Regulation of MCP-3 and BRCA2 mRNA expression levels by beta(1) integrins.

The integrin cytoplasmic domain has been shown to modulate several cellular functions, including cell proliferation, adhesion, migration, and intracellular signaling. The beta(1) integrin subunits beta(1C) and beta(1A), which contain variant cytoplasmic domains, differentially affect cancer and normal cell functions. To identify target genes selectively regulated by these beta(1) cytoplasmic variants, stable cell transfectants expressing either beta(1A) or beta(1C) under the control of a doxycycline-inducible promoter were obtained using murine beta(1)-deficient GD25 cells. Screening of 1176 murine cDNAs using first-strand cDNA of mRNA isolated from either beta(1C)- or beta(1A)-expressing cells showed a striking differential expression of few genes. The differential expression of two genes, MCP-3 and BRCA2 (monocyte chemoattractant protein-3 and breast cancer susceptibility gene 2, respectively), whose products are involved, respectively, in chemotaxis and embryonic proliferation, was confirmed by Northern blot analysis. Increased MCP-3 and decreased BRCA2 mRNA levels in cells expressing beta(1C) compared to those in cells expressing beta(1A) were observed. Since beta(1C) and beta(1A) stable cell transfectants showed comparable adhesion to fibronectin, upregulation of MCP-3 and downregulation of BRCA2 mRNA levels did not appear to be due to a differential ability of the beta(1C) cells to adhere to the beta(1) ligand fibronectin. Overall, our data show that beta(1) integrin cytoplasmic domain variants control expression of downstream target genes in a differential manner without affecting cell adhesion.     

Murine cytomegalovirus infection markedly reduces serum MCP-1 levels in MCP-1 transgenic mice

Monocyte chemoattractant protein-1 (MCP-1) is a pro-inflammatory chemokine believed to play a major role in atherogenesis. Injured endothelial cells express MCP-1, which attracts monocytes to the blood vessel wall and leads to the formation of atheromas. Cytomegalovirus infection may also play a role in atherogenesis and accelerates inflammation in tissues that overexpress MCP-1. To examine the relationship of cytomegalovirus infection and MCP-1, we infected MCP-1 transgenic mice with murine cytomegalovirus (MCMV) and collected serum 6 days post-infection to evaluate TH1-related cytokine levels by ELISA. Serum levels of IL-10, IL-12 and IFN-gamma were increased in MCP-1 transgenic mice on day 6 following MCMV infection, while levels of IL-1beta and TNF-alpha were undetectable. However, MCP-1 serum levels were reduced >50% in MCP-1 transgenic mice following MCMV infection compared to uninfected transgenic mice. This effect was not as dramatic when an M33 null MCMV was administered to MCP-1 transgenic mice. The mechanism by which MCMV lowers serum MCP-1 levels is unknown, but this effect may enhance the survival of the virus and thus allow CMV to contribute to the chronic inflammation of atherogenesis.     

Effect of siRNA on HSV-1 plaque formation and relative expression levels of UL39 mRNA

RNA interference (RNAi) is a process by which introduced small interfering RNA (siRNA) can cause the specific degradation of mRNA with identical sequences. In this study, we applied siRNAs targeting the UL39 gene of human herpes simplex virus type 1 (HSV-1), which encodes the large subunit of ribonucleotide reductase, ICP6. Using an ICP6 expression reporter plasmid and an in vitro model of infection, we showed that synthetic siRNA silenced effectively and specifically UL39 mRNA expression and inhibited HSV-1 replication. Our work offers new possibilities for RNAi as a genetic tool for inhibition of HSV-1 replication.     

Levels of MCP-1 and GM-CSF mRNA correlated with inflammatory cytokines mRNA levels in experimental autoimmune myocarditis in rats

Infiltration of monocytes and T cells is known to be an essential trigger for the progression of experimental autoimmune myocarditis (EAM) in rats. Monocyte chemotactic protein-1 (MCP-1) and granulocyte-macrophage colony-stimulating factor (GM-CSF) were shown to mediate the migration of monocytes and T cells into inflammatory sites and to proliferate monocytes. Thus, we evaluated levels of MCP-1 and GM-CSF mRNA in the myocardium of EAM in rats using a real time quantitative PCR method. We also examined the correlation of MCP-1 or GM-CSF mRNA levels with those of inflammatory cytokines such as tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta) and interleukin-6 (IL-6) in the same lesion. Levels of MCP-1, GM-CSF, TNF-alpha, IL-1beta and IL-6 mRNA increased with the progression of myocarditis which was accompanied by the accumulation of ED-1 positive cells. The MCP-1 and GM-CSF mRNA levels were positively correlated with TNF-alpha, IL-1beta and IL-6 mRNA levels in the same lesion of EAM. We also demonstrated that serum MCP-1 concentrations were increased during the active stage of EAM, and were correlated with MCP-1 mRNA levels in the myocardium of each rat. These findings suggest that elevated MCP-1 and GM-CSF may associate with the migration and proliferation of monocytes/macrophages in EAM. Thus, MCP-1 and GM-CSF may play an important role in the progression of EAM.     

MCP-1 is selectively expressed in the late phase by cytokine-stimulated human neutrophils: TNF-alpha plays a role in maximal MCP-1 mRNA expression.

Culture supernatants of phytohemagglutinin (PHA)-stimulated human peripheral blood mononuclear cells (PHA-sup) induced monocyte chemoattractant protein-1 (MCP-1) mRNA expression in human neutrophils. MCP-1 mRNA was first detected by Northern analysis at 8 h, and the peak level was detected at 16 h and sustained until 72 h. Cycloheximide and genistein, but not pertussis toxin, inhibited the expression of MCP-1 mRNA. Recombinant tumor necrosis factor alpha (TNF-alpha) induced a low level MCP-1 mRNA accumulation in neutrophils, and addition of anti-TNF-alpha IgG blocked 30-70% of MCP-1 mRNA expression induced with PHA-sup. PHA-sup-stimulated PMN synthesized and secreted 3.1+/-1.3 ng/5 x 10(6) PMN MCP-1 within the first 24 h. Hybridization of 32P-labeled cDNA preparations to an array of human cytokine cDNAs further indicated that MCP-1 mRNA was selectively up-regulated in the late phase after stimulation with the PHA-sup.     

MCP-1 in Alzheimer's disease patients: A-2518G polymorphism and serum levels

MCP-1 levels are increased in CSF of patients with Alzheimer's disease (AD) compared with controls, suggesting a role in the development of dementia. Recently, a biallelic A/G polymorphism in the MCP-1 promoter at position -2518 has been found, influencing the level of MCP-1 expression in response to an inflammatory stimulus. The distribution of the A-2518G SNP was determined in 269 AD patients and in 203 healthy age matched controls, showing no differences between the two groups. On the contrary, a significant increase of MCP-1 serum levels in AD patients carrying at least one G mutated allele was observed. Moreover, the highest peaks of MCP-1 serum levels were present in patients carrying two G alleles. Stratifying by ApoE genotype, gender or age at onset, no differences in both allele frequency and MCP-1 serum concentration were observed. The A-2518G polymorphism in MCP-1 gene does not seem to be a risk factor for the development of AD, but its presence correlates with higher levels of serum MCP-1, which can contribute to increase the inflammatory process occurring in AD.     

细菌脂多糖诱导单核细胞趋化蛋白-1在小鼠睾丸中表达

目的:正常睾丸间质组织中存在一定数量的巨噬细胞群和淋巴细胞,炎症时这些细胞大量增加,这一机制还未完全解明.本研究探讨正常睾丸和系统感染后睾丸中单核细胞趋化蛋白-1(Monocyte chenoattractant protein-1,MCP-1)的表达变化.方法:给予小鼠一次性腹腔注射0.4 μg/kg细菌脂多糖(Lipopolysaccharides,LPS),用半定量RT-PCR和Western blot方法检测3、6、12、24、48小时后单核细胞趋化蛋白-1(MCP-1)mRNA和蛋白质在睾丸中的表达情况,并用组织免疫荧光染色确定MCP-1在睾丸中的部位.结果:MCP-1 mRNA和蛋白质在正常小鼠睾丸中低剂量表达;注射LPS 3小时后MCP-1 mRNA表达水平迅速增加,一直延续到24小时;MCP-1蛋白质表达水平从染毒后12小时开始增加,48小时后仍处于高水平.MCP-1主要在睾丸的间质中表达.结论:正常睾丸中低剂量MCP-1可能起着维持巨噬细胞群在睾丸间质中滞留的作用;系统炎症中,MCP-1在睾丸中的高水平表达可能与吸引单核细胞和巨噬细胞进入间质组织有关.     

Protein and mRNA expression of serum and glucocorticoid-dependent kinase 1 in metanephrogenesis.

Abstract Expression of serum and glucocorticoid-dependent kinase 1 (SGK1) during development in mouse kidney (embryonic day [E] 14 to postnatal day [P] 1) was studied by in situ hybridization and immunofluorescence. In whole embryos, SGK1 mRNA was highly abundant in the developing metanephros, where SGK1 mRNA was expressed in the ureteric buds of the branching collecting duct system and in the mesenchymal blastema-derived comma- and s-shaped bodies. In E14 kidneys, SGK1 protein was below detection level, whereas at day E16, ureteric buds, s-shaped bodies and outgrowing loops of Henle expressed detectable amounts of SGK1 protein. SGK1 protein was also expressed in E16 primary tubules of the collecting duct system. In P1 kidneys, no or only faint SGK1 protein expression was apparent in comma- and s-shaped bodies, whereas SGK1 was continuously expressed by medullary collecting ducts. In conclusion, SGK1 is developmentally expressed in metanephrogenesis. High expression in developing collecting duct and in blastema-derived comma- and s-shaped bodies suggests a dual function of SGK1 in maturation of the reabsorbing collecting duct epithelium and in epithelial transition of the blastema cells.     

急性脊髓损伤患者血清中单核趋化蛋白-1的表达及其意义

目的 :观察急性不完全脊髓损伤患者血清中单核细胞趋化蛋白 1(MCP 1)的表达 ,探讨继发性脊髓损伤的可能机制。方法 :收集 2 8例急性不完全脊髓损伤患者、8例单纯脊柱压缩骨折患者及正常对照的血清 ,ELISA方法检测其中MCP 1的水平 ,并分析其入院时的MRI资料。结果 :与健康对照相比 ,急性不完全脊髓损伤患者血清中MCP 1的浓度明显增高 (P <0 0 1) ;且水平与脊髓受压程度及脊髓损伤的病理类型有相关性 (P <0 0 5 )。结论 :MCP 1可能参与脊髓损伤部位的继发性炎症反应 ,其血清浓度与诊断、预后有一定关系。     

人静脉移植物中MCP-1的表达

目的：探讨人静脉移植物中单核细胞趋化蛋白-1（MCP-1）的表达及其与巨噬细胞浸润的关系。方法：应用免疫荧光组织化学技术对30个静脉移植物再塞标本中MCP-1、CD68（巨噬细胞的标记物）、CD3l（内皮细胞的标记物）的表达进行了检测，用激光共聚焦显微镜拍片，图片用Silicon Graphics Octane进行处理。结果：在正常静脉血管中，MCP-1表达很弱；外膜中有少量CD68免疫阳性细胞，中膜和内膜中少见；CD31免疫阳性细胞仅在血管腔内皮细胞和外膜小血管中可见。在病变静脉血管，MCP-1在内皮细胞、平滑肌细胞中的表达呈强阳性；CD68免疫阳性细胞在外膜、中膜和内膜均可见到；CD31免疫阳性细胞不仅出现在血管腔内皮细胞和外膜小血管内皮细胞，且在中膜小血管内皮细胞也大量出现。免疫双重染色显示内皮细胞和巨噬细胞均表达MCP-1。结论：人静脉移植物MCP-1的表达上调，且和巨噬细胞的浸润关系密切，提示MCP-l对静物移植物炎症细胞的浸润及新内膜的形成有非常重要的作用。     

Risperidone reduces mRNA expression levels of Sulfonylurea Receptor 1 and TASK1 in PC12 cells

Electrophysiological and immunohistochemical studies have demonstrated that glucose-sensing neurons in the hypothalamus contain both ATP-sensitive K(+) (K(ATP)) and tandem-pore K(+) (TASK1 and TASK3) channels and that glucose-induced depolarization or hyperpolarization of these neurons function as an important link between glucose-excited or glucose-inhibited neurons and feeding behavior. Medication with atypical antipsychotics increases the appetite of schizophrenic patients and thus causes increases in body weight. Therefore, the present study investigates mRNA expression levels of the genes encoding the components of these K(+) channel subsets in PC12 cells cultured with risperidone (an atypical antipsychotic) and in the hypothalami of rats subcutaneously injected for 21 consecutive days with 0.1 or 0.01 mg/kg/day of risperidone. The mRNA expression levels of various genes were not obviously altered in rat hypothalami. However, the mRNA expression levels for sulfonylurea receptor 1, a component affording nucleotide-binding folds to K(ATP) channels, and TASK1 were down-regulated in PC12 cells cultured with 50 microM risperidone for 24h, but the amount of intracellular ATP in these cells was not affected by the drug. Collectively, these results indicate that the amplitude of the current through these K(+) channels in PC12 cells might be modulated as a pharmacological effect of risperidone.     

BRCA1 mRNA expression levels as an indicator of chemoresistance in lung cancer

Lung cancer is the most common cancer, with dismal outcome. Treatment approaches, including cisplatin-based chemotherapy and surgery, are currently based on the clinical classification of the tumor, without genetic assessment for predicting differential chemosensitivity. BRCA1 plays a central role in DNA repair, and decreased BRCA1 mRNA expression in the human breast cancer HCC1937 cell line caused cisplatin hypersensitivity, but the relation between BRCA1 and survival in lung cancer patients has never been examined. We used real-time quantitative polymerase chain reaction to determine BRCA1 mRNA levels in 55 surgically resected tumors of non-small-cell lung cancer patients who had received neoadjuvant gemcitabine/cisplatin chemotherapy, and divided the gene expression values into quartiles. When results were correlated with outcome, two cut-offs were observed; patients with levels <0.61 had better outcome, and those >2.45 had poorer outcome. Median survival was not reached for the 15 patients in the bottom quartile, whereas for the 28 in the two middle quartiles, it was 37.8 months (95% CI, 10.6-65), and for the 12 patients in the top quartile, it was 12.7 months (95% CI, 0.28-28.8) (P=0.01). Moreover, when patients were stratified by pathologic stage, those in the bottom quartile had a decreased risk of death (HR=0.206; 95% CI, 0.05-0.83; P=0.026) compared with those in the top quartile, and those in the two middle quartiles also had a decreased risk of death (HR=0.294; 95% CI, 0.10-0.83; P=0.020) compared with those in the top quartile. BRCA1 expression is potentially an important tool for use in cancer management and should be assessed for predicting differential chemosensitivity and tailoring chemotherapy in lung cancer.     

Levels of MCP-1 and GM-CSF mRNA Correlated with Inflammatory Cytokines mRNA Levels in Experimental Autoimmune Myocarditis in Rats

Infiltration of monocytes and T cells is known to be an essential trigger for the progression of experimental autoimmune myocarditis (EAM) in rats. Monocyte chemotactic protein-1 (MCP-1) and granulocyte-macrophage colony-stimulating factor (GM-CSF) were shown to mediate the migration of monocytes and T cells into inflammatory sites and to proliferate monocytes. Thus, we evaluated levels of MCP-1 and GM-CSF mRNA in the myocardium of EAM in rats using a real time quantitative PCR method. We also examined the correlation of MCP-1 or GM-CSF mRNA levels with those of inflammatory cytokines such as tumor necrosis factor- &#102 (TNF- &#102 ), interleukin-1 &#103 (IL-1 &#103 ) and interleukin-6 (IL-6) in the same lesion. Levels of MCP-1, GM-CSF, TNF- &#102, IL-1 &#103 and IL-6 mRNA increased with the progression of myocarditis which was accompanied by the accumulation of ED-1 positive cells. The MCP-1 and GM-CSF mRNA levels were positively correlated with TNF- &#102, IL-1 &#103 and IL-6 mRNA levels in the same lesion of EAM. We also demonstrated that serum MCP-1 concentrations were increased during the active stage of EAM, and were correlated with MCP-1 mRNA levels in the myocardium of each rat. These findings suggest that elevated MCP-1 and GM-CSF may associate with the migration and proliferation of monocytes/macrophages in EAM. Thus, MCP-1 and GM-CSF may play an important role in the progression of EAM.     

通心络对颈动脉粥样硬化性斑块ABCA1表达的影响

目的：探讨通心络对兔模型颈动脉粥样硬化性斑块的三磷酸腺苷结合盒运转体A1（ABCA1）、视黄酸X受体（RXRα）信使核糖核酸（mRNA）表达的影响及机制。方法：32只新西兰大白兔分为4组，其中空白对照组8只（A组），余24只兔于右侧颈动脉放置改良的硅橡胶圈，加1％高胆固醇喂养的方法建立粥样硬化性颈动脉狭窄动物模型。颈动脉狭窄模型而无药物干预的对照组8只（B组）；辛伐他丁治疗组8只，给予辛伐他丁5mg／kg，每天1次（C组）；通心络治疗组8只，给予通心络1g／kg，每日1次（D组）。药物干预前后分别检测兔模型静脉血的血清甘油三酯（TG）、总胆固醇（TC）、低密度脂蛋白胆固醇（LDL）及高密度脂蛋白胆固醇（HDL）水平，两种药物干预2周后处死动物取右侧颈动脉狭窄段及对侧相应段血管，以Western Blot法测定其ABCA1、RXRα蛋白质表达量。结果：给予通心络和辛伐他丁干预兔模型组（C组和D组）ABCA1、RXRα蛋白质表达水平均增高，血脂水平下降。结论：通心络可使模型兔颈动脉粥样硬化斑块的ABCA1 mRNA、RXRα mRNA表达水平和其蛋白质表达水平上调，可能有益于抗动脉粥样硬化斑块形成作用．     

Changes in serum and ascitic monocyte chemotactic protein-1 (MCP-1) and IL-10 levels in cirrhotic patients with spontaneous bacterial peritonitis.

Spontaneous bacterial peritonitis (SBP) is a prototypical infectious disease of cirrhotic patients. It has been suggested that cirrhotic patients' response to infection is less effective because of differences in the inflammatory and immune reactions. This study aimed to investigate the expression of the inflammatory cytokines monocyte chemotactic protein-1 (MCP-1) and interleukin-10 (IL-10) in cirrhotic patients with SBP. The MCP1 and IL-10 levels in the sera and ascitic fluids of cirrhotic patients with (n = 40) or without SBP (n=17) were serially analyzed by ELISA. In the non-SBP group, the mean MCP-1 levels in sera and ascites were 53.0 +/- 45.8 pg/mL and 197.5 +/- 109.5 pg/mL, respectively, and the IL-10 levels were 10.9 +/- 9.5 pg/mL and 77.6 +/- 79.7 pg/mL, respectively. In the SBP group, the mean MCP-1 levels in serum and ascites before treatment were 164.7 +/- 126.4 pg/mL and 365.3 +/- 583.0 pg/mL, respectively, and the IL-10 levels were 31.4 +/- 44.1 pg/mL and 188.1 +/- 189.5 pg/mL, respectively. The sera MCP-1 and ascites IL-10 levels differed significantly between the two groups. In the SBP group, sera and ascitic MCP-1 and IL-10 levels fell during treatment. The low MCP-1 and IL-10 levels on the seventh day of treatment were found to have a statistically significant relationship to patient survival. MCP-1 and IL-10 levels in sera and ascites may be related to the clinical course of SBP.     

LPS-induced murine systemic inflammation is driven by parenchymal cell activation and exclusively predicted by early MCP-1 plasma levels

Systemic inflammation remains a major cause of morbidity and mortality in the United States, across many disease processes. One classic murine model to study this syndrome is lipopolysaccharide (LPS)-induced Toll-like receptor 4 (TLR4)-dependent systemic inflammation. Although most studies have focused on inflammatory cell TLR4 responses, parenchymal cells also express TLR4. Our objective was to define the in vivo role of parenchymal- versus marrow-derived cell activation via TLR4 during LPS-induced inflammation. Mice bearing TLR4 on parenchymal cells only, marrow-derived cells only, both, or neither were generated using bone marrow transplantation. Mortality occurred only in mice that had TLR4 expression on their parenchymal cells. Before onset, virtually all major plasma cytokines and blood neutrophil responses were related to marrow-derived cell activation via TLR4. The only cytokine predictive of oncoming systemic inflammation was the chemokine monocyte chemoattractant protein-1. Late blood neutrophil responses were related to the presence of TLR4 on either parenchymal or marrow cells, whereas plasma cytokine elevations late in LPS-induced systemic inflammation were dependent on mice having TLR4 in both cell compartments. Parenchymal cell activation via TLR4 is a key component of LPS-induced systemic inflammation and mortality, although most plasma cytokine levels and blood neutrophil responses were not key components. Given its unique role, future studies into monocyte chemoattractant protein-1's exact role during systemic inflammation are warranted.     

Predicting skin test sensitivity and total serum IgE levels in family members

A total of 278 individuals in 42 randomly ascertained nuclear families were studied to determine correlations among family members for skin test response and total serum IgE levels. The major aim was to determine whether these measures of allergic response in family members could be used to predict whether the last child in the family would be skin test positive. There were significant correlations in total log[IgE] levels between parents and their children and an even higher correlation between siblings. For the measurement of skin test response (allergy index), the only significant correlation was between siblings. Discriminant analysis was performed with the fourth child in the family as the index case. This was done to determine how many of the index cases could be correctly predicted to be skin test positive or negative based on family information. With just the skin test results on the parents, only three of the 13 positive index cases were correctly predicted. However, when the mean value for the skin test results in the siblings (mean allergy index) was used, eight of the 13 skin test positive index cases were correctly predicted. These results suggest that, although there is a high degree of concordance for allergic disease within families, information from other siblings may be the most useful predictor of allergic status in another child.