Chondrogenesis of hMSC in affinity-bound TGF-beta scaffolds

Herein we describe a bio-inspired, affinity binding alginate-sulfate scaffold, designed for the presentation and sustained release of transforming growth factor beta 1 (TGF-β1), and examine its effects on the chondrogenesis of human mesenchymal stem cells (hMSCs). When attached to matrix via affinity interactions with alginate sulfate, TGF-β1 loading was significantly greater and its initial release from the scaffold was attenuated compared to its burst release (>90%) from scaffolds lacking alginate-sulfate. The sustained TGF-β1 release was further supported by the prolonged activation (14 d) of Smad-dependent (Smad2) and Smad-independent (ERK1/2) signaling pathways in the seeded hMSCs. Such presentation of TGF-β1 led to hMSC chondrogenic differentiation; differentiated chondrocytes with deposited collagen type II were seen within three weeks of in vitro hMSC seeding. By contrast, in scaffolds lacking alginate-sulfate, the effect of TGF-β1 was short-term and hMSCs could not reach a similar differentiation degree. When hMSC constructs were subcutaneously implanted in nude mice, chondrocytes with deposited type II collagen and aggrecan typical of the articular cartilage were found in the TGF-β1 affinity-bound constructs. Our results highlight the fundamental importance of appropriate factor presentation to its biological activity, namely - inducing efficient stem cell differentiation.     

Nicotine and cotinine affect the release of vasoactive factors by trophoblast cells and human umbilical vein endothelial cells

Objective

To examine nicotine (N) and cotinine (C) effects on trophoblast cells (TCs) and human umbilical vein endothelial cells (HUVEC) secretion of soluble fms-like tyrosine kinase (sFlt-1), soluble endoglin (sENG), placental growth factor (PlGF), transforming growth factor-beta (TGF-beta) and vascular endothelial growth factor (VEGF).

Study design

Human placentas and umbilical cords were collected from uncomplicated pregnancies at term from a total of 24 non-smoking women with a history of normal blood pressure. TCs and HUVEC were cultured for 24 h with C or N (from 10⁻¹² to 10⁻⁷ M).

Main outcome measures

sFlt-1, sENG, PlGF, TGF-beta and VEGF release and messenger RNA (mRNA) expression were evaluated by ELISA and real-time polymerase chain reaction (PCR), respectively.

Results

N and C reduced sFlt-1, sENG and PlGF release by TCs and TGF-beta release by HUVEC. Conversely, N and C increased PlGF secretion, while N alone increased sFlt-1 release by HUVEC. N and C were able to modulate VEGF mRNA expression in HUVEC.

Conclusions

Our results suggest that N and C affect the balance of some important vasoactive factors released by TCs and HUVEC. This might be one of the possible mechanism through which smoke reduces the risk of hypertensive disorders during pregnancy as well as contributes to the well known detrimental effects of smoking on fetal development.     

Do Regulatory T Cells Play a Role in the Control of Homeostatic Proliferation?

The control of peripheral lymphocyte numbers is a fundamental aspect of the immune system. Regulatory T cells are involved in the suppression of autoimmune, antitumor, allergic, and other inflammatory responses, as well as in facilitating graft acceptance. In this paper, we discuss whether the control of homeostatic proliferation is another facet of the immune system that is controlled by regulatory T cells. A review of the published data connecting regulatory T cells with the control of homeostatic proliferation indicates that several key questions remain open. One of these relates to the stage at which regulatory T cells could play a role (i.e., T-cell proliferation vs. survival).     

The differential expression of TGF-β1, ILK and wnt signaling inducing epithelial to mesenchymal transition in human renal fibrogenesis: an immunohistochemical study

Epithelial-to-mesenchymal transition (EMT) is a process for fully differentiated epithelial cells to undergo a phenotypic change to fibroblasts via diverse intracellular signaling pathways. While the pivotal role of fibroblasts in renal fibrosis is widely accepted, their origin remains undefined. In addition, although a large number of studies have provided evidence of EMT in human kidney diseases, specific signaling pathways leading to EMT have not yet been discovered in humans. To evaluate the origin of interstitial fibroblasts and signaling pathways involved in the EMT process, we analyzed the differential expression of EMT-related molecules in paraffin-fixed sections from 19 human fibrotic kidneys and 4 control kidneys. In human fibrotic kidneys, tubular epithelial cells (TECs) with intact tubular basement membrane (TBM) showed loss or down-regulation of an epithelial marker (E-cadherin), de novo expression of mesenchymal markers (vimentin and fibronectin), and significant up-regulation of inducers and mediators controlling the EMT process (transforming growth factor-β1 (TGF-β1), p-Smad2/3, β1-integrin, p38 mitogen-activated protein kinase (MAPK), WNT5B and β-catenin) in the areas of interstitial inflammation and fibrosis, compared with their expression in control kidneys. In conclusion, the type II EMT process in humans is thought to be an adaptive response of TECs to chronic injury and is regulated by interconnections of TGF-β/Smad, integrin/integrin-linked kinase (ILK) and wnt/β-catenin signaling pathways.     

生长因子β1对儿童哮喘的作用及孟鲁司特钠对其影响

目的 探讨生长因子β1在儿童哮喘中的作用及观察孟鲁司特钠对其的影响。方法 筛选2009年9月-2010年9月我院哮喘专病门诊轻度持续哮喘患儿60例及来院健康体检儿童30例,将哮喘患儿随机分成孟鲁司特钠组和安慰剂对照组;采用双抗夹心酶联免疫吸附试验( ELISA)、RT-PCR技术分别检测治疗前后患儿血浆中TGF-β1水平和外周血单个核细胞(PBMC)中TGF-β1mRNA表达;采用流式细胞技术,检测表达叉状头/翅膀状螺旋转录因子3的CD4T调节细胞(Foxp3+ CD4+ Treg)及各亚型的比例。结果 (1)血浆中TGF-β1水平：治疗前哮喘组[(11.51±1.12) ng/L]明显低于健康对照组[(47.92±1.52) ng/L](q=20.01,P＜0.01);治疗后,孟鲁司特钠组[ (20.03±1.14)ng/L]高于安慰剂组[(12.10±3.91) ng/L]（q=14.62,P＜0.05）,但均值仍低于健康对照组;(2)外周血单个核细胞中TGF-β1 mRNA表达：治疗前哮喘组(0.31 +0.07)明显低于健康对照组(0.61±0.2) (q =8.97,P＜0.05);治疗后,孟鲁司特钠组(0.46±0.13)表达高于安慰剂组(0.32±0.04)（q=8.25,P＜0.05）,但仍低于健康对照组;(3)流式细胞检测结果各组间比较差异有统计学意义(P＜0.05)：哮喘患儿与健康对照组相比,Foxp3+ CD4+ Treg细胞比例增加[(8.30±1.30)％,(6.05±1.80)％];其中CD45 RA+ Foxp3lo比例增高[(4.60±1.04)％,(3.27±1.03)％];CD45 RA - Foxp3h1比例降低[(0.75±0.13)％,(0.93±0.26)％];CD45 RA-Foxp3lo比例两组差异无统计学意义。治疗后,孟鲁司特钠组较安慰剂组,aTreg细胞占Foxp3+ CD4+ Treg比例增加[(1.16±0.24)％,(0.89±0.22)％],差异有统计学意义。结论哮喘儿童体内存在血浆及外周血单个核细胞中TGF-β1表达的降低,可能是导致哮喘发病的重要原因;孟鲁司特钠能有效改善TGF-β1的表达并通过调节Foxp3的表达来发挥治疗作用。     

局部晚期非小细胞肺癌患者血清TGF-β1及IL-6与放射性肺炎的相关性

目的评价局部晚期非小细胞肺癌(LA-NSCLC)同步放化疗患者放疗前、放疗中和放疗后血清转化生长因子-β1(TGF-β1)、白细胞介素-6(IL-6)的变化及调强放射治疗计划剂量体积直方图(DVH)参数与放射性肺炎的相关性。方法 49例行同步放化疗的LA-NSCLC患者,在治疗前、治疗过程中每周及治疗后第13周采用ELISA法检测血清TGF-β1和IL-6水平。应用Varian Eclipse DX计划系统,设计并评估调强放射治疗计划,并采集相关物理学参数。放射性肺炎按RTOG急性放射性肺炎标准评价,评价终点为≥2级放射性肺炎。结果 11例发生了放射性肺炎。放射性肺炎组放疗前血清TGF-β1浓度与无放射性肺炎组比较差异无统计学意义,有放射性肺炎组在放疗中各个时间点和放疗后均高于无放射性肺炎组(P<0.05);血清IL-6浓度在放疗前、放疗中各个时间点和放疗后有放射性肺炎组均高于无放射性肺炎组(P<0.05);放射性肺炎组DVH相关参数(包括肿瘤靶区的平均剂量、最大剂量、最小剂量,肺脏V5、V10、V20、平均剂量、正常组织并发症概率,食管V45,心脏平均受照剂量及脊髓最大受照剂量)与放射性肺炎组比较差异无统计学意义。结论 LA-NSCLC患者同步放化疗时,在使用DVH相关参数严格限制正常组织受照剂量的同时,血清TGF-β1和IL-6可以作为放射性肺炎的预测因子。     

Mucins and toll-like receptors: kith and kin in infection and cancer

Inflammation is underlying biological phenomenon common in infection and cancer. Mucins are glycoproteins which establish a physical barrier for undesirable entry of foreign materials through epithelial surfaces. A deregulated expression and an anomalous glycosylation pattern of mucins are known in large number of cancers. TLRs are class of receptors which recognize the molecular patterns of invading pathogens and activate complex inflammatory pathways to clear them. Aberrant expression of TLRs is observed in many cancers. A highly orchestrated action of mucins and TLRs is well evolved host defence mechanism; however, a link between the two in other non-infectious conditions has received less attention. Here we present an overview as to how mucins and TLRs give protection to the host and are deregulated during carcinogenesis. Further, we propose the possible mechanisms of cross-regulation between them in pathogenesis of cancer. As both mucins and TLRs are therapeutically important class of molecules, an understanding of the underlying molecular mechanisms connecting the two will open new avenues for the therapeutic targeting of cancer.     

转化生长因子-β受体抑制剂复合物C促进人胚胎干细胞向视网膜色素上皮细胞定向诱导

目的 观察转化生长因子-β（TGF-β）受体抑制剂复合物 C对人胚胎干细胞（hESC）向视网膜色素上皮（RPE）细胞定向诱导效率的影响。方法 将H1 hESC分为对照组和实验组。对照组在细胞过度融合后,以去除碱性成纤维细胞生长因子的血清替代物培养体系诱导RPE细胞定向分化。实验组于诱导分化前6 d在培养体系中加入1 μmol/L 复合物 C。诱导分化第1、3、5周,采用实时荧光定量逆转录聚合酶链反应（RT-PCR）检测两组人类配对盒基因（PAX6）、小眼球相关转录因子（MITF）、细胞视黄醛结合蛋白（CRALBP）、RPE65 mRNA的表达。将hESC来源的RPE（hESC-RPE）细胞分离纯化,采用RT-PCR、蛋白免疫印迹法（Western blot）和细胞免疫荧光法对纯化的hESC-RPE细胞进行鉴定。结果 诱导分化第4周,实验组肉眼可见色素团块,对照组无色素团块出现。将实验组的色素细胞分离纯化后,可见100%的细胞表现为多角形色素化。RT-PCR检测结果显示,实验组PAX6 mRNA表达在诱导分化第1、3周显著高于对照组,差异有统计学意义（t=28.498、3.141,P＜0.05）;诱导分化第3、5周,实验组MITF（t=8.866、10.111）、CRALBP（t=5.293、5.394）和RPE65（t=9.263、9.504）的表达均明显高于对照组,差异有统计学意义（P＜0.05）。hESC-RPE细胞PAX6、MITF、CRALBP、RPE65 mRNA表达均显著高于hESC（t=9.154、17.284、18.204、44.194）和ARPE-19（t=7.605、16.770、18.190、44.190）细胞,差异有统计学意义（P＜0.05）。Western blot检测结果显示,hESC-RPE细胞高水平表达RPE标志蛋白PAX6、RPE65。荧光显微镜观察发现,hESC-RPE细胞表达RPE细胞特异性标志蛋白PAX6、紧密连接蛋白-1。结论 TGF-β受体抑制剂复合物C能显著提高hESC向RPE定向诱导效率。     

Cryptotanshinone inhibits LPS-induced proinflammatory mediators via TLR4 and TAK1 signaling pathway

Cryptotanshinone (CTN), one of the major constituents of tanshinones, was investigated for anti-inflammatory activity in the murine macrophage cell line RAW 264.7. CTN inhibited the production of nitric oxide (NO) production, as well as expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2) in lipopolysaccharide (LPS)-stimulated macrophages. Since CTN was considered as inhibiting LPS-triggered phosphorylation of mitogen-activated protein kinase (MAPK) and nuclear factor (NF)-κB activation, we consequently evaluated the expression of toll-like receptor 4 (TLR4) and CD14, as well as phosphorylation of TGF-β-activated kinase 1 (TAK1). CTN reduced the expression of CD14 and TLR4, and suppressed LPS-induced phosphorylation of TAK1. Furthermore, CTN significantly increased the survival rate against LPS challenge in D-galactosamine-sensitized mice, which was in line with in vitro results. These results suggested that CD14/TLR4 and TAK1 might be the potential molecular targets for addressing the protective effects of CTN on LPS-induced inflammatory effects in macrophages.