Tumour necrosis factor-alpha receptor 1 polymorphisms and serum soluble TNFR1 in early spontaneous miscarriage

OBJECTIVES: The study investigated the association of TNFR1 gene polymorphism with early recurrent spontaneous miscarriage (ERSM) in Chinese women, and soluble TNFR1 (sTNFR1) expression in ERSM women. STUDY DESIGN: Two single nucleotide polymorphisms (SNPs) located at -383 (AGA to AGC) in the promoter region and +36 (CCA to CCG) in exon 1 of TNFR1 were investigated in 188 non-pregnant ERSM Chinese women. The serum sTNFR1 was measured by the ELISA method. RESULTS: Both SNPs were not associated with ERSM. The non-pregnant ERSM women had significantly higher levels of serum sTNFR1, compared with the non-pregnant, normal women (1.84+/-0.54 ng/ml versus 1.62+/-0.38 ng/ml; t=-2.053; p<0.05). CONCLUSIONS: The data do not provide evidence that TNFR1 gene polymorphism is etiologically important for ERSM in Chinese women. But, a significantly raised sTNFR1 level in non-pregnant ERSM women was recorded compared to women with normal pregnancies. The result suggests that pregnancy failure is associated with an increase of sTNFR1.     

Relationship between peripheral blood dendritic cells and cytokines involved in the pathogenesis of systemic lupus erythematosus

Recent studies indicate that dendritic cells (DC) and several cytokines are implicated in the induction of autoimmune diseases. In this study we investigated the relationship between the total number of DC (tDC), and their plasmacytoid (pDC) and myeloid (mDC) subpopulations, with serum concentrations of interferons (IFN-alpha and IFN-gamma) and selected cytokines (TNF-alpha, IL-4, IL-6), in patients with systemic lupus erythematosus (SLE) and healthy persons. Subpopulations of DC were determined by the following antigen expression profiles: BDCA-1+\CD11c+\HLA-DR+ (for mDC) and BDCA-2 +\CD123+\HLA-DR+ (for pDC), using flow cytometry. Serum levels of interferons and cytokines were assessed by an enzyme-linked immunosorbent assay (ELISA). The study was performed in 36 SLE patients and 19 healthy volunteers. The mean number of tDC was lower in SLE patients (13.9 +/- 6.4\microL) than in healthy persons (24.1 +/- 12.6\microL) (P < 0.001). The number of pDC was also significantly lower in SLE (6.6 +/- 3.6\microL) than in the control group (12.0 +/- 8.3\microL) (P < 0.02). Moreover, the mean pDC count was lower in active than in inactive disease (5.5 +/- 3.6\microL vs 7.6 +/- 3.4\microL; P < 0.04). The mean serum levels of IFN-alpha and IFN-gamma were significantly higher in SLE patients (63.8 pg\mL and 6.6 pg\mL, respectively) than in the control group (2.7 pg\mL and 0.5 pg\mL, respectively) (P < 0.008 and P < 0.001, respectively). Serum levels of TNF-alpha and IL-6 were also higher in SLE patients (mean 7.3 pg\mL and 18.4 pg\mL, respectively) than in healthy controls (4.2 pg\mL and 0.5 pg\mL, respectively) (P < 0.02 and P < 0.001, respectively). The mean serum IL-4 concentrations were similar in SLE and healthy persons (0.2 pg\mL and 0.31 pg\mL, respectively; P -/+ 0.119). A negative correlation was found between pDC number and the serum level of IFN-alpha (rho -/+ -- 0.386, P -/+ 0.02) and between mDC and IFN-gamma (rho -/+ -- 0.377, P -/+ 0.024). In conclusion, the correlation between peripheral blood DC subsets and serum levels of IFN-alpha and IFN-gamma suggests a possible relationship between these cytokines in the pathogenesis of SLE.     

TNF/TNFR1 signaling up-regulates CCR5 expression by CD8+ T lymphocytes and promotes heart tissue damage during Trypanosoma cruzi infection: beneficial effects of TNF-alpha blockade

In Chagas disease, understanding how the immune response controls parasite growth but also leads to heart damage may provide insight into the design of new therapeutic strategies. Tumor necrosis factor-alpha (TNF-alpha) is important for resistance to acute Trypanosoma cruzi infection; however, in patients suffering from chronic T. cruzi infection, plasma TNF-alpha levels correlate with cardiomyopathy. Recent data suggest that CD8-enriched chagasic myocarditis formation involves CCR1/CCR5-mediated cell migration. Herein, the contribution of TNF-alpha, especially signaling through the receptor TNFR1/p55, to the pathophysiology of T. cruzi infection was evaluated with a focus on the development of myocarditis and heart dysfunction. Colombian strain-infected C57BL/6 mice had increased frequencies of TNFR1/p55+ and TNF-alpha+ splenocytes. Although TNFR1-/- mice exhibited reduced myocarditis in the absence of parasite burden, they succumbed to acute infection. Similar to C57BL/6 mice, Benznidazole-treated TNFR1-/- mice survived acute infection. In TNFR1-/- mice, reduced CD8-enriched myocarditis was associated with defective activation of CD44+CD62Llow/- and CCR5+ CD8+ lymphocytes. Also, anti-TNF-alpha treatment reduced the frequency of CD8+CCR5+ circulating cells and myocarditis, though parasite load was unaltered in infected C3H/HeJ mice. TNFR1-/- and anti-TNF-alpha-treated infected mice showed regular expression of connexin-43 and reduced fibronectin deposition, respectively. Furthermore, anti-TNF-alpha treatment resulted in lower levels of CK-MB, a cardiomyocyte lesion marker. Our results suggest that TNF/TNFR1 signaling promotes CD8-enriched myocarditis formation and heart tissue damage, implicating the TNF/TNFR1 signaling pathway as a potential therapeutic target for control of T. cruzi-elicited cardiomyopathy.     

可溶性肿瘤坏死因子受体II-脂联素球部融合蛋白的真核表达及生物学活性检测

本研究设计和构建了一种人肿瘤坏死因子受体II胞外区与人脂联素球部的融合基因sTNFRII-gAD,且相应的融合蛋白在哺乳动物细胞BHK-21S的无血清培养体系中实现了表达,并对该融合蛋白进行了初步鉴定。首先,用RT-PCR方法从人的外周血淋巴细胞总RNA中扩增人肿瘤坏死因子II型受体胞外区基因片段,与脂联素球部基因片段融合,克隆至pAAV2neo表达载体中,构建成pAAV2neo-sTNFRII-gAD。随后,用pAAV2neo-sTNFRII-gAD转染BHK-21S细胞获得G418抗性细胞BHK-21S/pAAV2neo-sTNFRII-gAD;然后,将原来含有血清的培养液换成无血清的化学成分限定的培养液,细胞从贴壁培养方式转换成悬浮培养方式;最后,收集BHK-21S/pAAV2neo-sTNFRII-gAD无血清悬浮培养24h后的培养上清,进行sTNFRII-gAD融合蛋白的鉴定分析。酶切鉴定和测序结果显示,所构建的pAAV2neo-sTNFRII-gAD质粒结构正确,sTNFRII-gAD序列与预期一致;分别用抗人肿瘤坏死因子受体II和抗人脂联素球部的单克隆抗体检测pAAV2neo-sTNFRII-gAD瞬时转染的BHK-21S细胞,免疫荧光呈现阳性;免疫印迹分析在pAAV2neo-sTNFRII-gAD稳定转染的BHK-21S细胞上清中检测到sTNFRII-gAD融合蛋白的表达,并以单体、三聚体和三聚体以上的多聚体形式存在。活性测定结果表明,sTNFRII-gAD融合蛋白具有显著抑制TNFα杀伤L929细胞的活性。因此,本研究为下一步大量制备sTNFRII-gAD融合蛋白用于体内外功能研究提供了良好基础。     

Restoration of membrane TNF-like activity by cell surface targeting and matrix metalloproteinase-mediated processing of a TNF prodrug

Tumor necrosis factor (TNF) prodrugs are fusion proteins comprised of an N-terminal single-chain antibody variable fragment (scFv) targeting a TNF effector and a C-terminal TNF receptor (TNFR)1-derived inhibitor module. Introduction of matrix metalloproteinase (MMP)-2 recognition motifs between TNF and the TNFR1 fragment allowed activation by recombinant MMP-2 and MMP-expressing HT1080 cells. Processing by endogeneous MMPs required specific membrane binding of the TNF prodrug via the targeting scFv, ensuring strictly antigen-dependent activation. Interestingly, TNF bioactivity of the processed prodrug was approximately 1000-fold higher upon scFv-mediated targeting, and signaled juxtatropic cell death also to antigen-negative cells. Microscopical analyses of TNFR2 clustering and TNF receptor-associated factor 2 recruitment at contact sites to adjacent cells revealed the formation of stable TNFR complexes by target-bound, processed prodrug, resembling the increased signal capacity of natural, membrane-expressed TNF. MMP-2-sensitive TNF prodrugs represent novel cytokine-based reagents for targeted cancer therapy, which should be exploitable for MMP-overexpressing tumors.     

TNF-alpha and myocardial matrix metalloproteinases in heart failure: relationship to LV remodeling

The cytokine tumor necrosis factor (TNF)-alpha has been causally linked to left ventricular (LV) remodeling, but the molecular basis for this effect is unknown. Matrix metalloproteinases (MMPs) have been implicated in cardiac remodeling and can be regulated by TNF-alpha. This study tested the central hypothesis that administration of a TNF-alpha blocking protein would prevent the induction of MMPs and alter the course of myocardial remodeling in developing LV failure. Adult dogs were randomly assigned to the following groups: 1) chronic pacing (250 beats/min, 28 days, n = 12), 2) chronic pacing with concomitant administration of a TNF-alpha blocking protein (TNF block) using a soluble p75 TNF receptor fusion protein (TNFR:Fc; administered at 0.5 mg/kg twice a week subcutaneously, n = 7), and 3) normal controls (n = 10). LV end-diastolic volume increased from control with chronic pacing (83 +/- 12 vs. 118 +/- 10 ml, P < 0.05) and was reduced with TNF block (97 +/- 9 ml, P < 0.05). MMP zymographic levels (92 kDa, pixels) increased from control with chronic pacing (36,848 +/- 9,593 vs. 87,247 +/- 12,912, P < 0.05) and was normalized by TNF block. Myocardial MMP-9 and MMP-13 levels by immunoblot increased with chronic pacing relative to controls (130 +/- 10% and 118 +/- 6%, P < 0.05) and was normalized by TNF block. These results provide evidence to suggest that TNF-alpha contributes to the myocardial remodeling process in evolving heart failure through the local induction of specific MMPs.     

Plasma profiles of circulating granulocyte-macrophage colony-stimulating factor and soluble cellular adhesion molecules in acute myocardial infarction. Contribution to post-infarction left ventricular dysfunction

No in vivo data exist about the relationship of circulating granulocyte-macrophage colony stimulating factor (GM-CSF) and soluble adhesion molecules ICAM-1 and VCAM-1 (sICAM-1 and sVCAM-1) to the severity of acute myocardial infarction (AMI) and the pathophysiological events of post-infarction left ventricular dysfunction. We investigated the kinetics of these inflammatory mediators in the plasma of patients with AMI, and correlated the findings with the clinical severity of the disease during the first week of hospitalization as well as the degree of left ventricular dysfunction one month after the AMI. Plasma levels of inflammatory markers were determined in 41 AMI patients (all received thrombolytic treatment) by ELISA assays, serially during the first week of hospitalization and one month after hospital admission. Patients (n = 20) with uncomplicated AMI (Killip class I) were classified as group A, patients (n = 21) with AMI complicated by heart failure manifestations (Killip classes II and III) were classified as group B, while 20 age- and sex-matched volunteers were used as healthy controls. A sustained increase in GM-CSF, sICAM-1 and sVCAM-1 plasma concentrations was observed only in group B during the first week of the study. Patients from group B exhibited significantly higher levels of GM-CSF (P < 0.01), sICAM-1 (P < 0.05) and sVCAM-1 (P < 0.01) than patients from group A and the healthy controls (P < 0.001). In group B patients, significant correlations were observed between the peak of GM-CSF levels and the peak of serum creatine kinase-MB (r = 0.42; P < 0.05), white blood cell counts (r = 0.67; P < 0.001) and LVEF (r =- 0.51; P < 0.01). At one month follow-up, patients (n = 17) with severe post-infarction left ventricular dysfunction (LVEF 35%). Significant correlations were observed between GM-CSF levels and left ventricular end-diastolic volume index (r = 0.55; P < 0.001) or left ventricular end-systolic volume index (r = 0.49; P = 0.001). We have found a significant elevation of plasma GM-CSF and soluble adhesion molecules during the course of AMI, with the highest values in patients with AMI complicated by heart failure manifestations and severe left ventricular dysfunction. These monocyte-related inflammatory mediators may actively contribute to the pathophysiology of the disease and post-infarction cardiac dysfunction.     

Clinical significance of matrix metalloproteinases activity in acute myocardial infarction

Matrix metalloproteinases (MMP) degrade myocardial fibrillar collagen in acute myocardial infarction (MI) patients. Their activity is tightly controlled in normal myocardium by a family of closely related tissue inhibitors known as TIMP. An imbalance in their activity might contribute to post-MI remodeling. Plasma levels of MMP-1, TIMP-1 and MMP-1/TIMP-1 complex were measured, using relevant ELISA kits, in 24 (22 males-2 females), acute MI patients with a mean age 59 +/- 14 years. Blood samples were taken on admission (0 h), and 3 h, 6 h, 9 h, 18 h, 24 h, 36 h, 48 h, 3rd, 4th, 5th, 7th, 15th, 30th days after MI. All patients underwent coronary arteriography with ventriculography for estimation of left ventricular ejection fraction (LVEF) and extent of coronary artery diseases, and echocardiographic study for measuring end-diastolic diameter (EDD). Ten patients with an LVEF < 45%, an EDD > 47.5 mm, and heart failure symptoms were included in group A and compared against 12 patients with an LVEF > 45% an EDD < 47.5 mm in group B. Mean plasma concentrations of MMP-1 were higher by 21% in group A (1.3 +/- 0.2 ng/mL) compared to group B (1 +/- 0.1 ng/mL) over the total study period. TIMP-1 plasma concentrations showed very little difference between the 2 groups, (704 +/- 213 ng/mL versus 691 +/- 165 ng/mL, (6%)). Finally, plasma concentrations of MMP-1/TIMP-1 complex were lower by -36% in group A with a mean value of 2.7 +/- 0.6 ng/mL versus 3.7 +/- 0.5 ng/mL in group B. Mean values for the differences were significant at time points 0, 6, 18, 24 and 48 hours for MMP-1 (p < 0.036), and on 48 h and the 4th day for MMP-1/TIMP-1 complex (p < 0.031). Moreover, a good correlation was found between plasma concentrations of creatine kinase (CK) and MMP-1 at 18 h (r = 0.422, p = 0.041) and on the 4th day (r = 0.67, p = 0.046), and TIMP-1 on the 4th day (r = 0.67, p = 0.047). Additionally, mean values for LVEF were 35.8 +/- 8.8% in group A versus 51.2 +/- 1.8% (p = 0.00014) in group B. Also, the EDD in-group A was 52.1 +/- 6.9 mm versus 42.9 +/- 3.2 mm in group B (p = 0.00013). In acute MI patients, increased MMP-1, with no change in TIMP-1, is associated with left ventricular dysfunction and dilatation, suggesting that increased collagenolytic activity contributes to loss of LV function.     

基质细胞衍生因子-1 与围生期心肌病患者心力衰竭的相关性研究

目的:探讨基质细胞衍生因子-1与围生期心肌病患者心力衰竭的相关性。方法:采用前瞻性研究纳入59例围生期心肌病并发心力衰竭患者,33例单纯围生期心肌病患者作为对照组。患者均接受体检、实验室检查、心电图、心脏彩超评估。选取基质细胞衍生因子-1(SDF-1)、超敏C反应蛋白(hs-CRP)、氨基末端脑钠肽前体(NT-pro BNP)、血清肌钙蛋白(TNI)及心脏彩超相关参数为评价指标。结果:(l)围生期心肌病患者循环中基质细胞衍生因子-1水平明显高于对照组;(2)循环中基质细胞衍生因子-1与超敏C反应蛋白(CRP)、氨基末端脑钠肽前体(NT-pro BNP)呈正相关,与超声心动图左心室射血分数(LVEF)呈负相关。结论:基质细胞衍生因子-1与围生期心肌病患者心力衰竭具有显著相关性。     

Amelioration of Liver Injury by Continuously Targeted Intervention against TNFRp55 in Rats with Acute-on-Chronic Liver Failure

Background

Acute-on-chronic liver failure (ACLF) is an acute deterioration of established liver disease. Blocking the TNF (tumor necrosis factor)/TNFR (tumor necrosis factor receptor) 1 pathway may reduce hepatocyte apoptosis/necrosis, and subsequently decrease mortality during development of ACLF. We demonstrated that a long-acting TNF antagonist (soluble TNF receptor: IgG Fc [sTNFR:IgG-Fc]) prevented/reduced development of acute liver failure by blocking the TNF/TNFR1 (TNFRp55) pathway. However, it is still unclear if sTNFR:IgG-Fc can inhibit hepatocyte damage during development of ACLF.

Methodology

Chronic liver disease (liver fibrosis/cirrhosis) was induced in Wistar rats by repeatedly challenging with human serum albumin (HSA), and confirmed by histopathology. ACLF was induced with D-galactosamine (D-GalN)/lipopolysaccharide (LPS) i.p. in the rats with chronic liver disease. Serum and liver were collected for biochemical, pathological and molecular biological examinations.

Principal Findings

Reduced mortality was observed in sTNFR:IgG-Fc treated ACLF rats, consistent with reduced interleukin (IL)-6 levels in serum and liver, as well as reduced hepatic caspase-3 activity, compared to that of mock treated group. Reduced hepatic damage was confirmed with histopathology in the sTNFR:IgG-Fc treated group, which is consistent with reduced Bcl-2 and Bax, at mRNA and protein levels, but increased hepatocyte proliferation (PCNA). This is also supported by the findings that caspase-3 production was up-regulated significantly in ACLF group compared to the mock treated group. Moreover, up-regulated caspase-3 was inhibited following sTNFR:IgG-Fc treatment. Finally, there was up-regulation of hepatic IL-22R in sTNFR:IgG-Fc treated ACLF rats.

Conclusions

sTNFR:IgG-Fc improved survival rate during development of ACLF via ameliorating liver injury with a potential therapeutic value.     

Plasma membrane microdomains regulate TACE-dependent TNFR1 shedding in human endothelial cells

Upon stimulation by histamine, human vascular endothelial cells (EC) shed a soluble form of tumour necrosis factor receptor 1 (sTNFR1) that binds up free TNF, dampening the inflammatory response. Shedding occurs through proteolytic cleavage of plasma membrane-expressed TNFR1 catalysed by TNF-α converting enzyme (TACE). Surface expressed TNFR1 on EC is largely sequestered into specific plasma membrane microdomains, the lipid rafts/caveolae. The purpose of this study was to determine the role of these domains in TACE-mediated TNFR1 shedding in response to histamine. Human umbilical vein endothelial cells derived EA.hy926 cells respond to histamine via H1 receptors to shed TNFR1. Both depletion of cholesterol by methyl-β-cyclodextrin and small interfering RNA knockdown of the scaffolding protein caveolin-1 (cav-1), treatments that disrupt caveolae, reduce histamine-induced shedding of membrane-bound TNFR1. Moreover, immunoblotting of discontinuous sucrose gradient fractions show that TACE, such as TNFR1, is present within low-density membrane fractions, concentrated within caveolae, in unstimulated EA.hy926 endothelial cells and co-immunoprecipitates with cav-1. Silencing of cav-1 reduces the levels of both TACE and TNFR1 protein and displaces TACE, from low-density membrane fractions where TNFR1 remains. In summary, we show that endothelial lipid rafts/caveolae co-localize TACE to surface expressed TNFR1, promoting efficient shedding of sTNFR1 in response to histamine.     

TNFR-Fc fusion protein expressed by in vivo electroporation improves survival rates and myocardial injury in coxsackievirus induced murine myocarditis

Tumor necrosis factor-alpha (TNF-alpha) is one of the major cytokines that modulate the immune response in viral myocarditis, but its role has not yet been thoroughly evaluated. We antagonized TNF-alpha using the expressed soluble p75 TNF receptor linked to the Fc portion of the human IgG1 gene (sTNFR:Fc) by in vivo electroporation, and evaluated its effects on experimental coxsackieviral B3 (CVB3) myocarditis. A plasmid DNA encoding sTNFR:Fc (15microg/mouse) was injected into the gastrocnemius muscles of Balb/C male mice followed by electroporation (day -1). Control mice were injected with an empty vector. One day after electroporation, mice were infected with CVB3 (day 0). Serum levels of sTNFR:Fc increased from day 2 and peaked at day 5 following electroporation. The heart virus titers of sTNFR:Fc mice were higher than those of controls at day 3. However, subsequent to day 12, the survival rates of the sTNFR:Fc mice were significantly higher than those of the controls (36% versus 0% at day 27, P<0.01). Histopathological examination indicated that inflammation and myocardial fibrosis were significantly decreased in sTNFR:Fc mice at day 12. The expressed sTNFR:Fc could modulate the inflammatory process during the post-viremic phase of viral myocarditis.     

Inhibition of soluble tumor necrosis factor ameliorates synaptic alterations and Ca2+ dysregulation in aged rats

The role of tumor necrosis factor α (TNF) in neural function has been investigated extensively in several neurodegenerative conditions, but rarely in brain aging, where cognitive and physiologic changes are milder and more variable. Here, we show that protein levels for TNF receptor 1 (TNFR1) are significantly elevated in the hippocampus relative to TNF receptor 2 (TNFR2) in aged (22 months) but not young adult (6 months) Fischer 344 rats. To determine if altered TNF/TNFR1 interactions contribute to key brain aging biomarkers, aged rats received chronic (4-6 week) intracranial infusions of XPro1595: a soluble dominant negative TNF that preferentially inhibits TNFR1 signaling. Aged rats treated with XPro1595 showed improved Morris Water Maze performance, reduced microglial activation, reduced susceptibility to hippocampal long-term depression, increased protein levels for the GluR1 type glutamate receptor, and lower L-type voltage sensitive Ca(2+) channel (VSCC) activity in hippocampal CA1 neurons. The results suggest that diverse functional changes associated with brain aging may arise, in part, from selective alterations in TNF signaling.     

TNFR1 signaling resistance associated with female stem cell cytokine production is independent of TNFR2-mediated pathways

End-organ ischemia is a common source of patient morbidity and mortality. Stem cell therapy represents a novel treatment modality for ischemic diseases and may aid injured tissues through the release of beneficial paracrine mediators. Female bone marrow mesenchymal stem cells (MSCs) have demonstrated a relative resistance to detrimental TNF receptor 1 (TNFR1) signaling and are thought to be superior to male stem cells in limiting inflammation. However, it is not known whether sex differences exist in TNF receptor 2 (TNFR2)-ablated MSCs. Therefore, we hypothesized that 1) sex differences would be observed in wild-type (WT) and TNFR2-ablated MSC cytokine signaling, and 2) the production of IL-6, VEGF, and IGF-1 in males, but not females, would be mediated through TNFR2. MSCs were harvested from male and female WT and TNFR2 knockout (TNFR2KO) mice and were subsequently exposed to TNF (50 ng/ml) or LPS (100 ng/ml). After 24 h, supernatants were collected and measured for cytokines. TNF and LPS stimulated WT stem cells to produce cytokines, but sex differences were only seen in IL-6 and IGF-1 after TNF stimulation. Ablation of TNFR2 increased VEGF and IGF-1 production in males compared with wild-type, but no difference was observed in females. Female MSCs from TNFR2KOs produced significantly lower levels of VEGF and IGF-1 compared with male TNFR2KOs. The absence of TNFR2 signaling appears to play a greater role in male MSC cytokine production. As a result, male, but not female stem cell cytokine production may be mediated through TNFR2 signaling cascades.     

Elevated CSF levels of TACE activity and soluble TNF receptors in subjects with mild cognitive impairment and patients with Alzheimer's disease

We recently reported that expression levels of tumor necrosis factor (TNF) receptors, TNFR1 and TNFR2, are significantly changed in the brains and cerebrospinal fluid (CSF) with Alzheimer's disease (AD). Moreover, we also found that, in an Alzheimer's mouse model, genetic deletion of TNF receptor (TNFR1) reduces amyloid plaques and amyloid beta peptides (Aβ) production through β-secretase (BACE1) regulation. TNF-α converting enzyme (TACE/ADAM-17) does not only cleave pro- TNF-α but also TNF receptors, however, whether the TACE activity was changed in the CSF was not clear. In this study, we examined TACE in the CSF in 32 AD patients and 27 age-matched healthy controls (HCs). Interestingly, we found that TACE activity was significantly elevated in the CSF from AD patients compared with HCs. Furthermore, we also assayed the CSF levels of TACE cleaved soluble forms of TNFR1 and TNFR2 in the same patients. We found that AD patients had higher levels of both TACE cleaved soluble TNFR1 (sTNFR1) and TNFR2 (sTNFR2) in the CSF compared to age- and gender-matched healthy controls. Levels of sTNFR1 correlated strongly with the levels of sTNFR2 (rs = 0.567-0.663, p < 0.01). The levels of both sTNFR1 and sTNFR2 significantly correlated with the TACE activity (rs = 0.491-0.557, p < 0.05). To examine if changes in TACE activity and in levels of cleaved soluble TNFRs are an early event in the course of AD, we measured these molecules in the CSF from 47 subjects with mild cognitive impairment (MCI), which is considered as a preclinical stage of AD. Unexpectedly, we found significantly higher levels of TACE activity and soluble TNFRs in the MCI group than that in AD patients. These results suggest that TACE activity and soluble TNF receptors may be potential diagnostic candidate biomarkers in AD and MCI.     

Acute changes in dietary omega-3 fatty acid intake lowers soluble interleukin-6 receptor in healthy adult normal weight and overweight males

OBJECTIVE: To determine the effect of a short-term isocaloric exchange of alpha-linolenic acid (ALA, 18:3n3) for linoleic acid (LA, 18:2n6) on fasting levels of soluble interleukin-6 receptor (sIL6R), and soluble tumor necrosis factor-alpha receptors 1 and 2 (sTNFR1 and sTNFR2) in healthy normal weight and overweight/obese adult males. DESIGN: Four-day clinical intervention study with 0.5% or 5% of total energy from ALA. Fasting (10 h) blood samples were obtained on the morning of day 5 in both diet treatments to measure sTNFR1, sTNFR2, and sIL6R. SUBJECTS: Nine normal weight (BMI < 25 kg/m2) and seven overweight (BMI > or = 25 kg/m2) healthy males. RESULTS: Fasting sIL6R decreased significantly from the control (C) diet following four days on the high ALA isocaloric (ISO) diet in normal weight and overweight/obese subjects (normal weight: C = 34.89 +/- 3.17 ng/ml, ISO = 30.91 +/- 2.24 ng/ml, p < 0.05; overweight/obese: C = 38.19 +/- 3.92 ng/ml, ISO = 33.57 +/- 2.47 ng/ml, p , 0.05). The dietary intervention did not have a significant effect on fasting sTNFR1 or sTNFR2. CONCLUSIONS: The results suggest that an isocaloric exchange of ALA for LA can reduce fasting sIL6R concentration by approximately 11% after a four-day dietary intervention in both overweight/obese and normal weight subjects. The data also suggest that longer exposure to a similar diet may have the potential to reduce inflammatory burden and thus lower the risk of both cardiovascular disease as well as diabetes.     

Selective disruption of MMP-2 gene exacerbates myocardial inflammation and dysfunction in mice with cytokine-induced cardiomyopathy

Tumor necrosis factor-alpha (TNF-alpha) plays a pathophysiological role in the development and progression of heart failure. Matrix metalloproteinase (MMP)-2 is involved in extracellular matrix remodeling. Recent evidence suggests a protective role for this protease against tissue inflammation. Although MMP-2 is upregulated in the failing heart, little is known about its pathophysiological role. We thus hypothesized that ablation of the MMP-2 gene could affect cardiac remodeling and failure in TNF-alpha-induced cardiomyopathy. We crossed transgenic mice with cardiac-specific overexpression of TNF-alpha (TG) with MMP-2 knockout (KO) mice. Four groups of male and female mice were studied: wild-type (WT) with wild MMP-2 (WT/MMP(+/+)), WT with MMP-2 KO (WT/MMP(-/-)), TNF-alpha TG with wild MMP-2 (TG/MMP(+/+)), and TG with MMP-2 KO (TG/MMP(-/-)). The upregulation of MMP-2 zymographic activity in TG/MMP(+/+) mice was completely abolished in TG/MMP(-/-) mice, and other MMPs and tissue inhibitors of metalloproteinase were comparable between groups. Survival was shorter for male TG/MMP(-/-) than TG/MMP(+/+) mice. Female TG/MMP(-/-) mice were more severely affected than TG/MMP(+/+) mice with diminished cardiac function. Myocardial TNF-alpha and other proinflammatory cytokines were increased in TG/MMP(+/+) mice, and this increase was similarly observed in TG/MMP(-/-) mice. The extent of myocardial infiltrating cells including macrophages was greater in TG/MMP(-/-) than in TG/MMP(+/+) mice. Selective ablation of the MMP-2 gene reduces survival and exacerbates cardiac failure in association with the increased level of myocardial inflammation. MMP-2 may play a cardioprotective role in the pathogenesis of cytokine-induced cardiomyopathy.     

Elevated levels of soluble TNF receptors 1 and 2 correlate with Plasmodium falciparum parasitemia in pregnant women: potential markers for malaria-associated inflammation

Plasmodium falciparum-infected erythrocytes (IEs) sequester in the intervillous space (IVS) of the placenta causing placental malaria (PM), a condition that increases a woman's chances of having a low-birth-weight baby. Because IEs sequester, they frequently are not observed in peripheral blood smears, resulting in women with PM being misdiagnosed and thus not treated. Because sequestered IEs induce inflammation in the IVS, detection of inflammatory mediators in the peripheral blood may provide an approach for diagnosing PM. Two counterregulatory molecules, TNF-αR (TNFR) 1 and TNFR2, modulate the pathological effects of TNF-α. Levels of these soluble TNFRs (sTNFRs) are reported to be elevated in children with severe malaria, but it is unclear if they are increased in the peripheral blood of PM-positive women with asymptomatic infections. In this study, sTNFR levels were measured throughout the course of pregnancy, as well as at delivery, in women with asymptomatic infections and those who remained uninfected. Results showed that both sTNFRs were significantly increased in the peripheral blood of women with asymptomatic malaria (p < 0.0001) and were positively correlated with parasitemia (p < 0.0001 for sTNFR1 and p = 0.0046 for sTNFR2). Importantly, levels of sTNFR2 were elevated in the peripheral blood of women who were PM-positive but peripheral blood-smear negative (p = 0.0017). Additionally, sTNFR2 levels were elevated in the blood of malaria-positive women who delivered low-birth-weight babies. In vitro studies demonstrated that syncytiotrophoblasts were not a major source of sTNFR. These data suggest that sTNFR2 may be a valuable biomarker for detection of malaria-associated inflammation.     

Differential Expression of Dicer, miRNAs, and Inflammatory Markers in Diabetic Ins2+/− Akita Hearts

Diabetic cardiomyopathy is a leading cause of morbidity and mortality, and Insulin2 mutant (Ins2+/?) Akita is a genetic mice model of diabetes relevant to humans. Dicer, miRNAs, and inflammatory cytokines are associated with heart failure. However, the differential expression of miRNAs, dicer, and inflammatory molecules are not clear in diabetic cardiomyopathy of Akita. We measured the levels of miRNAs, dicer, pro-inflammatory tumor necrosis factor alpha (TNFα), and anti-inflammatory interleukin 10 (IL-10) in C57BL/6J (WT) and Akita hearts. The results revealed increased heart to body weight ratio and robust expression of brain natriuretic peptide (BNP: a hypertrophy marker) suggesting cardiac hypertrophy in Akita. The multiplex RT-PCR, qPCR, and immunoblotting showed up regulation of dicer, whereas miRNA array elicited spread down regulation of miRNAs in Akita including dramatic down regulation of let-7a, miR-130, miR-142-3p, miR-148, miR-338, miR-345-3p, miR-384-3p, miR-433, miR-450, miR-451, miR-455, miR-494, miR-499, miR-500, miR-542-3p, miR-744, and miR-872. Conversely, miR-295 is induced in Akita. Cardiac TNFα is upregulated at mRNA (RT-PCR and qPCR), protein (immunoblotting), and cellular (immunohistochemistry and confocal microscopy) levels, and is robust in hypertrophic cardiomyocytes suggesting direct association of TNFα with hypertrophy. Contrary to TNFα, cardiac IL-10 is downregulated in Akita. In conclusion, induction of dicer and TNFα, and attenuation of IL-10 and majority of miRNA are associated with cardiomyopathy in Akita and could be used for putative therapeutic target for heart failure in diabetics.