肿瘤坏死因子超家族基因在慢性心力衰竭外周血单核细胞的表达

目的:应用基因芯片技术分析比较慢性心力衰竭（CHF）患者和健康对照组外周血单核细胞（PBMC）中细胞因子和其它炎症相关基因表达差异,以识别CHF细胞因子网络的不平衡。方法:分别抽提CHF和健康对照组PBMC总RNA并纯化mRNA,反转录合成单链、双链cDNA后,体外转录合成cRNA。分别用cy3-dCTP和cy5-dCTP标记对照和CHF组cRNA。将基因芯片和杂交探针变性后杂交、洗涤。用基因芯片扫描仪进行图像扫描,Im aGene3.0软件分析cy3、cy5两种荧光信号的强度和比值。结果:cDNA表达谱芯片中含365个炎性细胞因子相关基因,其中CHF组上调34个基因,下调2个。上调基因包括肿瘤坏死因子（TNF）超家族成员,趋化因子/趋化因子受体,转化生长因子超家族成员等。TNF超家族成员中,淋巴毒素（LTB）、TNF超家族成员5、7、10,TNF受体超家族成员13b,TNF相关凋亡诱导配体受体4（TRAIL-R4）在CHF组均上调。而TNF受体超家族成员10C（TRAIL-R3）在CHF显著下调。结论:CHF与正常人PBMC细胞因子相关基因的表达有显著差异,CHF存在细胞因子网络的不平衡;尤其是多个TNF超家族基因的高表达可能在CHF发生发展中起着重要作用。     

Increased expression of tumor necrosis factor-alpha converting enzyme and tumor necrosis factor-alpha in peripheral blood mononuclear cells in patients with advanced congestive heart failure

BACKGROUND: Tumor necrosis factor-alpha converting enzyme (TACE) has recently been identified as a metalloproteinase-disintegrin, which converts pro-tumor necrosis factor-alpha (TNF-alpha) to the mature form, and is an important mediator in the pathogenesis of CHF. AIMS: In order to establish the importance of TACE in the regulation of TNF-alpha synthesis in peripheral blood mononuclear cells (PBMC), we analyzed mRNAs and protein-positive cells of both TACE and TNF-alpha in PBMC obtained from patients with congestive heart failure (CHF). METHODS AND RESULTS: PBMC were obtained from 46 patients with CHF and 22 controls. PBMC were activated by phorbol 12-myristate 13-acetate and ionomycin and assessed for TACE and TNF-alpha mRNAs by real-time RT-PCR, intracellular TACE and TNF-alpha levels by flow cytometry, and TNF-alpha secretion by supernatant ELISA. Levels of TACE and TNF-alpha mRNAs, intracellular TACE and TNF-alpha, and supernatant TNF-alpha were higher in CHF than in controls (P<0.001). There was a positive correlation between TACE and TNF-alpha levels in CHF patients (mRNA: r=0.60, P<0.001, intracellular protein levels: r=0.76, P<0.001). When the CHF group was divided into two subgroups by NYHA functional class (I and II vs. III and IV), levels of TACE and TNF-alpha were significantly higher in severe CHF patients (NYHA III or IV) than in mild CHF patients (NYHA I or II) (mRNA: P<0.001; intracellular protein levels: P<0.001). CONCLUSION: These results demonstrate that in patients with CHF, and especially those with severe CHF, TACE expression in PBMC increases with TNF-alpha expression. These observations suggest that TACE in PBMC is an important regulator of TNF-alpha maturation, meaning that TACE may be a potential target for the inhibition of cellular TNF-alpha production in CHF.     

Elevated tumor necrosis factor alpha of blood mononuclear cells in patients with congestive heart failure

To study the changes of tumor necrosis factor-alpha (TNF-alpha) produced by peripheral blood mononuclear cells (PBMC) of patients with congestive heart failure (CHF), we determined the TNF-alpha concentrations of plasma and culture supernatant of PBMCs in 113 patients with various heart diseases. The results showed that plasma levels of TNF-alpha were significantly higher in CHF patients (n=50, 13.36+/-6.09 pg/ml) and in cachetic CHF patients (n=26, 19.20+/-10.42 pg/ml) than in the controls (n=37, 6.73+/-3.91 ng/ml). The supernatant levels of TNF-alpha in cultured PBMC (PBMC-TNF-alpha) were also significantly elevated in CHF patients (5.77+/-2.11 ng/ml), and even higher in cachetic CHF patients (7.01+/-5.11 ng/ml), as compared with the controls (2.27+/-2.24 ng/ml). There was a significant correlation between levels of PBMC-TNF-alpha and severity of heart failure (r=0.48, P<0.01). But no correlation between PBMC-TNF-alpha and plasma TNF-alpha was observed. These results demonstrate that the expression of TNF-alpha in PBMC is increased in patients with CHF, especially in patients with CHF accompanied by cachexia, suggesting that PBMC-TNF-alpha may be a potential biochemical indicator to evaluate the patients with CHF.     

Effect of interleukin-10 on the production of tumor necrosis factor-alpha by peripheral blood mononuclear cells from patients with chronic heart failure

Chronic heart failure (HF) is a state of inflammatory immune activation characterized by elevated circulating levels of tumor necrosis factor-alpha (TNF-alpha). Interleukin-10 (IL-10) is a potent anti-inflammatory cytokine that inhibits TNF-alpha production and lessens endotoxin bioactivity. It is not known whether IL-10 reduces lipopolysaccharide (LPS) stimulated TNF-alpha production of peripheral blood mononuclear cells (PBMCs) from patients with chronic HF. PBMCs were isolated from 15 patients with chronic HF (New York Heart Association functional class 3.0 +/- 0.2, left ventricular ejection fraction 30 +/- 2%, peak oxygen consumption 18.1 +/- 0.8 ml/kg/min) and 15 healthy control subjects and stimulated with 1 and 10 ng/ml LPS for 24 hours with or without prior addition of IL-10 (10 ng/ml). TNF-alpha was quantified in cell-free supernatants by an enzyme-linked immunosorbent assay. TNF-alpha, soluble TNF receptors, IL-10, and LPS were quantified in plasma. LPS stimulated TNF-alpha production was highest in those patients in New York Heart Association class II (p <0.01 vs New York Heart Association class III and IV, p <0.001 vs control subjects). IL-10 reduced PBMC TNF-alpha production in all stimulated samples at 1 and 10 ng/ml LPS (mean reduction 43% at 1 ng/ml, p <0.01 and 55% at 10 ng/ml, p <0.0001). The percentage reduction in TNF-alpha release did not differ significantly between patients and control subjects or with respect to severity of chronic HF or baseline immune parameters. Independently of clinical severity, IL-10 profoundly inhibits TNF-alpha release from PBMCs isolated from patients with chronic HF. IL-10 is, therefore, a potential therapy for use in chronic HF associated with inflammatory immune activation.     

Increased production of tumor necrosis factor-alpha by peripheral blood mononuclear cells in the patients with aplastic anemia

The activity of tumor necrosis factor-alpha (TNF-alpha) in the supernatant of cultured peripheral blood mononuclear cells (PBMC) was measured in patients with aplastic anemia. It was significantly higher in patients with aplastic anemia than in normal controls, both when PBMC were unstimulated or when they were stimulated with PHA. Results from aplastic anemia patients were also significantly higher than patients who had received allogeneic bone marrow transplants. In aplastic anemia patients, the TNF-alpha value produced by PBMC upon stimulation and the platelet count were inversely correlated, as well those patients who had high TNF-alpha values tended to have lower hemoglobin and leukocyte values although this was not significant statistically. These results suggest that the increased production of TNF-alpha by PBMC plays a role in the severe suppression of hematopoiesis in aplastic anemia.     

Tumor necrosis factor alpha production by peripheral blood mononuclear cells of patients with chronic liver disease

We investigated the production of tumor necrosis factor alpha by peripheral blood mononuclear cells of patients with chronic liver disease and its association with hepatitis activity. Tumor necrosis factor alpha production was measured with an enzyme-linked immunosorbent assay. Tumor necrosis factor alpha production by peripheral blood mononuclear cells stimulated with recombinant gamma-interferon of patients with chronic active hepatitis (5.8 +/- 4.0 units per ml, p less than 0.05) and patients with cirrhosis (4.1 +/- 2.1 units per ml, p less than 0.05) was significantly increased when compared with controls (2.5 +/- 1.6 units per ml). Tumor necrosis factor alpha production by peripheral blood mononuclear cells stimulated with a combination of recombinant gamma-interferon and recombinant interleukin 2 of patients with chronic persistent hepatitis (5.8 +/- 3.8 units per ml, p less than 0.05), patients with chronic active hepatitis (8.9 +/- 3.0 units per ml, p less than 0.001) and patients with cirrhosis (6.7 +/- 3.2 units per ml, p less than 0.05) was significantly increased in comparison with controls (3.3 +/- 1.8 units per ml). Tumor necrosis factor alpha production of patients with chronic active hepatitis was significantly higher than that of patients with chronic persistent hepatitis (p less than 0.05). There was a significant correlation (r = 0.5699, p less than 0.005) between tumor necrosis factor alpha production and histologic activity index in patients with chronic persistent hepatitis or chronic active hepatitis. These findings show that tumor necrosis factor alpha production is increased in chronic liver disease and that the increased tumor necrosis factor alpha production is related to hepatitis activity.     

Differential gene expression profiles of peripheral blood mononuclear cells in childhood asthma

Objective: Asthma is a common childhood disease with strong genetic components. This study compared whole-genome expression differences between asthmatic young children and healthy controls to identify gene signatures of childhood asthma. Methods: Total RNA extracted from peripheral blood mononuclear cells (PBMC) was subjected to microarray analysis. QRT-PCR was performed to verify the microarray results. Classification and functional characterization of differential genes were illustrated by hierarchical clustering and gene ontology analysis. Multiple logistic regression (MLR) analysis, receiver operating characteristic (ROC) curve analysis, and discriminate power were used to scan asthma-specific diagnostic markers. Results: For fold-change>2 and p?<?0.05, there were 758 named differential genes. The results of QRT-PCR confirmed successfully the array data. Hierarchical clustering divided 29 highly possible genes into seven categories and the genes in the same cluster were likely to possess similar expression patterns or functions. Gene ontology analysis presented that differential genes primarily enriched in immune response, response to stress or stimulus, and regulation of apoptosis in biological process. MLR and ROC curve analysis revealed that the combination of ADAM33, Smad7, and LIGHT possessed excellent discriminating power. Conclusions: The combination of ADAM33, Smad7, and LIGHT would be a reliable and useful childhood asthma model for prediction and diagnosis.     

Increased spontaneous osteoclastogenesis from peripheral blood mononuclear cells in phenylketonuria

Phenylketonuria (PKU) is commonly complicated by a progressive bone impairment of uncertain aetiology. The therapeutic phenylalanine (Phe)-restricted diet and the possible noxious effects of high plasma Phe concentrations on bone have previously been suggested as possible determinant factors. Since osteoclasts are involved in bone reabsorption, they could play a role in determining bone damage in PKU. The reported increased excretion of bone resorption markers in PKU patients is consistent with this hypothesis. Although different diseases characterized by bone loss have been related to increased spontaneous osteoclastogenesis from peripheral blood mononuclear cells (PBMCs), to date there is no evidence of increased osteoclast formation in PKU. In this study, we compared the spontaneous osteoclastogenesis from PBMCs in 20 patients affected by PKU with that observed in age- and sex-matched healthy subjects. Phenylketonuric patients showed the number of osteoclasts to be almost double that observed in controls (159.9 ± 79.5 and 87.8 ± 44.7, respectively; p = 0.001). Moreover, a strict direct correlation between the spontaneous osteoclastogenesis in PKU patients and the mean blood Phe concentrations in the preceding year was observed (r = 0.576; p = 0.010). An imbalance between bone formation and bone resorption might explain, at least in part, the pathogenesis of bone loss in this disease. These findings could provide new insights into the biological mechanisms underlying bone damage in PKU.     

肿瘤坏死因子与心力衰竭关系的研究

肿瘤坏死因子为炎性细胞因子标志之一,其在心血管疾病中的作用越来越受到重视。许多研究提示肿瘤坏死因子与心力衰竭的发生、发展有一定相关性,其对心力衰竭的作用机制被逐步深入了解。传统观念认为肿瘤坏死因子与心力衰竭的治疗也有相关作用,但RENAISSANCE、ATTACH等研究显示抗肿瘤坏死因子治疗效果不尽如人意。该文主要对肿瘤坏死因子在心力衰竭中的作用、相关药物治疗等最新研究进行综述。     

慢性心力衰竭患者血清瘦素与肿瘤坏死因子α浓度变化

目的 :通过分析慢性心力衰竭 (HF)恶病质患者血清瘦素 (leptin)及肿瘤坏死因子α(TNF α)的浓度变化 ,探讨其在心源性恶病质发病过程中的可能作用及临床意义。方法 :测定 4 8例慢性HF患者和 2 0例健康对照者的体重指数 (BMI)、腰臀比 (WHR)、皮下脂肪厚度 (TSF)、左室射血分数 (LVEF)、leptin及TNF α等指标。leptin及TNF α用放射免疫法测定 ,并分析leptin与其他参数的相关性。结果 :①全部受试者的血清leptin浓度与BMI、TSF、TNF α呈显著正相关 (P <0 .0 5 ) ,与WHR及LVEF无显著相关性。而恶病质组的血清leptin浓度还与TNF α呈显著相关关系 (P <0 .0 5 )。②恶病质组与非恶病质组相比 ,BMI、leptin及TSF差异有显著性意义 ,均P <0 .0 1。结论 :leptin作为调节能量平衡的重要激素 ,可能与TNF α共同作用导致心源性恶病质的发生     

The importance of tumor necrosis factor and lipoproteins in the pathogenesis of chronic heart failure

Elevated levels of proinflammatory cytokines, such as tumor necrosis factor (TNF) and interleukin-6 (IL-6), have been demonstrated in patients with chronic heart failure (CHF). Evidence suggests that cytokines such as these may play a central role in the pathogenesis of this syndrome. TNF has several properties that are particularly detrimental in CHF, such as negatively inotropic effects, the promotion of left ventricular remodelling, and the induction of dilated cardiomyopathy in humans. Furthermore, TNF can cause skeletal muscle wasting and apoptosis, and, therefore, may be important in the development of cardiac cachexia. Although the precise stimulus for immune activation in CHF is unknown, one hypothesis is that endotoxin may be a significant trigger for cytokine release. This is supported by the finding that decompensated CHF patients have elevated endotoxin levels that normalize on diuretic therapy. The factors that influence endotoxin responsiveness in patients with CHF, in particular the potential importance of serum lipoproteins, will be discussed in this review.     

Increased productivity of tumor necrosis factor-alpha in helper T cells in patients with systolic heart failure

BACKGROUND AND AIMS: Whereas increased circulating proinflammatory cytokines, such as tumor necrosis factor-alpha (TNF-alpha), play an important role in heart failure, where and how TNF-alpha production is upregulated remains largely unknown. We studied the productivity of TNF-alpha in peripheral lymphocytes and underlying mechanisms in patients with heart failure. METHODS: Symptomatic NYHA II-IV patients with chronic heart failure with systolic dysfunction (n=39, aged 74+/-11, ejection fraction [EF]<==50%) were compared with asymptomatic NYHA I patients (n=18, aged 72+/-10, EF>50%) and normal subjects (n=15, aged 67+/-11). Lymphocyte subsets (CD3, CD4, and CD8) and intracellular production of TNF-alpha in peripheral leukocytes were quantified by immunofluorescent flow cytometry, and relationships between these parameters and circulating proinflammatory cytokines were analyzed. RESULTS: Subpopulation of TNF-alpha-producing CD4 was larger in NYHA II-IV patients (23.7% [18.0-28.6]) than in normal subjects (17.1% [6.5-19.5], p<0.05) and was correlated with plasma TNF-alpha levels (r=0.26, p<0.05), EF (r=-0.26, p<0.05), CD4/CD8 ratios (r=0.42, p<0.001), and subpopulation of TNF-alpha-producing monocytes (r=0.47, p<0.0001). Plasma levels of soluble CD14 and interleukin-12 (IL-12) were significantly higher in NYHA II-IV patients than in normal subjects (1971 ng/mL [1740-2375] vs. 1607 ng/mL [1530-1930], p<0.01; and 121 pg/mL [62-230] vs. 62 pg/mL [54-99], p<0.05, respectively), and plasma IL-12 levels were correlated with plasma TNF-alpha levels (r=0.41, p<0.001). CONCLUSIONS: Increased productivity of TNF-alpha in helper T cells, associated with their dominance over cytotoxic T cells, may partially contribute to an increase in circulating TNF-alpha levels in heart failure.