氯离子通道阻滞剂对人眼小梁细胞增殖的影响

目的 探讨氯离子通道阻滞剂5-硝基-2-(3-苯丙胺)苯甲酸[5-nitro-2-(3-phenyl-propylamino)-benzoic acid,NPPB]对体外培养人眼小梁细胞增殖的影响.方法 RT-PCR检测体外培养人眼小梁细胞ClC-3 mRNA表达,用不同浓度的NPPB作用体外培养的人眼小梁细胞,台盼蓝染色细胞计数法检测细胞存活率,四甲基偶氮唑蓝法(MTT)检测其增殖能力.结果 体外培养人眼小梁细胞存在ClC-3 mRNA表达;不同浓度NPPB作用后小梁细胞存活率无显著差异;在一定浓度范围内(50～100 μmol/L),NPPB均可使体外培养的人眼小梁细胞增殖能力下降,其中100 μmol/L NPPB的作用最强.结结论一定浓度的NPPB可降低体外培养的人眼小梁细胞增殖能力.     

干扰CLC-2基因表达对人眼小梁细胞细胞周期进程的影响

目的 观察抑制CLC-2基因的表达对人眼小梁细胞细胞周期进程的影响.方法 构建针对CLC-2的小干扰(siRNA)重组表达载体,脂质体LipofectamineTM2000介导转染人眼小梁细胞后,应用RT-PCR半定量检测小梁细胞CLC-2 mRNA表达量的变化;流式细胞仪检测干扰CLC-2表达后,人眼小梁细胞细胞周期进程.结果 与空载体组比较,重组载体组的CLC-2 mRNA表达明显降低,且有效抑制小梁细胞由G0期到G1的转位.结论 抑制CLC-2的表达可以干扰人眼小梁细胞正常细胞周期进程.     

抑制PI3K/AKt信号通路对帕金森病炎症细胞模型反应的影响

目的探讨LY294002(LY)抑制磷脂酰肌醇-3-激酶(PI3K)/蛋白激酶B(AKt)信号通路后对帕金森病(PD)细胞模型中炎症反应的影响。方法体外培养小鼠小胶质瘤细胞(BV2),分为对照组、脂多糖(LPS)组、LY+LPS组及LY组。LPS刺激BV2细胞来诱导炎症反应,运用Western印迹检测PI3K/AKt信号通路标志物磷酸化(p)-AKt蛋白表达情况;LY抑制PI3K/AKt信号通路后,运用荧光定量PCR (Q-PCR)检测炎症因子白细胞介素(IL)-1β、肿瘤坏死因子(TNF)-α的mRNA的表达情况,Western印迹检测诱导型一氧化氮合酶(iNOS)蛋白表达情况。结果与对照组相比,LPS组p-AKt蛋白表达显著下降(P0.01);与LPS组相比,LY+LPS组p-AKt蛋白下降更明显(P0.01);与对照组相比,LPS组IL-1β、TNF-αmRNA及iNOS蛋白表达均显著升高(均P0.01);与LPS组相比,LY+LPS组中IL-1β和TNF-αmRNA及iNOS蛋白表达均显著下降(均P0.01)。结论 LY抑制PI3K/AKt信号通路对BV2细胞中的LPS诱导的炎症反应有一定的抑制作用。     

Stat3活性降低对人胃癌5-氟尿嘧啶耐药细胞的影响

目的探讨Stat3信号在人胃癌5-氟尿嘧啶(5-Fu)耐药细胞及其亲本细胞中活化水平的差异与胃癌耐药机制的关系。方法分别采用凝胶阻滞电泳法和Western blot法检测人胃癌细胞株SGC7901及其5-Fu耐药细胞株SGC7901／R中Stat3-DNA结合活性的变化和磷酸化Stat3蛋白的表达;半定量逆转录-PCR法检测耐药细胞及其亲本细胞中Stat3和血管内皮生长因子(VEGF)mRNA的表达;免疫细胞化学染色法显示VEGF蛋白在2种细胞内的表达情况。结果耐药细胞及其亲本细胞中有不同程度的Stat3组成性激活和磷酸化Stat3蛋白的表达;与亲本细胞SGC7901相比,耐药细胞SGC7901／R中Stat3-DNA的结合活性水平较低,磷酸化Stat3蛋白的表达减少;耐药细胞中Stat3 mRNA的表达下降,核因子-κB1 mRNA的表达显著上升,而VEGF mRNA及其编码的蛋白水平却较其亲本细胞降低。结论人胃癌5-FU耐药细胞株SGC7901／R中Stat3活性水平降低与其耐药机制相关,且可能与耐药细胞中VEGF表达减少有关。     

芹菜素对非小细胞肺癌细胞HCC827增殖、凋亡的影响及机制

目的探究芹菜素(API)对非小细胞肺癌HCC827细胞增殖、凋亡的影响。方法体外常规培养HCC827细胞,取对数细胞进行实验,API的浓度分别设为0、10、20、40、80μmol/L。噻唑蓝(MTT)法测定不同浓度的API对细胞的生长抑制作用;流式细胞仪Annexin V-FITC/PI双染色法检测细胞凋亡率; Western印迹方法检测API对环氧化酶(COX)-2蛋白表达的影响。结果 API能够有效抑制非小细胞肺癌HCC827细胞的增殖,随着芹菜素浓度的增加、作用时间的延长其作用效果更明显(均P<0. 05)。不同浓度的API作用24 h后,细胞的凋亡率逐渐增加,呈现一定浓度依赖性(均P<0. 05); API处理细胞组COX-2蛋白的表达量显著降低,且呈浓度依赖性(均P<0. 05)。结论 API能够抑制非小细胞肺癌HCC827细胞的增殖,并诱导其凋亡,其机制可能与调控COX-2蛋白的表达有关。     

氯离子通道阻滞剂对心肌低氧复氧损伤的影响

目的研究氯离子流在心肌缺氧、缺氧再灌注、缺氧预处理中的病理生理作用。方法利用培养的心肌细胞，制作低氧复氧、缺氧预处理和低渗预处理模型，并使用氯离子通道阻滞剂SITS和Tamoxifen进行干预，最后检测细胞存活率及培养细胞上清液中乳酸脱氢酶(LDH)、丙二醛(MDA)和超氧化物歧化酶(SOD)的活性变化。结果使用氯离子通道阻滞剂后的低氧复氧组，其细胞存活率及上清液中LDH、MDA及SOD活性与对照组相比无明显差异；加入氯离子通道阻滞剂后再进行缺氧预处理、低渗预处理的各组，细胞存活率及LDH、MDA和SOD的活性与单纯低氧复氧组相比无显著性差异。结论氯离子通道阻滞剂、缺氧预处理、低渗预处理对心肌缺氧损伤均具有保护作用，而缺氧预处理、低渗预处理的这一保护作用能被氯离子通道阻滞剂阻断。     

脑出血患者外周血干扰素-γ、细胞免疫球蛋白黏蛋白分子-3 mRNA表达及意义

目的探讨脑出血患者外周血干扰素(IFN)-γ、细胞免疫球蛋白黏蛋白分子(TIM)-3的表达及意义。方法 96例脑出血患者均为发病6 h内来诊,按发病距采血时间分为≤6 h组、>6 h且≤24 h组、>24 h且≤72 h组和>72 h组。另选24例正常人为对照组。检测各组IFN-γ的水平及外周血中单个核细胞TIM-3 mRNA的表达。结果各实验组IFN-γ的表达与对照组均具有统计学差异(P<0.05)。>24 h且≤72 h组IFN-γ的表达显著高于其他实验组(P<0.05)。各实验组TIM-3 mRNA的表达与对照组均具有统计学差异(P<0.05)。>24 h且≤72 h组TIM-3mRNA的表达显著高于其他实验组(P<0.05)。结论脑出血患者发病6 h内,IFN-γ、TIM-3的表达增加,IFN-γ和TIM-3参与脑出血后的病理过程。     

活性氧在COPADG诱导食管癌Eca-109细胞凋亡中的作用

不同浓度2-（3-羧基-1-丙酰氨基）-2-脱氧-D-葡萄糖（COPADG）作用于人食管癌Eca-109细胞24h。结果显示随COPADG浓度的升高Eca-109细胞的抑制率增加,Eca-109细胞凋亡率与COPADG浓度呈正相关,r=1．0,P〈0．01;Eca-109细胞内活性氧（ROS）水平与Eca-109细胞凋亡率呈正相关,r=1.0,P〈0．01。认为COPADG可能通过提高Eca-109细胞内ROS水平诱导Eca-109细胞凋亡。     

ω-3多不饱和脂肪酸对HepG2细胞胆固醇代谢的影响

目的胆固醇是维持哺乳动物细胞存活及功能所不可缺少的成分,然而高水平的血清胆固醇是动脉硬化性心血管疾病的独立危险因素。体内胆固醇代谢的平衡取决于以下三个主要代谢途径:体内胆固醇的合成、小肠对胆固醇的吸收以及胆汁/粪便胆固醇的分泌等。因此,降低体内胆固醇的合成,减少胆固醇的吸收,以及增加胆固醇的分泌都是降低体内胆固醇水平的有效手段。已有研究报道ω-3多不饱和脂肪酸(ω-3 PUFA)可以增加小鼠巨噬细胞胆固醇的流出而促进胆固醇的逆向转运。但ω-3 PUFA对肝细胞胆固醇代谢的影响至今未见报道。本研究旨在探讨ω-3 PUFA在肝细胞胆固醇合成和分泌中的作用。方法应用50μmol/Lω-3 PUFA(DHA或EPA)处理人肝癌细胞系HepG2 48 h后,收集细胞,用甲醇/氯仿(2∶1)溶液抽提细胞总脂质,在表面活性剂TritonX-100的作用下将脂质溶解于蒸馏水中,并用酶法测定细胞胆固醇浓度;提取细胞总蛋白,用Western blot检测与胆固醇合成相关基因SREBP2和HMG-CoA还原酶,胆固醇向胆酸转化的限速酶CYP7a1,以胆固醇分泌相关基因ABCG8的蛋白表达情况。结果经50μmol/LDHA或EPA处...     

CXC趋化因子配体5对前列腺癌细胞生长的作用

目的研究CXC趋化因子配体(CXCL)5受体(CXCR2)蛋白在前列腺癌细胞株PC-3、DU145和LNCaP细胞中的表达情况及CXCL5对LNCaP细胞生长的影响。方法应用Western印迹技术检测CXCR2在3种前列腺癌细胞株PC-3、DU145和LNCaP细胞中的表达情况,筛选出表达量最高的细胞株,然后用MTT法检测5、10、20 ng/ml浓度CXCL5对CXCR2蛋白表达量最高的前列腺癌细胞生长的影响。结果在PC-3、DU145和LNCaP细胞中均有CXCR2蛋白的表达,LNCaP细胞中最高(1.65±0.089),DU145细胞次之(1.44±0.107),PC-3细胞中最低(0.36±0.056)(P<0.05);5、10、20 ng/ml CXCL5浓度组作用LNCaP细胞6 d后,吸光度值分别为0.55±0.06,0.61±0.08和0.73±0.10,与对照组(0.52±0.14)比较,20 ng/ml CXCL5浓度组增长率明显增高(P<0.05)。结论 CXCL5可通过作用于其自身受体CXCR2进而促进LNCaP细胞生长。     

Primary cilia and the reciprocal activation of AKT and SMAD2/3 regulate stretch-induced autophagy in trabecular meshwork cells

Activation of autophagy is one of the responses elicited by high intraocular pressure (IOP) and mechanical stretch in trabecular meshwork (TM) cells. However, the mechanosensor and the molecular mechanisms by which autophagy is induced by mechanical stretch in these or other cell types is largely unknown. Here, we have investigated the mechanosensor and downstream signaling pathway that regulate cyclic mechanical stretch (CMS)-induced autophagy in TM cells. We report that primary cilia act as a mechanosensor for CMS-induced autophagy and identified a cross-regulatory talk between AKT1 and noncanonical SMAD2/3 signaling as critical components of primary cilia-mediated activation of autophagy by mechanical stretch. Furthermore, we demonstrated the physiological significance of our findings in ex vivo perfused eyes. Removal of primary cilia disrupted the homeostatic IOP compensatory response and prevented the increase in LC3-II protein levels in response to elevated pressure challenge, strongly supporting a role of primary cilia-mediated autophagy in regulating IOP homeostasis.

The trabecular meshwork (TM) is a pressure-sensitive tissue located in the anterior segment of the eye and key regulator of intraocular pressure (IOP). Malfunction of this tissue results in improper drainage of aqueous humor outflow, leading to ocular hypertension, the major risk factor for developing glaucoma (1–3). The TM consists of an irregular lattice of collagen beams lined by TM endothelial-like cells, followed by a zone of loose connective tissue containing TM cells, through which aqueous humor must pass before leaving the eye. Changes in pressure gradients and fluid flow associated with eye movement, circadian rhythm, or the ocular pulse cause small and high variations in IOP, which are translated in continuous cycles of tissue deformation and relaxation. Cells in the TM are known to be able to sense these deformations as mechanical forces and respond to them by eliciting a variety of responses, including reorganization of actin cytoskeleton, changes in gene expression, secretion of cytokines, modulation of matrix metalloproteinases, and extracellular-matrix remodeling (reviewed in ref. 4). It is believed that these mechanoresponses are critical regulators of IOP homeostasis; however, the mechanosensors and the downstream mechanotransduction signaling in TM have still not been characterized.Our laboratory has identified the activation of macroautophagy (hereafter autophagy) as one of the responses elicited in TM cells following application of static or cyclic mechanical stretch (CMS) (5–7). Activation of autophagy was also observed quickly after pressure elevation in porcine perfused eyes (5), which prompted us to propose autophagy as a crucial physiological response to adapt to mechanical forces and maintain cellular homeostasis. The exact roles of autophagy in TM cells and tissue function are still under investigation. Most recently, we have shown the CMS-induced translocation of the autophagy marker LC3 (microtubule-associated protein 1 light chain 3) to the nuclear compartment, where it associates with the nucleolus and interacts with the ribophagy receptor NUFIP1 (nuclear FMR1 interacting protein 1), suggesting a potential role of CMS-induced autophagy in surveillance of the nucleolus activity (6). Furthermore, we have also recently provided direct evidence demonstrating autophagy as a regulator of TGF-β/SMAD-induced fibrogenesis in TM cells (8).Autophagy is a fundamental process for degradation or recycling of intracellular components, which is essential to maintain cellular homeostasis. Autophagy occurs constitutively at basal levels, but it is quickly activated upon several types of stress, such as nutrient depletion, pathogen infections, drug treatment, accumulation of aggregated proteins and damaged organelles, and mechanical stress (7, 9–11). The molecular mechanisms by which cells recognize stress and regulate autophagic activity are very complex and differ based on the stimuli. A variety of components, for example, receptor tyrosine kinases, second messengers (Ca²⁺ or cAMP [cyclic adenosine monophosphate]), protein kinases, and downstream autophagy-related (ATG) complexes, participate in the regulation of autophagy (10). Among them, the best characterized is MTOR (mechanistic target of rapamycin kinase), a negative regulator of autophagy (10, 12, 13). Although MTOR acts as a core sensor in autophagic regulation, numerous studies have shown the MTOR-independent autophagy activation upon different stresses (10). Indeed, our own study and that of King et al. showed that the induction of autophagy in response to mechanical stress occurs in an MTOR-independent manner (5, 14). The mechanosensor and the downstream signaling pathway responsible for the activation of autophagy in response to stretching have still not been identified.Primary cilium (PC) is a nonmotile cell-surface projection found almost ubiquitously in vertebrate cells, which acts as a cellular antenna that senses a wide variety of signals, including chemical and mechanical stimuli (15–19). PC plays a critical role in smell, sight, and mechanosensation. PC defects are associated with a number of human diseases termed ciliopathies. The most common feature in patients affected with ciliopathies include visual dysfunction (16). In particular, Lowe syndrome patients often develop ocular hypertension and glaucoma (20). Structurally, the PC consists of a microtubule-based core structure, called axoneme, and a basal body, which is a derivative of the centriole of centrosome from which axoneme is extended and surrounded by ciliary membrane (21). The ciliary membrane is a specialized domain extension of the plasma membrane enriched on signaling receptors and channels, including hedgehog (Hh) and Ca²⁺ pathways, which enables the PC to function as a signaling hub (16, 22, 23). Cargo trafficking into and out of the cilium is mediated by a specialized form of vesicle trafficking, named intraflagellar transport (IFT), that is composed of a multiprotein complex (16, 23).Recent studies have demonstrated the reciprocal relationship between PC and autophagy. Autophagy has been shown to both positively and negatively regulate ciliogenesis. Under nutrient-rich conditions, basal autophagy inhibits cilia growth by limiting trafficking of PC components to the basal body through direct degradation of IFT20 (24). In contrast, nutrient starvation triggers the autophagic degradation of oral-facial-digital syndrome 1 and promotes cilia growth (24, 25). Conversely, functional PC are required for activation of autophagy in response to starvation and fluid flow. In both cases, autophagy was initiated by the recruitment of ATG16L to the basal body, suggesting that this event is a hallmark for PC-induced autophagy. Intriguingly, the signaling pathway mediating PC-induced autophagy activation differed based on the stimuli. While Hh/smoothened (SMO) was reported to mediate activation of autophagy in response to starvation (24), the LKB1–AMPK–MTOR signaling pathway was found to regulate PC-induced autophagy and cell volume in kidney epithelial cells under shear stress (26, 27). Whether PC are also involved in the regulation of autophagy triggered by mechanical stretching has not yet been explored.The purpose of this study is to investigate a potential role of PC in stretch-induced autophagy in TM cells. Here, we report that PC acts as a mechanosensor for CMS-induced autophagy, and we identified AKT1 and SMAD2/3 as critical components of the signal mechanotransduction.     

Impaired TRPV4-eNOS signaling in trabecular meshwork elevates intraocular pressure in glaucoma

Primary Open Angle Glaucoma (POAG) is the most common form of glaucoma that leads to irreversible vision loss. Dysfunction of trabecular meshwork (TM) tissue, a major regulator of aqueous humor (AH) outflow resistance, is associated with intraocular pressure (IOP) elevation in POAG. However, the underlying pathological mechanisms of TM dysfunction in POAG remain elusive. In this regard, transient receptor potential vanilloid 4 (TRPV4) cation channels are known to be important Ca²⁺ entry pathways in multiple cell types. Here, we provide direct evidence supporting Ca²⁺ entry through TRPV4 channels in human TM cells and show that TRPV4 channels in TM cells can be activated by increased fluid flow/shear stress. TM-specific TRPV4 channel knockout in mice elevated IOP, supporting a crucial role for TRPV4 channels in IOP regulation. Pharmacological activation of TRPV4 channels in mouse eyes also improved AH outflow facility and lowered IOP. Importantly, TRPV4 channels activated endothelial nitric oxide synthase (eNOS) in TM cells, and loss of eNOS abrogated TRPV4-induced lowering of IOP. Remarkably, TRPV4-eNOS signaling was significantly more pronounced in TM cells compared to Schlemm’s canal cells. Furthermore, glaucomatous human TM cells show impaired activity of TRPV4 channels and disrupted TRPV4-eNOS signaling. Flow/shear stress activation of TRPV4 channels and subsequent NO release were also impaired in glaucomatous primary human TM cells. Together, our studies demonstrate a central role for TRPV4-eNOS signaling in IOP regulation. Our results also provide evidence that impaired TRPV4 channel activity in TM cells contributes to TM dysfunction and elevated IOP in glaucoma.

Glaucoma is a heterogenic group of multifactorial neurodegenerative diseases characterized by progressive optic neuropathy. It is the leading cause of irreversible vision loss with more than 70 million people affected worldwide (1), and the prevalence is estimated to increase to 111.6 million by the year 2040 (2). Primary open angle glaucoma (POAG) is the most common form of glaucoma, accounting for ∼70% of all cases (1). POAG is characterized by progressive loss of retinal ganglion cell axons that leads to an irreversible loss of vision (1, 3). Elevated intraocular pressure (IOP) is a major, and the only treatable, risk factor associated with POAG (4). The trabecular meshwork (TM), a molecular sieve-like structure, maintains homeostatic control over IOP by constantly adjusting the resistance to aqueous humor (AH) outflow. In POAG, there is increased resistance to AH outflow, elevating IOP (5). This increase in AH outflow resistance is associated with dysfunction of the TM (6–8).The TM has an intrinsic ability to sense the AH flow and regulate outflow facility to maintain IOP homeostasis (6), although the precise flow-sensing mechanisms in TM cells are unclear. In this regard, transient receptor potential vanilloid 4 (TRPV4) cation channels have emerged as a major flow-activated Ca²⁺ entry pathway in multiple cell types (9–12). Upon activation, TRPV4 channels allow localized Ca²⁺ influx (termed as TRPV4 sparklets), which influences a variety of cellular homeostatic processes (13, 14). TRPV4 sparklets are spatially restricted signals with a spatial spread (maximum width at half maximal amplitude) of ∼11 microns (13). Treatment with a selective TRPV4 channel activator GSK1016790A (GSK101) lowered IOP in rats and mice (15). Furthermore, baseline IOP was higher in global TRPV4^−/− mice compared to their wild-type (WT) littermates (15). However, the exact cell type responsible for these IOP-lowering effects is not known. Previous studies have shown that TRPV4 channel protein is expressed in TM cells and tissues (15, 16). The physiological roles of TRPV4 channels in TM cells (TRPV4_TM) and downstream signaling mechanisms remain unknown. TM constitutively expresses Ca²⁺-sensitive endothelial nitric oxide synthase (eNOS) (17), a known regulator of outflow facility and IOP (18–22). In vascular endothelial cells, TRPV4 channels are important regulators of eNOS activity (23–26). We, therefore, hypothesized that TRPV4_TM-eNOS signaling promotes outflow facility and reduces IOP.Glaucoma-associated pathological changes are known to impair physiological function of TM (8). One of the hallmarks of the glaucomatous TM is its inability to maintain normal IOP and AH outflow resistance (6). Here, we postulated that impaired TRPV4_TM-eNOS signaling contributes to TM dysfunction and elevated IOP in glaucoma. In this report, our studies in human TM cells and TM tissue showed shear stress–mediated activation of TRPV4-eNOS signaling. Moreover, reduced AH outflow and elevated IOPs were observed in TM-specific TRPV4^−/− (TRPV4_TM^−/−) mice and eNOS^−/− mice. Importantly, TRPV4_TM activity and shear stress–mediated activation of TRPV4_TM-eNOS signaling are compromised in human glaucomatous TM cells. Our results provide direct evidence for a physiological role of TRPV4_TM-eNOS signaling and indicate that impaired TRPV4_TM-eNOS signaling may underlie TM dysfunction and IOP dysregulation in glaucoma.     

Aging of trabecular meshwork cells of the human eye in vitro

A monolayer cell line established from the trabecular meshwork of a human eye exhibited a limited proliferation potential with about 18 population doublings (PD). There was a gradual increase of the average PD time with increasing PD level. Finally, there was complete growth cessation. These non-replicating cells were larger and more flattened (decrease of the saturation density); they became granulated and finally died off in the course of several months. This growth pattern resembled the senescence phenomenon, as originally described by Hayflick and Moorhead for cultured human fibroblasts. In comparison to proliferating phase-II cultures, senescent cultures revealed reduced glycosaminoglycan synthesis rates with a relative decrease of hyaluronic acid and increase of heparan sulfate. Exogenous (medium-supplied) hyaluronic acid or ascorbic acid stimulated hyaluronic acid synthesis of phase-II cultures.     

Flow cytometric assay of phagocytic activity of human neutrophils and monocytes in whole blood by neutral red uptake

In a new, simple, and fast flow-cytometric method for the simultaneous measurement of phagocytic activity of human neutrophils and monocytes in whole blood, the fluorescence capability of the well-known vital stain, neutral red was used. The incubation of 0.5 ml heparinized human blood with the phagocytic stimulus of zymosan dose- and time-dependently increased the percentage and the red fluorescence intensity of both neutrophils (4.3±1.2 times) and monocytes (2.7±0.7 times) measured cytofluorimetrically. Decreased uptake of neutral red was observed in a patient with phagocytic disorder, based upon impaired engulfment of particles and production of reactive oxygen species. In a patient with chronic granulomatosis, however, no decrease of neutral red uptake was measured. Platelet activating factor and phorbol myristate acetate were also able to increase the uptake of neutral red by both monocytes and neutrophils, but to a lesser extent than zymosan. The advantage of this method is the possibility for the simultaneous measurement of phagocytic activities of monocytes and neutrophils stimulated by either particles or soluble activators. This method is suitable for the selective measurement of activation processes not related to the production of free radicals in the phagocytes.     

Dendritic cells in human aging

Dendritic cells (DCs) play a critical role in linking innate and adaptive immunity. A role of DCs in immunosenescence and chronic inflammation associated with aging has not been investigated in detail. In this article, we will briefly review DCs biology and changes associated with human aging.     

In vitro cultures of trabecular meshwork cells of the human eye as a model system for the study of cellular aging

Cells from the human trabecular meshwork providing a drainage system for the outflow of aqueous humour in the eye were isolated and propagated in monolayer culture. Following serial subcultivation of the primary cultures, there was a gradual decline in the fraction of dividing cells with increasing population doubling level (PDL) resulting finally in growth cessation and disintegration of these 'senescent' cultures. The number of population doublings was at most 20. Senescent cultures revealed reduced glycosaminoglycan synthesis rates (as measured by [14C]glucosamine incorporation) with a relative decrease of hyaluronic acid and increase of heparan sulfate. Medium-supplied (exogenous) hyaluronic acid enhanced hyaluronic acid synthesis of trabecular meshwork cells cultured in a defined, serum-free medium. Ascorbic acid (25-200 micrograms/ml), which is found in very high concentration in the ocular aqueous humour, stimulated hyaluronic acid synthesis of confluent cultures, also. The functional significance of decreased hyaluronic acid (and elevated heparan sulfate) synthesis in the process of cellular aging in vitro (and in vivo?), as well as the importance of hyaluronic acid for the structural integrity and functional activity of the trabecular meshwork were discussed.     

The influence of synovial fibroblasts on the phagocytosis of Staphylococcus by polymorphonuclear cells

Summary It has been suggested that the reduced resistance of patients with rheumatoid arthritis (RA) to bacterial joint infections may be due in part to polymorphonuclear cell (PMN) function. To obtain further insight into the mechanis that contribute to the increased susceptiblity of RA patients to such infections we investigated the influence of different solid surfaces on the ingestion of various bacterial strains by PMN. Both in the presence and absence of serum, phagocytosis of bacteria by PMN was significantly lower on monolayers of synovial fibroblasts as compared to monolayers of endothelial cells and embryonic fibroblasts. It could be shown that the relative influence of the solid surfac on the results of the phagocytosis assay increased when decreasing concentrations of purified IgG were used. The results of this study sugpurified IgG were used. The results of this study suggested that the effect of synovial fibroblasts on PMB may lead to reduced clearance of bacteria from the joint.