protein, interferes with this mechanism, and this polymorphism is in our assay system the only genetic determinant of IL6-induced promoter activity among six polymorphisms in the fibrinogen beta promoter.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Fibrinogênio/química , Fibrinogênio/genética , Interleucina-6/biossíntese , Proteínas Nucleares/metabolismo , Polimorfismo Genético , Regiões Promotoras Genéticas , Fatores de Transcrição/metabolismo , Alelos , Motivos de Aminoácidos , Sequência de Bases , Sítios de Ligação , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Análise Mutacional de DNA , Primers do DNA/química , Proteínas de Ligação a DNA/química , Relação Dose-Resposta a Droga , Eletroforese , Fibrinogênio/metabolismo , Genes Reporter , Variação Genética , Vetores Genéticos , Fator 3-alfa Nuclear de Hepatócito , Fator 3-beta Nuclear de Hepatócito , Fator 3-gama Nuclear de Hepatócito , Humanos , Inflamação , Interleucina-6/metabolismo , Luciferases/metabolismo , Dados de Sequência Molecular , Mutação , Proteínas Nucleares/química , Ligação Proteica , Interferência de RNA , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Fatores de Risco , Fatores de Transcrição/química , Ativação TranscricionalRESUMO
The exact pathogenesis of hypertrophic scar and keloid formation is still unknown and a good therapy to prevent or treat these scars is lacking. Because immunological processes seem to be important in excessive scar formation, immunological cells and parameters were studied in a standardized breast reduction wound-healing model in the present study. Standardized scar samples were taken from infra-mammary breast reduction scars, 3 and 12 months following surgery. The samples were investigated for their number of mast cells, Langerhans cells, T-lymphocytes, and macrophages, and the presence of interleukin-4 (IL-4) and counter-regulating interferon-gamma (gamma-IFN), in relation to the scar's clinical appearance--normal or hypertrophic. In this study, hypertrophic scar formation was significantly associated with an increased number of epidermal Langerhans cells (p=0.0001) and significantly (p<0.05) increased expression of epidermal IL-4. No relationship was found between mast cell, T-lymphocyte and macrophage numbers or gamma-IFN staining and the formation of normal or hypertrophic scars. These results, combined with previous observation of abnormal keratinocyte behaviour in this context, indicate that the epidermal immune barrier plays an important role in the development of hypertrophic scars.
Assuntos
Cicatriz Hipertrófica/patologia , Células de Langerhans/patologia , Adolescente , Adulto , Idoso , Mama/patologia , Mama/cirurgia , Contagem de Células , Epiderme/patologia , Feminino , Humanos , Interferon gama/análise , Interleucina-4/análise , Macrófagos/patologia , Mastócitos/patologia , Pessoa de Meia-Idade , Período Pós-Operatório , Linfócitos T/patologia , Cicatrização/fisiologiaRESUMO
We hypothesized that possession of either of 2 functional coagulation factor XIII polymorphisms, one within subunit A (Val34Leu) and one within subunit B (His95Arg), might modulate the prothrombotic effects of estrogen and help to explain the variation in incidence of arterial thrombotic events among postmenopausal women using hormone replacement therapy. In a population-based case-control study of 955 postmenopausal women, we assessed the associations of factor XIII genotypes and their interactions with estrogen therapy on risk of nonfatal myocardial infarction (MI). The presence of the factor XIIIA Leu34 allele was associated with a reduced risk of MI (odds ratio [OR] = 0.70, 95% confidence interval [95% CI] = 0.51-0.95). The presence of the factor XIIIB Arg95 allele had little association with MI risk. Neither factor XIII polymorphism alone significantly modified the association between the risk of MI and current estrogen use. In exploratory analyses, however, there was a significant factor XIII subunit gene-gene interaction. Compared to women homozygous for both common factor XIII alleles, the Arg95 variant was associated with a reduced risk of MI in the presence of the Leu34 variant (OR = 0.36, 95% CI = 0.17-0.75) but not in the absence of the Leu34 variant (OR = 1.11, 95% CI = 0.69-1.79). Moreover, among women who had at least 2 copies of the variant factor XIII alleles and were current estrogen users, the risk of MI was reduced by 70% relative to estrogen nonusers with fewer than 2 factor XIII variant alleles (P value for interaction =.03). If confirmed, these findings may permit a better assessment of the cardiovascular risks and benefits associated with postmenopausal estrogen therapy.