Immunoregulatory functions of paf-acether. VI. Inhibition of T cell activation via CD3 and potentiation of T cell activation via CD2

In the present report we further explored the role of paf-acether (paf), a phospholipid cytokine, in the modulation of T cell activation induced via the CD2 and the CD3 pathways. Evidence was obtained that paf inhibited T cell proliferation induced by immobilized CD3 mAb (OKT3i), but potentiated that induced by a combination with the CD2 mAb, anti-(T11.1 + D66). Both effects were dose-dependent between 2 and 10 microM paf, and specific in that lysoPC, a phospholipid closely related to paf, had no effect. The inhibition became apparent after 48 h and was maintained up to 144 h of culture, whereas the enhancement was observed only by 96 h of culture. Interestingly, paf was able to inhibit OKT3i mAb response when added to cultures as late as 24-48 h after the initiation of a 96 h incubation. By contrast, paf enhanced the proliferative response only when added concomitantly with anti-(T11.1 + D66) mAb, suggesting that it modulates an early event of T cell activation. paf, which enhanced T cell proliferation induced via the CD2 pathway, also led to a substantial up-regulation of IL-2 secretion and CD25 expression. Moreover, paf markedly augmented IL-4 secretion upon CD2 mAb stimulation. Finally, when T cells were triggered via the CD3 molecule, paf inhibited the proliferative response but also down-modulated CD25 expression without impairing IL-2 secretion. When considered together, these data demonstrate that paf, a phospholipid cytokine released during inflammatory reactions, play a differential regulatory role in T cell activation induced via the CD3 and CD2 (T11.1 + D66) pathways.     

Increased interleukin-10 production and Th2 skewing in the absence of 5-lipoxygenase

Eicosanoids (prostaglandins and leukotrienes) are important mediators of inflammatory responses. These lipid mediators may also regulate the production of peptide mediators of the immune system. In this study, we investigated the effect of the absence of 5-lipoxygenase (5-LO)-derived leukotrienes on interleukin (IL)-10 production. IL-10 is a key regulator of immune and inflammatory responses, and previous studies have suggested that prostaglandins effect their immunosuppressive functions in part by stimulation of IL-10 production. We therefore investigated whether leukotriene production would have a similar role in regulation of IL-10 production. We have made the striking observation that absence of 5-LO-derived leukotrienes results in increased IL-10 production with a concomitant decrease in the production of pro-inflammatory cytokines, including tumour necrosis factor (TNF)-alpha and IL-12. Moreover, T-cell cytokine production in the absence of 5-LO-derived leukotrienes results in increased IL-4 production and decreased interferon (IFN)-gamma production. This may be in part secondary to increased IL-10 production and its effects on dendritic cell function resulting in altered T-cell differentiation. These findings indicate that, in addition to the central role leukotrienes play in the acute inflammatory response, endogenous leukotrienes are also important regulators of inflammatory cytokine production, via regulation of IL-10 production and in vivo differentiation of T cells.     

肝细胞生长因子激活因子抑制因子-1的表达与功能

肝细胞生长因子激活因子抑制因子-1(hepatocyte growth factor activator inhibitor type1,HAI-1)是一种Kunitz型丝氨酸蛋白酶抑制因子,定位于细胞的基底侧,具有膜型和分泌型两种形式,能有效抑制肝细胞生长因子激活因子HGFA和丝氨酸蛋白酶Matriptase的活性,参与HGF/c-Met信号传导途径调节。HAI-1在包括妊娠、再生及肿瘤等各种正常生理及病理状态下均有不同水平的表达,其表达水平的变化以及其与靶蛋白酶表达比例的变化直接影响到靶蛋白酶的活性,从而在调控个体发育、血管生成、组织损伤修复以及抑制肿瘤的侵袭性生长等生理和病理过程中发挥重要作用。     

Intracellular adhesion molecule-1 up-regulation on thyrocytes by iodine of non-obese diabetic.H2(h4) mice is reactive oxygen species-dependent

Intracellular adhesion molecule-1 (ICAM-1) expression on the thyroid follicular cells of non-obese diabetic (NOD).H2(h4) mice is enhanced by iodide treatment, which correlates with autoimmune thyroid disease in genetically susceptible NOD.H2(h4) mice. The current study examines the mechanism of iodine-enhanced up-regulation of ICAM-1 on the surface of thyroid cells. We hypothesized that the up-regulation of ICAM-1 is due to a transient increase in production of reactive oxygen species (ROS). ROS may initiate signalling of the ICAM-1 gene promoter, enhancing up-regulated ICAM-1 protein on the cell surface. Single-cell suspensions of thyroid follicular cells from thyroiditis-susceptible NOD.H2(h4) or non-susceptible BALB/c mice were treated in vitro with sodium iodide. Extracellular and intracellular ROS were assessed by luminol-derived chemiluminescence and flow cytometry assays respectively. Our results demonstrate that thyroid follicular cells of NOD.H2(h4) generate higher levels of ROS compared with cells from non-susceptible strains of mice. Expression of a subunit protein of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, p67(phox), was analysed by Western blot immunoassay. A constitutive expression of the p67(phox) subunit protein was observed in NOD.H2(h4) mice prior to iodine treatment. No such expression was found in BALB/c mice. Treatment of NOD.H2(h4) thyroid cells with diphenyleneiodium, an inhibitor of NADPH oxidase, reduced generation of ROS and of ICAM-1 protein expression. Thus, thyrocytes from NOD.H2(h4) mice produce enhanced levels of ROS that may be mediated by NADPH oxidase. Consequently, in NOD.H2(h4) mice the ROS-induced signal for ICAM-1 up-regulation may contribute to mononuclear cellular infiltration of the thyroid gland and the progression of autoimmune thyroid disease.     

蛇毒金属蛋白酶抑制剂基因的合成及杆状病毒表达载体的构建

研究蛇毒金属蛋白酶抑制剂（BJ46a）基因的合成及杆状病毒表达载体的构建。根据GenBank中查找的BJ46a基因序列（AF294836），将其分为20个片段，通过缓慢退火PCR法使之拼接为完整的BJ46a基因，经SacⅠ及XhoⅠ双酶切后定向克隆到载体pET-42a（＋）中，PCR扩增、酶切及测序鉴定；根据测序结果用定点突变法逐一改正错误位点。随后将该基因插入到转座载体pFastBac HT C的MCS中，转化大肠杆菌DH5 α-T1，以LB／AP平板筛选阳性重组子，PCR扩增、酶切鉴定，提取pFastBac HT C-BJ46a重组体进一步转化DH10Bac感受态细胞，48h后通过蓝白筛选，挑取单个白色菌落划线，证实所得菌落均为白斑，挑克隆PCR鉴定。结果表明成功构建杆状病毒重组转座载体、重组穿梭载体。含BJ46a基因的重组杆状病毒感染Sf9昆虫细胞，获得目的蛋白的表达。BJ46a基因的合成及杆状病毒表达载体的成功构建，为进一步研究其生物学功能奠定了坚实的基础。     

Expression of the Bcl-2 family member A1 is developmentally regulated in T cells.

During T cell development, cells that fail to meet stringent selection criteria undergo programmed cell death. Thymocyte and peripheral T cell susceptibility to apoptosis is influenced by expression of Bcl-2 family members, some of which are expressed in a developmentally patterned manner. We previously showed developmentally regulated expression of A1, an anti-apoptotic Bcl-2 family member, among B cell developmental subsets. Here we show that cells of the T lineage also express A1 in a developmentally regulated manner. Both A1 mRNA and A1 protein are readily detectable in the thymus, and while present among DN cells, A1 mRNA is up-regulated to very high levels among double-positive (DP) thymocytes. It is then down-regulated to moderate levels among single-positive (SP) thymocytes, and finally expressed at approximately 25-fold lower levels among mature SP CD4(+) and CD8(+) lymph node T cells than among DP thymocytes. Furthermore, we find that in vitro TCR ligation up-regulates A1 expression among both DP and SP thymocytes. Together, these data show that A1 expression is developmentally regulated in T lymphocytes and is responsive to TCR signaling, suggesting that A1 may play a role in maintaining the viability of DP thymocytes.     

Proviral integration site 2 is required for interleukin-6 expression induced by interleukin-1, tumour necrosis factor-�� and lipopolysaccharide

PIM (proviral integration site) kinases are a distinct class of serine/threonine‐specific kinases consisting of PIM1, PIM2 and PIM3. PIM2 is known to function in apoptosis pathways. Expression of PIM2 is highly induced by pro‐inflammatory stimuli but the role of PIM2 in the expression of pro‐inflammatory cytokines is unclear. In this study, we showed that over‐expression of PIM2 in HeLa cells as well as in human umbilical vein endothelial cells enhanced interleukin‐1β (IL‐1β) ‐induced and tumour necrosis factor‐α‐induced IL‐6 expression, whereas over‐expression of a kinase‐dead PIM2 mutant had the opposite effect. Studies with small interfering RNA specific to PIM2 further confirmed that IL‐6 expression in HeLa cells requires PIM2. To investigate the function of PIM2 further, we generated PIM2‐deficient mice. It was found that IL‐6 production was significantly decreased from PIM2‐deficient spleen cells after stimulation with lipopolysaccharide. Taken together, we demonstrated an important function of PIM2 in controlling the expression of the pro‐inflammatory cytokine IL‐6. PIM2 inhibitors may be beneficial for IL‐6‐mediated diseases such as rheumatoid arthritis.     

Expression of Hand2 is sufficient for neurogenesis and cell type-specific gene expression in the enteric nervous system.

The basic helix-loop-helix DNA binding protein Hand2 is expressed in neural crest-derived precursors of enteric neurons and has been shown to affect both neurogenesis and neurotransmitter specification of noradrenergic sympathetic ganglion neurons. In the current study, our goal was to determine whether Hand2 affects neurogenesis and/or expression of vasoactive intestinal polypeptide and choline acetyltransferase in developing enteric neurons. Gain-of-function of Hand2 in HNK-1(+) immmunoselected precursor cells resulted in increased neurogenesis. The number of neurons expressing vasoactive intestinal polypeptide increased in response to Hand2 overexpression although choline acetyltransferase was not affected. Targeted deletion of Hand2 in neural crest cells resulted in loss of all neurons expressing vasoactive intestinal polypeptide along the length of the gastrointestinal tract, patterning defects in the myenteric plexus of the stomach, and altered number and morphology of neurons expressing TH. Our data demonstrate that expression of Hand2 is sufficient and necessary for neurogenesis and expression of a subset of cell type-specific markers in the developing enteric nervous system.     

Regulation of the Caenorhabditis elegans posterior hox gene egl‐5 by microRNA and the polycomb‐like gene sop‐2

In Caenorhabditis elegans, the domains of Hox gene expression are controlled by the novel global regulatory gene sop‐2. We identified a region located 3′ of the Hox gene egl‐5 that promotes ectopic expression of an egl‐5 reporter gene in a sop‐2 mutant. SOP‐2 could directly block positive regulatory factors acting in this region, or it could block their expression. We identified three possible miRNA binding sites within the egl‐5 3′ untranslated region (UTR). Cognate microRNAs are expressed in relevant tissues and can block egl‐5 expression when expressed from a transgene. Mutation of the putative binding sites in the egl‐5 3′UTR resulted in a modest degree of misexpression of a minimal egl‐5 reporter gene, suggesting that microRNAs may contribute to the tight restriction of egl‐5 expression to particular cell lineages. Developmental Dynamics 238:595–603, 2009. © 2009 Wiley‐Liss, Inc.     

Effect of AA-861, a 5-Lipoxygenase Inhibitor, on Leukotriene Synthesis in Human Polymorphonuclear Leukocytes and on Cyclooxygenase and 12-Lipoxygenase Activities in Human Platelets

AA-861, a selective inhibitor of 5-lipoxygenase of arachidonic acid, was tested for ability to inhibit leukotriene C4 and leukotriene B4 synthesis in human polymorphonuclear leukocytes after calcium ionophore stimulation. AA-861 dose-dependently inhibited leukotriene B4 and leukotriene C4 generation in human polymorphonuclear leukocytes; the concentration required to inhibit generation by 50% (IC50) was 3 X 10(-7) M for leukotriene B4 and 1 X 10(-8) M for leukotriene C4. BW-755C inhibited the generation of leukotriene C4 with an IC50 of about 10(-5) M, indicating that AA-861 is about 1,000 times more potent than BW-755C. AA-861 did not affect the activity of either cyclooxygenase or 12-lipoxygenase at a concentration up to 10(-5) M in human platelets. AA-861 did not inhibit histamine release from human basophils. These results indicate that AA-861 selectively inhibits 5-lipoxygenase but not cyclooxygenase or 12-lipoxygenase in human specimens.