Effects of PKI55 protein, an endogenous protein kinase C modulator, on specific PKC isoforms activity and on human T cells proliferation

PKI55 protein, coded by the recently identified KI55 gene [R. Selvatici, E. Melloni, M. Ferrati, C. Piubello, F.C. Marincola, E. Gandini, J. Mol. Evol. 57 (2003) 131-139] is synthesized following protein kinase C (PKC) activation and acts as a PKC modulator, establishing a feedback loop of inhibition. In this work, PKI55 was found to inhibit recombinant alpha, beta(1), beta(2), gamma, delta, zeta and eta PKC isoforms; the effect on conventional PKC was lost in the absence of calcium. Confocal immunofluorescence experiments showed that PKI55 can penetrate into peripheral blood mononuclear cells (PBMC), following a coordinated movement of calcium ions. The addition of PKI55 protein down-regulated the PKC enzyme activity in phytohaemagglutinin-activated PBMC, decreasing the activity of alpha, beta(1) and beta(2) PKC isoforms. Moreover, inhibition in PBMC proliferation was observed. Similar effects were detected in Jurkat T cells transfected with a plasmid containing the coding sequence of PKI55. The PKI55 protein functional role could be to control the pathological over-expression of specific PKC isoforms and to regulate proliferation.     

The effect of acute exercise on GLUT4 levels in peripheral blood mononuclear cells of sled dogs

Using sled dogs as exercise model, our objectives of this study were to (1) assess the effects of one acute bout of high-intensity exercise on surface GLUT4 concentrations on easily accessible peripheral blood mononuclear cells (PBMC) and (2) compare our findings with published research on exercise induced GLUT4 in skeletal muscle. During the exercise bout, dogs ran 5 miles at approximately 90% of VO₂ max. PMBC were collected before exercise (baseline), immediately after exercise and after 24 h recovery.GLUT4 was measured via ELISA. Acute exercise resulted in a significant increase on surface GLUT4 content on PBMC. GLUT4 was increased significantly immediately after exercise (~50%; p<0.05) and reduced slightly by 24 h post-exercise as compared to baseline (~22%; p>0.05). An effect of acute exercise on GLUT4 levels translocated to the cell membrane was observed, with GLUT4 levels not yet returned to baseline after 24 h post-exercise. In conclusion, the present investigation demonstrated that acute high-intensity exercise increased GLUT4 content at the surface of PBMC of sled dogs as it has been reported in skeletal muscle in other species. Our findings underline the potential use of peripheral blood mononuclear cell GLUT4 protein content as minimally invasive proxy to investigate relationships between insulin sensitivity, insulin resistance, GLUT4 expression and glucose metabolism.     

Immune cell activation and subsequent epithelial dysfunction by Staphylococcus enterotoxin B is attenuated by the green tea polyphenol (-)-epigallocatechin gallate

Bacterial superantigens (SAg) are potent T cell activators and when delivered systemically elicit a self-limiting enteropathy in mice. Also, SAg-stimulated human peripheral blood mononuclear cells (PBMC) increase enteric epithelial cell monolayer permeability in vitro. Epigallocatechin gallate (EGCG), the major polyphenol component of green tea (Camilla sinesis) leaf, has been presented as an anti-inflammatory agent. We tested the hypothesis that EGCG (10-100 microM) would block PBMC activation by the SAg, Staphylococcus aureus enterotoxin B (SEB, 1 microg/ml), thus preventing disruption of the epithelial barrier. Pretreatment or co-treatment of human PBMC or murine lymphnode cells with EGCG significantly reduced SEB-induced proliferation and IL-2, IFNgamma, and TNFalpha production. ConA-induced proliferation was also inhibited by EGCG (50 microM) co-treatment. These effects of EGCG were not due to induction of immune cell apoptosis, and were independent of EGCGs anti-oxidant activity, and inhibition of NF-kappaB or AP-1 activation. Moreover, addition of exogenous IL-2 (20 ng/ml) to the cultures could not overcome the immunosuppressive effect of EGCG. Culture supernatant from PBMC stimulated in the presence of EGCG failed to increase the permeability of T84 epithelial cell monolayers: a finding consistent with the reduced IFNgamma and TNFalpha production by SAg+EGCG treated PBMC. These data promote EGCG as a suppressor of T cell activation, and given the prominent role that bacteria and T cells play in inflammatory disease we suggest that EGCG could be a useful addition to current treatments for enteric immune disorders and T cell driven immunopathologies.     

乙型肝炎免疫指标全阴的原发性肝细胞癌患者体内HBV-DNA的检测及研究

本文以聚合酶链式反应(PCR)对35例乙型肝炎免疫指标(HBVm)全阴性的原发性肝细胞癌(PHC)患者血清、外周血单核细胞(PBMC)、肝活检标本进行乙型肝炎病毒HBV-DNA 检测,其检出率分别为34.29%、60.00%、68.57%。说明一部分血清 HBVm 全阴性的 PHC 患者血清并非无传染性,ELISA 法具有一定的局限性。PBMC 内有相当的HBV-DNA 存在,这对于研究 PHC 患者体内 HBV 的存在情况、各型乙型肝炎患者的传染性及疗效的评价提出了新课题,具有十分重要的意义。HBVm 全阴的 PHC 患者肝细胞内 HBV-DNA 有很高的分布,二者有明显的相关关系,支持 HBV 对 PHC 有较高致病性研究。本文结果还显示 PCR 法具有较高的敏感度,对研究 HBV 对人类的感染、在患者体内的分布情况及血清传染性等都具有较高的价值。     

Differential gene expression profiling in blood from patients with digestive system cancers

To develop a non-invasive and sensitive diagnostic test for cancer using peripheral blood, we evaluated gene expression profiling of blood obtained from patients with cancer of the digestive system and normal subjects. The expression profiles of blood-derived total RNA obtained from 39 cancer patients (11 colon cancer, 14 gastric cancer, and 14 pancreatic cancer) was clearly different from those obtained from 15 normal subjects. By comparing the gene expression profiles of cancer patients and normal subjects, 25 cancer-differentiating genes (p < 5.0 × 10⁻⁶ and fold differences >3) were identified and an “expression index” deduced from the expression values of these genes differentiated the validation cohort (11 colon cancer, 8 gastric cancer, 18 pancreatic cancer, and 15 normal subjects) into cancer patients and normal subjects with 100% (37/37) and 87% (13/15) accuracy, respectively. Although, the expression profiles were not clearly different between the cancer patients, some characteristic genes were identified according to the stage and species of the cancer. Interestingly, many immune-related genes such as antigen presenting, cell cycle accelerating, and apoptosis- and stress-inducing genes were up-regulated in cancer patients, reflecting the active turnover of immune regulatory cells in cancer patients. These results showed the potential relevance of peripheral blood gene expression profiling for the development of new diagnostic examination tools for cancer patients.     

外周血单个核细胞TNF-α 基因表达与哮喘相关表型*

目的:探讨外周血单个核细胞(PBMC)TNF-α基因mRNA表达与哮喘严重程度、临床病理及相关影响因素的关系。方法:采用实时荧光定量PCR技术检测67例哮喘病人和25例健康对照PBMC中TNF-α mRNA表达水平,分析其与哮喘控制程度、血浆TNF-α浓度、嗜酸性粒细胞百分比(EOS%)、血浆总IgE浓度和哮喘相关影响因素的关系。结果:经方差分析和SNK-q检验,哮喘未控制组PBMC中TNF-α mRNA表达水平高于正常组(P<0.01)、控制组(P<0.01)和部分控制组(P<0.05)。相关分析显示哮喘病人TNF-α mRNA表达与血浆TNF-α浓度和EOS%呈正相关,相关系数分别为r=0.584(P<0.01)和r=0.29(P<0.05),有吸烟史的哮喘病人TNF-α mRNA表达水平高于非吸烟病人(P<0.05)。结论:哮喘病人PBMC中TNF-α mRNA表达水平与哮喘的控制程度呈负相关,与血浆TNF-α浓度呈正相关,PBMC中TNF-α mRNA表达水平与血浆TNF-α浓度可作为哮喘控制程度的参考指标。     

Human T-cell leukemia virus type 1 Tax transactivates the matrix metalloproteinase 7 gene via JunD/AP-1 signaling

Adult T-cell leukemia (ATL) is a T-cell malignancy associated with human T-cell leukemia virus type 1 (HTLV-1) and characterized by visceral invasion. Degradation of the extracellular matrix by matrix metalloproteinases (MMPs) is a crucial process in invasion of tumors and metastasis. MMP-7 (or matrilysin), is a “minimal domain MMP” with proteolytic activity against components of the extracellular matrix. To determine the involvement of MMP-7 in visceral spread in ATL, this study investigated MMP-7 expression in ATL. MMP-7 expression was identified in HTLV-1-infected T-cell lines, peripheral blood ATL cells and ATL cells in lymph nodes, but not in uninfected T-cell lines or normal peripheral blood mononuclear cells. MMP-7 expression was induced following infection of a human T-cell line with HTLV-1, and specifically by the viral protein Tax. Functionally, MMP-7 promoted cell migration of HTLV-1-infected T cells. The MMP-7 promoter activity was increased by Tax and reduced by deletion of the activator protein-1 (AP-1) binding site. Electrophoretic mobility shift assay showed high levels of AP-1 binding proteins, including JunD, in HTLV-1-infected T-cell lines and ATL cells, and Tax elicited JunD binding to the MMP-7 AP-1 element. Tax-induced MMP-7 activation was inhibited by dominant negative JunD and augmented by JunD/JunD homodimers. Short interfering RNA against JunD inhibited MMP-7 mRNA expression in HTLV-1-infected T-cell lines. These results suggest that the induction of MMP-7 by Tax is regulated by JunD and that MMP-7 could facilitate visceral invasion in ATL.     

The acute-phase protein serum amyloid A induces endothelial dysfunction that is inhibited by high-density lipoprotein

The acute-phase protein serum amyloid A (SAA) is elevated during inflammation and may be deposited in atheroma where it promotes atherosclerosis. We investigated the proatherogenic effects of SAA on the vascular endothelium and their regulation by high-density lipoprotein (HDL). Exposure of human aortic endothelial cells (HAEC) to SAA (0.25-25 μg/ml) decreased nitric oxide (^•NO) synthesis/bioavailability, although the endothelial NO synthase monomer-to-dimer ratio was unaffected. SAA (10 μg/ml) stimulated a Ca²⁺ influx linked to apocynin-sensitive superoxide radical anion (O₂^•−) production. Gene expression for arginase-1, nuclear factor κB (NF-κB), interleukin-8, and tissue factor (TF) increased within 4 h of SAA stimulation. Enzymatically active Arg-1/2 was detected in HAEC cultured with SAA for 24 h. Therefore, in addition to modulating ^•NO bioavailability by stimulating O₂^•− production in the endothelium, SAA modulated vascular l-Arg bioavailability. SAA also diminished relaxation of preconstricted aortic rings induced by acetylcholine, and added superoxide dismutase restored the vascular response. Preincubation of HAEC with HDL (100 or 200, but not 50, μg/ml) before (not after) SAA treatment ameliorated the Ca²⁺ influx and O₂^•− production; decreased TF, NF-κB, and Arg-1 gene expression; and preserved overall vascular function. Thus, SAA may promote endothelial dysfunction by modulating ^•NO and l-Arg bioavailability, and HDL pretreatment may be protective. The relative HDL to SAA concentrations may regulate the proatherogenic properties of SAA on the vascular endothelium.