Complex regulation of the Csk homologous kinase (Chk) by IL-4 family cytokines and IFN-gamma in human peripheral blood monocytes

Csk homologous kinase (Chk) is a tyrosine kinase that shares homology with Csk and, like Csk, has the potential to inhibit src-family kinase function through phosphorylation. In myeloid lineage cells, Chk expression is dependent on monocytic differentiation. IL-4 and IL-13 are cytokines involved in monocytic differentiation that have recently been shown to induce Chk expression in peripheral blood monocytes (PBMs). In this study, we show that two other members of the IL-4 family, IL-3 and GM-CSF, can also induce Chk expression at RNA and protein levels. Interestingly, Chk induction is both blocked and reversed by IFN-gamma treatment. Additionally, a short pretreatment with IFN-gamma is sufficient to prevent Chk induction, and the effects of IFN-gamma are dependent on protein synthesis. Collectively, these results suggest that activation of Chk expression and signaling may have a role in the IL-4 family-mediated differentiation of myeloid cells, and inhibition of Chk activation may be one mechanism by which IFN-gamma alters IL-4-mediated affects.     

Ischaemia/reperfusion induced cardiac stem cell homing to the injured myocardium by stimulating stem cell factor expression via NF-κB pathway

Ischaemia/reperfusion (I/R) is a major cause of heart failure. Recently cardiac stem cells (CSCs) were proposed as the most appropriate cell type for heart disease therapy. However, it is still unclear whether I/R can stimulate the CSCs homing to the injured myocardium. Male Sprague–Dawley rats were subjected to a 30-min ischaemia followed by reperfusion of different intervals. RT-PCR, western blotting and immunohistochemistry were performed to detect stem cell factor (SCF) expression at mRNA and protein levels respectively. Activation of nuclear factor-κB (NF-κB) was determined by electrophoretic mobility shift assay. To assess the homing of CSCs in vivo , BrdU-labelled CSCs were injected into AV-groove before induction of ischaemia and examined by immunofluorescent staining in the injured myocardium after I/R. From day 3 to day 6 after reperfusion, the accumulation of CSCs was significantly elevated in the injured area, which was matched with the increased SCF expression during I/R. Pretreatment of rats with NF-κB inhibitor, N -acetyl- l -cysteine (NAC) not only suppressed NF-κB activation induced by I/R but also attenuated SCF expression. Further analysis revealed that I/R induced phosphorylation of IκBα after 15 min of reperfusion, and the raised phosphor-IκBα returned to the basal level at 2 h of reperfusion. In simulated I/R(SI/R) in vitro , it enhanced NF-κB activation and SCF expression in cultured neonatal rat cardiomyocytes, which was markedly inhibited by NF-κB decoy oligodeoxynucleotide or NAC. Taken together, our results demonstrated that I/R induced CSCs homing to the injured myocardium by stimulating myocardial SCF expression via activation of NF-κB.     

Authentic 17kDa tumour necrosis factor α is synthesized and released by canine mast cells and up-regulated by stem cell factor

Background Previous studies have suggested varying molecular weights for mast cell derived tumour necrosis factor alpha (TNFa) and little data exist upon the factors which may regulate the control of this cytokine in these cells. Objective To determine the molecular weight of canine mastocytoma-derived TNFrt. to determine whether it is pre-formed within the granule and whether its expression could be up-regulated by stem cell factor (SCF). Methods Molecular sizing was assessed by immunoblot. The cellular localization of the TNFα was determined by immunocytochemistry before and after stimulation by A23187 and passive sensitization. Subcellular localization was performed by immuno-gold immunocytochemistry. Changes in the level of mastocytoma mRNA for TNFα in response to stimulation with SCF or fibroblast conditioned media for up to 12 weeks were studied using Northern analysis and changes in the level of TNFα protein expression on immunoblot and immunocytochemistry. Results Mast cells contained authentic 17 kDa TNFα as identified by immunoblotting. Immunocytochemical studies demonstrated preformed TNFα which was released by stimulation with antigen after passive sensitization. or by the calcium ionophore A23187. Further confirmation of the preformed nature of this TNFa was provided by immunogold electron microscopy which localized this cytokine to the granule of the inactive mast cell. Northern blotting revealed a constitutive message for TNFα. which increased in response to fibroblast conditioned media (FCM) and to recombinant human SCF. Immunocytochemical studies of mast cells cultured long-term with FCM or with recombinant SCF showed increased expression of TNFa over the course of 12 weeks incubation with these stimuli. Conclusion Mastocytoma derived—TNFα is a preformed, granule—associated 17 kDa cytokine which is released on stimulation with A23187 or passive sensitization. It is up-regulated by stem cell factor and by FCM over the course of 12 weeks.     

NGF对乳腺癌细胞系MDA-MB-231和MCF-7增殖和存活的影响

目的:研究神经生长因子(NGF)对乳腺癌细胞系MDA-MB-231和MCF-7细胞增殖与存活的影响。方法:运用免疫荧光方法检测NGF及其受体酪氨酸蛋白激酶A(TrkA)的表达;运用酶联免疫吸附测定(ELISA)方法检测细胞的NGF自分泌情况;运用蛋白质印迹(Western blotting)方法检测TrkA蛋白的表达情况;运用NGF阻断剂Ro 08-2750对细胞进行NGF剥夺,通过单核细胞直接细胞毒性测定法(MTT)检测细胞增殖的情况;运用流式细胞仪检测细胞凋亡情况以及细胞周期分布的变化。结果:2株乳腺癌细胞系均表达NGF及其受体TrkA,NGF阻断剂Ro 08-2750能够明显抑制2种细胞的增殖,并具有剂量依赖性;流式细胞仪显示Ro 08-2750处理的MDA-MB-231细胞和MCF-7细胞的S期细胞比例明显增加,G2/M期细胞比例明显降低,MDA-MB-231细胞出现凋亡峰。结论:NGF剥夺明显抑制乳腺癌细胞系MDA-MB-231和MCF-7的增殖,并引起MDA-MB-231细胞凋亡。     

Role of c-kit receptor tyrosine kinase in the development, survival and neoplastic transformation of mast cells

The c-kit gene is allelic with the dominant spotting ( W ) locus on mouse chromosome 5 and encodes a receptor tyrosine kinase. The llgand for c-klt receptor is stem cell factor (SCF), which is the principal growth factor for mast cells. The loss-of-function mutations of c-kit receptor affect the development of mast cells, thereby resulting in a depletion of mast cells. The abundant expression of c-ktt receptor is indispensable for the survival of mast cells. In addition, the galn-of-function mutations of c-kit receptor were found in several tumor mast cell lines. When these galn-of-function mutations were introduced to cells of murine interleukin (IL)-3-dependent cell lines, the expression of c-kit receptor with constitutive tyrosine kinase activity not only abrogated the IL-3 requirement of the cells, but also caused them to become tumorlgenic in nude athymic mice. The gain-of-function mutations of c-kit receptor appear to result in the malignant transformation of mast cells. Taken together, the signals from the c-ktt receptor are essential for the development, survival, and malignant transformation of mast cells.     

A combination of stem cell factor and granulocyte colony-stimulating factor enhances the growth of human progenitor B cells supported by murine stromal cell line MS-5

We have developed a long-term culture system using the murine bone marrow stromal cells MS-5 to support the growth of progenitor B cells with CD34^–, CD10⁺, CD19⁺, and cytoplasmic μ chain (Cμ)-negative surface phenotype from human CD34⁺ cells purified from umbilical cord blood (CB). When 10³ CB CD34⁺ cells/well were seeded on MS-5 stromal cells at the beginning of culture in the absence of exogenously added cytokines, progenitor B cells first appeared after 14 days, and the maximal cell production was achieved during the 6th week of culture. Intriguingly, the addition of recombinant human stem cell factor (rhSCF) and granulocyte colony-stimulating factor (rhG-CSF), but not rhIL-7, strikingly enhanced the growth of progenitor B cells from CB CD34⁺ population cultured on MS-5 stromal cells. The culture of progenitor B cells could be maintained until the 6th week of culture when some cells were revealed to have a Cμ⁺ phenotype, and a small number of cells had immunoglobulin μ chain on their cell surface in the presence of both rhSCF and rhG-CSF. When CD34⁺ cells were cultured physically separated from the stromal layer by membrane, supportive effects of MS-5 stromal cells for the growth of progenitor B cells were not observed. These results suggest that the present culture system could generate progenitor B cells to proliferate from CB CD34⁺ cells, that some of these progenitor B cells could differentiate into immature B cells in conjunction with rhSCF and rhG-CSF, and that a species-cross-reactive membrane-bound factor(s), which stimulates early human B lymphopoiesis, may exist in MS-5 stromal cells. Further studies are required to investigate the mechanism how rhG-CSF acts on progenitor B cells to allow their proliferation and differentiation.     

The effect of epidermal growth factor on production of vascular endothelial growth factor by amnion-derived (WISH) cells

Our objective was to clarify the physiological role of vascular endothelial growth factor (VEGF) by amnion-derived (WISH) cells. WISH cells were cultured, and the effect of epidermal growth factor (EGF), mitogen-activated protein (MAP) kinase kinase or extracellular signal-regulated kinase (ERK) kinase (MEK) inhibitors (U0126) or phosphatidylinositol (PI) 3-kinase on the production of VEGF was examined. VEGF was assayed by ELISA. The activation of MAP kinase and akt, which is phosphorylated by PI 3-kinase, were detected by Western blot analysis using anti-phosphorylated MAP kinase antibody and anti-phosphorylated akt antibody. In the time course of VEGF production following EGF treatment, VEGF production showed a significant increase only after 16 (p < 0.01)–32?h (p < 0.01). EGF increased the production of VEGF by WISH cells in a dose-dependent manner. The MAP kinase and akt activity were determined by treatment with EGF. VEGF production was significantly decreased following pretreatment with U0126 or wortmannin for two hours before treatment with EGF (p < 0.01, p < 0.01). WISH cells appeared to produce VEGF via a mechanism involving tyrosine kinase activation of EGF receptor and MAP kinase or PI 3-kinase. It is suggested that VEGF may contribute to the neovascularization and proliferation of the placenta and gestational tissue, and EGF may play an important role in regulation of VEGF production in the placenta.     

表皮生长因子对高转移人卵巢癌细胞生长和播散的影响

目的：探讨表皮生长因子（ＥＧＦ）对高转移人卵巢癌细胞（ＨＯ－８９１０ＰＭ）生长和播散的影响。方法：用细胞生长曲线，形态变化，细胞迁移集落观察ＥＧＦ对细胞生长和迁移的影响。用免疫细胞化学和Ｗｅｓｔｅｒｎ印迹方法观察ＥＧＦＲ和Ｎｅｕ癌基因蛋白在细胞的表达。结果：当培养液中加ＥＧＦ０．０１ｍｇ·Ｌ－１培养７天，细胞数量增加１１倍，对照细胞数量增加４．９倍。细胞形态变梭形，边界清楚，排列松散，有６３％的细胞长径大于１０μｍ。当加ＥＧＦ０．０１ｍｇ·Ｌ－１培养６天后，可见原始种植面积半径增至０．５０８ｃｍ，有９３２个细胞迁移到原始种植区外。对照细胞未发生迁移现象。免疫细胞化学显示均有ＥＧＦＲ和Ｎｅｕ癌基因蛋白表达。Ｗｅｓｔｅｒｎ印迹方法显示均有ＥＧＦＲ和Ｎｅｕ癌基因蛋白条带。结论：ＥＧＦ能促进高转移人卵巢癌细胞生长，并且随着ＥＧＦ量的增加癌细胞迁移能力增强，该细胞ＥＧＦＲ和Ｎｅｕ癌基因蛋白表达也增强     

干细胞因子反义寡核苷酸对小鼠成纤维细胞-肥大细胞相互作用的影响

目的探讨干细胞因子介导的成纤维细胞-肥大细胞相互作用在哮喘病理生理过程中的作用。方法以SCF反义寡核苷酸(SCF ASON)转染成纤维细胞NIH3T3,免疫组化和RT-PCR法检测SCF的表达;从小鼠骨髓中分离肥大细胞,建立与NIH3T3共育体系,加入10 mg/L SCF ASON,通过ELISA法和荧光测定法检测上清液中组织胺和Eotaxin的含量;绘制NIH3T3和肥大细胞生长曲线;丫啶橙染色、流式细胞仪检测肥大细胞的凋亡。结果SCFASON可显著抑制NIH3T3产生SCF;以SCF ASON干预共培养体系后,成纤维细胞生长受抑,肥大细胞出现凋亡(14.0%±0.81%,96 h);组胺[3.08±0.38]μg/L比较(3.83±0.4)μg/L,P<0.05]和Eotaxin的产生[(4.40±0.33)μg/L比较(5.79±0.40)μg/L,P<0.05]减少。结论SCF可能通过抑制肥大细胞凋亡,促进肥大细胞激活和成纤维细胞增殖,参与哮喘的发生、发展。     

Akt cross-links IL-4 priming, stem cell factor signaling, and IgE-dependent activation in mature human mast cells

Challenge of human mast cells with both stem cell factor (SCF) and IL-4 enhances antigen-dependent mediator release raising the assumption of intracellular crosstalk between the IL-4, SCF, and Fc?RI signaling pathways. Here, we analyzed the intracellular crosstalk of IL-4-, SCF-, and IgE-dependent activation pathways in mucosal mast cells isolated from human intestine. The release of β-hexosaminidase, leukotriene C₄, and IL-8, but not IL-6, was strongly enhanced in response to sequential challenge of mast cells with IL-4, SCF and Fc?RI cross-linking compared to stimulation by Fc?RI cross-linking alone. Previous studies revealed that MAPK and other serine/threonine kinases are involved in mast cell activation processes. Here we found that activation of mast cells by Fc?RI cross-linking alone results in phosphorylation of ERK and p38, but not of Akt. Stimulation with SCF alone also induced phosphorylation of ERK and p38, and additionally of Akt. IL-4 priming enhanced activation of ERK, but blocked activation of p38. Activation of p38 was required for IL-6 production explaining the inhibitory effect of IL-4 on IL-6 expression in human mast cells. Moreover, IL-4 priming that anteceded Fc?RI cross-linking induced activation of Akt. The combined challenge of mast cells with IL-4, SCF and Fc?RI cross-linking substantially up-regulated activation of Akt, whereas blocking of Akt inhibited the pronounced production and release of IL-8 in response to the three mast cell agonists. In summary, our data demonstrate that ERK, p38, and especially Akt play an important role in cross-linking IL-4 priming, SCF signaling, and IgE-dependent activation of mature human mast cells.     

Improved bioassay for the detection of porcine tumor necrosis factor using a homologous cell line: PK(15)

Close similarities of various physiological parameters makes the pig one of the preferred animal models for the study of human diseases, especially those involving the cardiovascular system. Unfortunately, the use of pig models to study diseases such as viral hemorrhagic fevers and endotoxic shock syndrome have been hampered by the lack of the necessary immunological tools to measure important immunoregulatory cytokines such as tumor necrosis factor (TNF). Here we describe a TNF-bioassay which is based on the porcine kidney cell line PK(15). Compared to the widely used murine fibroblastoid cell line L929, the PK(15) cell line displays a 100–1000-fold higher sensitivity for porcine TNF-, a higher sensitivity for human TNF-, and a slightly lower sensitivity for murine TNF-. Using a PK(15) bioassay we can detect recombinant TNF- as well as cytotoxic activity in the supernatants of lipopolysaccharide (LPS)-activated porcine monocytes at high dilutions. This suggests that the sensitivity of the test should permit the detection of TNF in biological specimens such as pig serum.     

脐血干细胞移植与神经生长因子联合治疗小儿脑性瘫痪的安全性分析

背景：脐血干细胞移植联合神经生长因子治疗小儿脑性瘫痪是临床研究热点。 目的：分析脐血干细胞移植联合神经生长因子治疗小儿脑性瘫痪疗效及安全性。 方法：选取天津红桥医院儿科2011年11月至2013年2月收治的脑性瘫痪患儿80例,按治疗方法分别分为试验组(n=40)和对照组(n=40),分别接受脐血干细胞移植与神经生长因子联合治疗和单独神经生长因子治疗。 结果与结论：与治疗前相比,2组患儿治疗后脑性瘫痪综合功能评定表及粗大运动功能测量量表分值均有所提高(P < 0.05),且白细胞计数、中性粒细胞分数、总胆红素、丙氨酸氨基转移酶、天冬氨酸氨基转移酶明显升高(P < 0.05),且试验组患儿的治疗效果优于对照组(P < 0.05)。所有患儿未见严重不良反应。说明脐血干细胞移植联合神经生长因子治疗小儿重症脑性瘫痪疗效显著,安全性高。中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程全文链接：     

Characterization of a newly established human acinic cell adenocarcinoma cell line (HACC) originating from the salivary gland: Morphological features and role of various growth factors on the growth of the HACC cell line

Human acinic cell adenocarcinoma cell (HACC) line was established from the pleural effusion that contains meta-static tumor cells of acinic cell adenocarcinoma of papillary and microcystic type originating from the parotid gland. The HACC cells grew in an adherent monolayer with a doubling time of 66 h. Implanted tumor of SCID mice revealed similar histologlcal findings to that of the primary tumor. The HACC cells produced mucin and expressed epithelial markers as well as α₁-antitrypsin and lysozyme, whereas salivary peptide P-C was expressed in cultured HACC cells but not In the primary and Implanted HACC cell tumors. S-100 protein was also expressed in both the primary tumor and HACC cell line. Neither amplification of common oncogenes nor expression of p53 was observed. The receptor for epidermal growth factor (EGF) was expressed, indicating EGF and transforming growth factor-α (TGF-α) enhanced the growth of the HACC line. Unexpectedly, tumor necrosis factor-α (TNF-α) also enhanced the growth of the HACC line significantly. However, there was no evidence of autocrine growth using these growth factors. In contrast, TGF-β1 inhibited the growth of the HACC cell line through apoptosis. The HACC cell line has features similar to both acinar and intercalated ductal cells of the salivary gland. Epidermal growth factor, TGF-α and TNF-α are potential growth factors for the HACC cell line. The HACC cell line may be a good model for studying the biological behavior of salivary gland neoplasms.     

Relationship between a novel human cytotoxin (factor 2) produced by a B cell line (Karpas 160) and phorbol-myristate-acetate-associated cytotoxicity.

We found that 160b cells, a subclone of Karpas 160 human B cell line, spontaneously secreted a novel cytotoxin, factor 2 (F2). F2 was also extracted from the cells by 60% ammonium sulphate, 0.5% CHAPS and 0.28% Triton X-114. We were able to show that phorbol myristate acetate (PMA) greatly enhanced the production of F2, and PMA may also account for part of the putative F2 cytotoxic activity to K562 cells in crude preparations. We compared the cytotoxic effect of F2 with PMA-associated F2-like cytotoxicity to K562 cells as well as the adequacy of our schemes to purified F2 with regard to its separation from PMA. We found that it was possible to separate PMA from F2 preparations by gel filtration and Rotofor preparative isoelectric focusing. The fate of PMA was also monitored with 3H-PMA and chromatographic profiles of 3H-PMA were studied using DE52 and gel filtration chromatography. We were able to establish that less than 2.9% of the cytotoxicity to K562 was due to PMA. We also found that the radioactive peaks and cytotoxicity peaks to K562 were not well correlated, indicating that the cytotoxicity was not mainly due to remaining PMA. Activated charcoal removed virtually all F2 and PMA but not tumour necrosis factor activity. Our results also showed that cytotoxicity to K562 resulting from F2 or PMA-associated proteins had different physicochemical properties, indicating that they are different molecular entities. These findings are consistent with the earlier observation that 160 cells produce F2 spontaneously and that PMA can amplify its production significantly.     

抗KDR单克隆抗体的制备

目的：研制针对肿瘤新生血管内皮细胞生长因子受体（KDR）的特异性单克隆抗体（mAb）。方法：以谷胱甘肽转硫酶耦联的KDR（GST-KDR）可溶性融合蛋白免疫BALB／c小鼠,采用杂交瘤技术制备分泌抗KDRmAb的杂交瘤细胞。结果：获得1株稳定分泌抗KDRmAb的杂交瘤细胞株,命名为3E9B2G11。结论：成功获得能分泌抗KDRmAb杂交瘤细胞,为以后将该mAb改造成小分子酪氨酸激酶抑制剂,使其能进入细胞内与受体KDR特异性结合并阻断血管内皮细胞生长因子的强大生物学功能奠定了重要基础。     

Levels of glial cell line‐derived neurotrophic factor are decreased,but fibroblast growth factor 2 and cerebral dopamine neurotrophic factor are increased in the hippocampus in Parkinson’s disease

Growth factors can facilitate hippocampus‐based learning and memory and are potential targets for treatment of cognitive dysfunction via their neuroprotective and neurorestorative effects. Dementia is common in Parkinson’s disease (PD), but treatment options are limited. We aimed to determine if levels of growth factors are altered in the hippocampus of patients with PD, and if such alterations are associated with PD pathology. Enzyme‐linked immunosorbent assays were used to quantify seven growth factors in fresh frozen hippocampus from 10 PD and nine age‐matched control brains. Western blotting and immunohistochemistry were used to explore cellular and inflammatory changes that may be associated with growth factor alterations. In the PD hippocampus, protein levels of glial cell line‐derived neurotrophic factor were significantly decreased, despite no evidence of neuronal loss. In contrast, protein levels of fibroblast growth factor 2 and cerebral dopamine neurotrophic factor were significantly increased in PD compared to controls. Levels of the growth factors epidermal growth factor, heparin‐binding epidermal growth factor, brain‐derived neurotrophic factor and mesencephalic astrocyte‐derived neurotrophic factor did not differ between groups. Our data demonstrate changes in specific growth factors in the hippocampus of the PD brain, which potentially represent targets for modification to help attenuate cognitive decline in PD. These data also suggest that multiple growth factors and direction of change needs to be considered when approaching growth factors as a potential treatment for cognitive decline.