LAP~+CD4~+T细胞在肝癌微环境中的分布特点及意义

目的研究肝癌患者肿瘤组织中LAP~+CD4~+T细胞的分布特点,探讨LAP~+CD4~+T细胞在肝癌发生发展中的作用。方法抽取北京大学深圳医院2011年10月到2012年12月期间28例肝癌患者的手术肝组织标本与28例因良性病变行肝切除的正常肝组织标本,采用双重酶标免疫组化染色法检测LAP~+CD4~+T细胞在肝癌组织、癌旁组织及正常肝组织中的分布特点,并取单个高倍视野平均数进行半定量分析。结果肝癌患者肿瘤组织中LAP定位于CD4~+T细胞的胞浆和胞膜上;正常肝组织及癌旁组织中LAP~+CD4~+T细胞以散在方式分布,而在癌组织中多数以簇丛状方式聚集分布于肿瘤间质内,与CD4~+T细胞伴行并与之紧密接触。肝癌组、癌旁组以及正常对照组中LAP~+CD4~+T细胞单个高倍视野平均数分别为(11.25±3.00)、(5.75±1.00)及(2.61±0.83);肝癌组与癌旁组比较、肝癌组与正常对照组比较、癌旁组与正常对照组比较,三组比较差异均有统计学意义(P=0.000,P=0.000,P=0.000,P0.05)。结论肝癌患者肿瘤组织中LAP定位于CD4~+T细胞的胞浆和胞膜上,肝癌微环境中LAP~+CD4~+T细胞明显聚集,多以簇丛状方式分布,与其它淋巴细胞紧密接触,分布数量较癌旁组织及正常肝组织均增多,提示LAP~+CD4~+T细胞在肝癌微环境中浸润,并有可能通过细胞接触的方式抑制肿瘤局部免疫,促使肝癌细胞逃避免疫监视。     

肝细胞癌微环境中CD4+CD25+调节性T细胞的分布及其临床病理意义

目的研究CD4^＋CD25^＋调节性T细胞（Treg）与肝细胞癌（HCC）临床病理之间的关系；探讨其在评价HCC侵袭性和癌症进展以及预后中的价值。方法用双重酶标免疫组织化学的方法测定52例HCC组织及癌旁组织中Treg数量分布状况。分析其临床病理学资料，研究在HCC微环境中Treg数量分布与其各项临床病理学指标间的关系。结果8例正常肝组织中未发现Treg分布，52例HCC病人的肝癌及癌旁组织标本中，在HCC组织中Treg数量明显高于癌旁组织，差异有显著意义。HCC组织的Treg数量与病人性别、年龄、肝硬化、病理学分级、包膜、术前AFP浓度无显著关系（P〉0．05），而与肿瘤的大小、癌栓、子灶、TNM分期有显著关系。25例小肝癌组和27例大肝癌组Treg细胞单个高倍视野平均数分别为7．1440±1．8535、8．0815±1．5122，两组比较在统计学上有显著差异（P〈0．001）；14例有血管癌栓组和38例无血管癌栓组Treg细胞单个高倍视野平均数分别为8．1143±2．4487、7．4526±1．8794，两组比较差异有统计学意义（P〈0．05，P=0．014）；19例癌周有子灶组和33例癌周无子灶组Treg细胞单个高倍视野平均数分别为8．2211±2．4516、7．2909±1．8217，两组比较在统计学上有显著差异（P〈0．001）；不同临床病理分期18例Ⅰ期组、22例Ⅱ期组和12例Ⅲ期组中Treg细胞单个高倍视野平均数分别为6．7333±1．5980、7．8818±2．4171、8．5167±2．2480，在统计学上有显著差异（P〈0．001）。结论肝细胞癌微环境中Treg数量分布与肿瘤大小、血管癌栓、子灶，和临床分期有明显关系，而与其它指标无显著关系。HCC微环境中Treg数量与肝癌的侵袭、进展以及预后有密切关系，HCC微环境中Treg数量增多可以作为判断HCC侵袭性、进展以及预后的一个潜在重要指标。除去或减少肝癌微环境中的Treg细胞有可能提高肿瘤的免疫治疗效果。     

乳腺癌患者外周血CD4+CD25+Foxp3+调节性T细胞水平的检测及意义

目的：探讨乳腺癌患者外周血CD4+CD25+Foxp3+调节性T细胞（Treg）水平检测的意义。 方法：流式细胞术检测74例乳腺癌患者与30例健康对照者外周血CD4+CD25+Foxp3+Treg占CD4+T细胞百分比,分析CD4+CD25+Foxp3+Treg细胞水平与乳腺癌患者临床病理特征及相关免疫组化指标的关系。 结果：乳腺癌患者外周血CD4+CD25+Foxp3+Treg占CD4+T细胞的百分比高于健康对照者[（9.15± 2.24）% vs.（2.29±1.36）%],差异有统计学意义（P<0.05）。统计分析显示,乳腺癌患者外周血CD4+CD25+Foxp3+Treg细胞水平与肿瘤组织学分级、淋巴结转移、pTNM分期以及HER-2、pS2、nm23的表达有关（均P<0.05）,而与肿瘤大小、病理类型以及雌激素受体（ER）、孕激素受体（PR）、p53、Ki-67表达无关（均P>0.05）。进一步相关性分析显示,CD4+CD25+Foxp3+Treg细胞水平与肿瘤组织学分级、淋巴结转移数、pTNM分期、HER-2的表达呈正相关（r=0.583,r=0.333,r=0.919,r=0.604,均P<0.05）而与pS2、nm23表达呈负相关（r=-0.229,r=-0.401,均P<0.05）。 结论：乳腺癌患者外周血CD4+CD25+Foxp3+Treg细胞水平升高,并与与乳腺癌的进展、转移密切相关,对其检测可能有助于患者预后及治疗效果的评估。

     

乳腺癌患者CD4+CD25+Foxp3+调节性T细胞的变化及意义

目的:探讨乳腺癌患者CD4+CD25+Foxp3+调节性T细胞(简称Foxp3+Treg)的变化及意义。方法:选择40例乳腺癌患者和32例乳腺良性肿瘤患者,采用流式细胞术检测外周血Foxp3+Treg、CD8+CD28+T细胞、NK细胞水平;用Western blot和RT-PCR病变乳腺组织Foxp3蛋白与m RNA表达。结果:乳腺癌患者外周血中Foxp3+Treg比例较乳腺良性肿瘤患者明显升高,而CD8+CD28+T细胞、NK细胞比例明显降低(均P0.05),且乳腺癌患者外周血Foxp3+Treg水平与CD8+CD28+T细胞和NK细胞水平呈负相关(r=-0.631,r=-0.578,均P0.05);乳腺癌患者术后外周血Foxp3+Treg水平较术前明显降低(P0.05)。乳腺癌组织中Foxp3蛋白与m RNA的表达均较乳腺良性肿瘤组织明显升高(均P0.05)。结论:Foxp3+Treg和其标记分子Foxp3在乳腺癌患者中的表达增加,且可能通过抑制CD8+CD28+T细胞和NK细胞而产生肿瘤免疫抑制。     

原发性肝癌患者肿瘤浸润T淋巴细胞亚群分析

目的:了解原发性肝癌患者肿瘤浸润T淋巴细胞中CD4+及CD8+的表达特点。方法:收集30例原发性肝癌患者(均有乙型肝炎病毒感染背景)的癌组织(乙肝肝癌组)和癌旁组织(乙肝癌旁组)以及30例因良性病变而行肝切除的患者的新鲜肝组织(对照组),分离组织浸润淋巴细胞,用抗CD3、CD4和CD8单克隆抗体同时荧光染色,用流式细胞仪检测各表面标志的表达情况。结果:1)乙肝肝癌组、乙肝癌旁组及对照组CD3+CD4+T细胞占浸润淋巴细胞的比例分别为(22.31±3.68)%、(10.69±2.47)%及(4.21±4.26)%。乙肝肝癌组显著高于乙肝癌旁组及对照组,差异有统计学意义(P=0.000,P=0.001),乙肝癌旁组高于对照组,差异亦有统计学意义(P=0.019)。2)乙肝肝癌组、乙肝癌旁组及对照组CD3+CD8+T细胞占浸润淋巴细胞的比例分别为(26.10±5.82)%、(21.82±2.70)%及(41.31±14.01)%,乙肝肝癌组及乙肝癌旁组显著低于对照组,差异有统计学意义(P=0.014,P=0.004),而乙肝肝癌组与乙肝癌旁组之间差异无统计学意义(P=0.651)。3)乙肝肝癌组组织浸润淋巴细胞中CD3+CD4+T细胞与CD3+CD8+T细胞的比值为0.91±0.30,显著高于乙肝癌旁组(0.47±0.11,P=0.003)及对照组(0.11±0.13,P=0.000),CD3+CD4+与CD3+CD8+T细胞的比值出现失衡。结论:原发性肝癌患者肿瘤浸润淋巴细胞中T细胞亚群失调,表现为CD3+CD4+T细胞所占比例升高,CD3+CD8+T细胞所占的比例下降,CD3+CD4+/CD3+CD8+明显升高。     

原发性肝癌患者微环境中CD8+CD28-调节性T细胞表达变化及意义

目的：探讨原发性肝癌患者微环境中CD8+CD28-调节性T细胞(CD8+CD28-Treg)的表达变化及其意义。 方法：采用流式细胞检测技术对60例原发性肝癌患者及其50例健康患者的外周血中CD8+CD28-Treg进行检测,对于顺利手术切除的46例患者的肝癌组织及癌旁组织进行CD8+CD28-Treg的检测,再选取30例肝癌石蜡标本,检测其肝癌组织和癌旁组织中的CD8+CD28-Treg的表达。 结果：通过对肝癌组与健康对照组检测结果比较发现：CD8+CD28-Treg在原发性肝癌患者的外周血中测定值明显高于健康对照组[（15.55±3.14）% vs.（10.12±3.16）%],肝癌组织的CD8+CD28-Treg表达较癌周组织中的CD8+CD28-Treg表达增高[（19.34±3.12）% vs.（13.59±2.47）%],石蜡标本中肝癌组织CD8+CD28-Treg表达高于癌旁组织中CD8+CD28-Treg的表达[（13.58±2.46）% vs. （9.47±3.04）%],差异均有统计学意义（均P<0.05）。 结论：CD8+CD28-调节性T细胞在原发性肝癌患者的微环境中表达明显增加,其可能在机体对原发性肝癌的免疫应答中有重要的意义。     

原发性肝癌患者体内的CD4+CD25+ T细胞变化及其意义

目的:探讨原发性肝细胞癌(HCC)患者体内CD4+CD25+T细胞变化的及其意义。 方法:实验分HCC组（n=20）与正常人对照组(n=10)，采用免疫荧光标记法，流式细胞仪分离出外周血CD4+CD25+T细胞，分析两组CD4+CD25+T细胞所占T细胞的比例。计算并比较癌组织与癌旁组织中CD4+CD25+T细胞所占T细胞的比例。结果:HCC组机体的CD4+CD25+T 细胞比例为(19.3±3.0) %，明显高于对照组(5.2±1.6) % (P<0.05)。癌组织中CD4+CD25+T 细胞比例为(6.5±2.9) %，癌旁组织中CD4+CD25+T 细胞比例为(5.8 ±2.1) %， (P>0.05)。结论:原发性肝细胞癌患者机体免疫功能降低的原因之一可能是外周血CD4+CD25+T 细胞数明显增加。     

人肝癌浸润淋巴细胞的分离培养及其CD8阳性亚群特性研究

目的从原发性肝癌（HCC）患者术后的肿瘤组织中提取、培养肿瘤浸润性淋巴细胞（TILs），进一步分选、扩增其CD8阳性亚群（CD8+TILs），并研究其特性。 方法标本取自38例肝癌根治术后的肿瘤组织，免疫组织化学染色观察癌巢组织和癌旁组织TILs的分布，通过机械剪碎、混合酶消化和不连续密度梯度离心法分离提取TIL前体细胞。采用重组人白介素2进行激活，抗CD3、抗CD28抗体进行共刺激扩增，用磁珠分选出CD8+TILs，CCK8法观察CD8+TILs对肝癌细胞Hep3B、HepG2生长的影响。 结果TILs富集于癌旁组织，12例成功提取TILs，6例培养并实现大量扩增，扩增17~50倍后细胞数为（1.2~2.5）×10⁸个，CD3+细胞比例为（77.53±16.37）%，CD3+CD4+比例为（27.08±21.56）%，CD3+CD8+比例（44.55±12.73）%，细胞活力为（90.5±3.0）%，CD8+TILs对肿瘤细胞系Hep3B、HepG2有较强的生长抑制作用。 结论本研究成功建立高效提取、培养CD8+TILs的方法，获得具有高抗肿瘤活性的CD8+TILs，为晚期肝癌等实体瘤的过继免疫治疗提供研究基础。     

膀胱尿路上皮癌组织中Snail蛋白表达与E-cadherin蛋白、T细胞亚群的相关性分析

目的 研究膀胱尿路上皮癌组织中Snail蛋白表达与E-cadherin蛋白、T细胞亚群的相关性。 方法 采用免疫组化SP法检测156例膀胱尿路上皮癌组织和80例癌旁组织中Snail蛋白、E-cadherin蛋白的表达情况,比较二者在不同病理组织中的阳性表达率,并分析二者表达的相关性;分析膀胱尿路上皮癌组织中Snail蛋白阳性表达和CD4+、CD8+细胞数量及CD4+/CD8+的相关性。结果膀胱尿路上皮癌组织Snail蛋白阳性表达率65.4％(102/156)显著高于癌旁组织的48.8％(39/80),差异有统计学意义(P＜0.05);其表达与膀胱尿路上皮癌的临床分期、病理分级、肿瘤数量、远处转移及复发有关（P值均＜0.05）。膀胱尿路上皮癌中Snail蛋白与E-cadherin蛋白表达呈负相关（r= -0.186,P＜0.05）;Snail蛋白阳性表达与CD4+细胞数量以及CD4+/CDt+值呈负相关(r=-0.313,P＜0.05;r=-0.305,P＜0.05),而与CDs+细胞数量无关（r= -0.250,P＞0.05）。 结论 Snail蛋白可能通过抑制E-cadherin蛋白表达及诱导膀胱肿瘤局部免疫抑制,促进膀胱尿路上皮癌的浸润、转移。     

CD4+CD25+FOXP3+T细胞在肝癌肝移植肿瘤复发中的作用

目的 探讨肝癌肝移植病人移植前后外周血和肿瘤组织中CD4+CD25+FOXP3+T细胞比例变化及其临床意义.方法 用流式细胞仪检测肝癌肝移植病人和其他肝移植病人术后外周血中CD4+CD25+FOXP3+T细胞的比例,并采用正常人作对照.用免疫组化法检测肝癌病人和非肝癌病人肿瘤组织中FoxP3的表达及CD8+T细胞浸润的比例.观察肝癌肝移植病人术后及肿瘤复发后调节性T细胞的变化及其对肿瘤复发的影响.结果 流式细胞检测显示肝癌肝移植、非肝癌肝移植的病人术后外周血中CD4+CD25+FOXP3+T细胞占CD4+T细胞的比例较正常人明显升高,分别为(10.15±1.00)%、(5.30±1.64)%和(3.20±1.18)%,P<0.05.肝癌肝移植肿瘤复发病人较未复发病人外周血CD4+CD25+FOXP3+T比例明显升高,分别为(15.15±1.50)%和(6.80±1.50)%,P<0.01.免疫组化检测显示肿瘤组织中FOXP3+T细胞增多,CD8+T细胞浸润明显减少.结论 肝癌肝移植肿瘤复发的病人外周血中CD4+CD25+FOXP3+T细胞比例升高.调节性T细胞可能通过减少CD8+T细胞浸润,加速肿瘤复发.     

Strong depletion of CD14(+)CD16(+) monocytes during haemodialysis treatment.

BACKGROUND: The immune defect in haemodialysis (HD) patients is associated with a monocytic dysfunction, including an increased production of proinflammatory cytokines. Monocytes fall into subpopulations comprising CD14(++)CD16(-) and CD14(+)CD16(+) cells. Circulating numbers of the latter can rapidly increase during infectious episodes and inflammation. METHODS: We determined the amount of CD14(+)CD16(+) monocytes in HD patients and characterized their fate during HD treatment. In 34 HD patients and 17 healthy controls, the distinct cell populations were determined by differential blood counts and flow cytometry. Cells from 14 HD patients were analysed at the start, 10, 30 and 120 min thereafter, and at the end of HD treatment. RESULTS: Before HD, patients show a monocytosis with a strongly increased CD14(+)CD16(+) subpopulation. Early during HD treatment, circulating leukocyte numbers decrease, with monocytes being most profoundly influenced. Interestingly, among them, sequestration is most pronounced in the CD14(+) CD16(+) subpopulation. After 30 min, approximately 83+/-9% of CD14(+)CD16(+) cells are removed from circulation. This sequestration does not differ between patients treated with polyamide or haemophan membranes. The sequestration is a short-lived temporary effect and cell numbers are replenished within 120 min of treatment for the entire monocyte population. Beyond that time point, cellular activation by the dialyser membrane becomes visible. Reappearence kinetics of CD14(+)CD16(+) monocytes is slower; however, initial numbers are reached by the end of treatment. CONCLUSION: Haemodiaysis leads to temporary removal of monocytes from the bloodstream followed by the reappearance of activated cells. This might contribute to the state of chronic microinflammation, which is reflected by high levels of CD14(+)CD16(+) monocytes.     

Inhibition by uraemic sera of CD4+ T-cell adhesion to extracellular matrix components

BACKGROUND.: T-cell-mediated immune responses are impaired in patients withchronic renal failure. The migration, proliferation, differentiation,biological functioning, and interaction with other T cells aremediated by cell surface adhesion proteins, which include integrins. METHODS.: To elucidate how uraemia can impair T-cell-mediated responsesin vivo, the effects of sera from uraemic patients on T-cellproliferation and adhesion to extracellular matrix (ECM) componentswere examined. RESULTS.: Preincubation of human CD4⁺ T cells with sera from undialysedand dialysed (haemodialysis or peritoneal dialysis) uraemicpatients inhibited the capacity of the cells to be stimulatedby phytohaemmagglutinin and by anti-CD3 monoclonal antibodyplus immobilized fibronectin (FN). Sera from uraemic and dialysedpatients, but not from healthy individuals, inhibited significantly,and in a dose-dependent fashion, human CD4⁺ T cell adhesionto immobilized FN and laminin (LN). The degree of inhibitionof adhesion was similar whether the sera were continuously present,even during the adhesion assay, or removed by washing. The adhesioninhibiting capacity of the uraemic sera was not due to modificationof the expression of ß1 integrins on the surfacesof the T cells. CONCLUSIONS.: These results suggest that uraemia can impair the proliferativecapacity and adhesion of immune cells, and thus may affect normalimmune processes and contribute to the overall immune deficiencyobserved in patients with renal failure.     

Effects of isoflurane and xenon on Ba2+-currents mediated by N-type calcium channels

Background. Isoflurane and xenon are inhalation general anaestheticswith differing clinical profiles and contrasting synaptic actions.Both agents have been shown to depress excitatory synaptic responses.Whether this is via pre-synaptic or post-synaptic mechanismshas not been determined clearly. N-type calcium channels area putative pre-synaptic target for these agents. We tested whetherN-type calcium channels were sensitive to isoflurane and xenonand whether there was any stereoselectivity in the effect ofisoflurane. Methods. We used patch-clamp electrophysiology on isolated HEK293cells stably expressing N-type calcium channels to investigatethe effects of isoflurane and xenon on barium currents mediatedby N-type calcium channels. Results. Racemic isoflurane caused a concentration-dependentreduction (11–35%) in the peak current through the N-typechannels in the concentration range 0.15–1.22 mM. In theclinically relevant concentration range the inhibition was small.At an isoflurane concentration of 0.31 mM (equivalent to 1 MAC),the peak N-type current was inhibited by 14 (1)%. The opticalisomers of isoflurane were found to be equally potent at inhibitingcurrents through N-type channels. The inert gas anaestheticxenon was found to have no measureable effect on N-type channelsat a concentration of 3.4 mM (1 MAC). Conclusions. These results suggest that N-type calcium channelsare not the targets mediating general anaesthesia with thesetwo inhalation agents. Declaration of interest. Professor Franks is a board memberof an Imperial College spin-out company, Protexeon Ltd, thatis interested in developing clinical applications for medicalgases, including xenon. Professor Franks is a paid consultantin this activity. In addition Air Products have funded workin the authors' laboratories that bears on the actions of xenonas an anaesthetic and neuroprotectant. Air Products has a financialstake in Protexeon Ltd.     

Distinct Ca2+ channels mediate transmitter release at excitatory synapses displaying different dynamic properties in rat neocortex

To study the type of presynaptic calcium channels controlling transmitter release at synaptic connections displaying depression or facilitation, dual whole cell recordings combined with biocytin labelling were performed in acute slices from motor cortex of 17- to 22-day-old rats. Layer V postsynaptic interneurons displayed either fast spiking (FS) (n = 12) or burst firing (BF) (n = 12) behaviour. The axons of FS cells ramified preferentially around pyramidal cell somata, while BF cell axons ramified predominately around pyramidal cell dendrites. Synapses between pyramidal cells and FS cells displayed brief train depression (n = 12). Bath application of omega-Agatoxin IVA (0.5 microM), blocking P/Q-type calcium channels, decreased mean peak amplitudes of the EPSPs to 40% of control EPSPs (n = 8). Failure rate of the EPSPs after the first presynaptic action potential increased from 9 +/- 11 to 28 +/- 15%. This was associated with an increase in paired pulse ratio of 152 +/- 44%. Omega-conotoxin GVIA (1-10 microM), selectively blocking N-type calcium channels, had no effect on peak amplitudes or frequency dependent properties of these connections (n = 5). Synapses from pyramidal cells to BF cells displayed brief train facilitation (n = 8). Application of omega-Conotoxin in these connections decreased peak amplitudes of the EPSPs to 15% of control EPSPs (n = 6) and decreased the paired pulse ratio by 41 +/- 30%. Omega-agatoxin did not have any significant effect on the EPSPs elicited in BF cells. This study indicates that P/Q-type calcium channels are associated with transmitter release at connections displaying synaptic depression, whereas N-type channels are predominantly associated with connections displaying facilitation.     

Mg2+ dependence of Ca2+ release from the sarcoplasmic reticulum induced by sevoflurane or halothane in skeletal muscle from humans susceptible to malignant hyperthermia

Background. In normal resting muscle, cytosolic Mg²⁺ exertsa potent inhibitory influence on the sarcoplasmic reticulum(SR) Ca²⁺ release channel (ryanodine receptor, RyR1). ImpairedMg²⁺-regulation of RyR1 has been proposed as a causal factorin malignant hyperthermia (MH). The aim of this study was tocompare the effects of cytosolic Mg²⁺ on SR Ca²⁺ release inducedby halothane or sevoflurane in normal (MHN) and MH susceptible(MHS) human skeletal muscle fibres. Methods. Samples of vastus medialis muscle were obtained frompatients under investigation for MH susceptibility. Single fibreswere mechanically skinned and perfused with solutions mimickingthe intracellular milieu. Changes in [Ca²⁺]_i were detected usingfura-2 fluorescence after application of equimolar halothaneor sevoflurane. Results. In MHN fibres, concentrations of sevoflurane or halothaneas high as 10 mM typically failed to induce SR Ca²⁺ releaseat physiological free [Mg²⁺] (1 mM). However, when [Mg²⁺] wasdecreased to 0.4 mM, SR Ca²⁺ release occurred in 51% (16/33)and 6% (2/33) of MHN fibres after the addition of 1 mM halothaneor 1 mM sevoflurane, respectively. Further decreases in [Mg²⁺]increased the proportion of responsive fibres. In the presenceof 0.1 mM [Mg²⁺], Ca²⁺ release occurred in all fibres (33/33)after the introduction of 1 mM halothane or 1 mM sevoflurane.In MHS fibres, 1 mM halothane or 1 mM sevoflurane-induced Ca²⁺release in 54% (7/13) or 15% (2/13) of fibres, respectively,at 1 mM Mg²⁺. A decrease in [Mg²⁺] to 0.2 mM Mg²⁺ was sufficientto render 100% of MHS fibres (13/13) responsive to 1 mM halothaneor 1 mM sevoflurane. Conclusions. In both MHS and MHN fibres (i) halothane is a morepotent activator of SR Ca²⁺ release than sevoflurane and (ii)as with halothane, the efficacy of sevoflurane-induced SR Ca²⁺release exhibits a marked dependence on cytosolic [Mg²⁺]. Themarked potentiation of SR Ca²⁺ release after a moderate reductionin cytosolic [Mg²⁺] suggests that conditions which cause hypomagnesaemiawill increase the probability and possibly severity of an MHevent. Conversely, maintenance of a normal or slightly increasedcytosolic [Mg²⁺] may reduce the probability of MH.     

Increase in deoxyribonuclease activity in uraemic lymphocytes is caused by the cleavage of the largest polymerase I subunit

Deoxyribonucleases and DNA-dependent RNA polymerase activitiesin T and B lymphocytes isolated from patients with chronic renalfailure and control subjects were studied. The data clearlyshows that the nuclease activity in T and B cells isolated fromuraemic patients is remarkably enhanced when compared to thecontrol cells. Concomitant with the enhancement in enzyme activity,the reduction in RNA polymerase I activity and quantity wasobserved. It was found that the increase in nuclease activityand quantity was limited to the group of relatively small nucleaseswith molecular weights ranging from 14kDa to 18kDa. It has beenreported previously that these nucleases are among the cleavageproducts of the largest subunit of DNA-dependent RNA polymeraseI. Thus we suggest that the depressed metabolic activity isa characteristic feature of the uraemic lymphocyte cells andthe observed increased in DNase activity in those cells is aresult of polymerase I degradation.     

Transmembrane signalling in human monocyte/mesangial cell co-cultures: role of cytosolic Ca(2+).

BACKGROUND: Adhesion of monocytes triggers apoptosis, cytotoxicity, cytokine release, and later proliferation of cultured human mesangial cells (HMC). In the search for transmembrane signals transducing the interaction of HMC adhesion molecules with leukocyte counterreceptors, we measured variations of cytosolic Ca(2+) ([Ca(2+)](i)) in HMC and monocytes of the U937 cell line during 6-h co-cultures. METHODS: Monolayer cultures of HMC and suspensions of U937 cells were loaded with the fluoroprobe fura 2-AM and subsequently co-cultured for 6 h while separately monitoring by microfluorometry the Ca(2+)-dependent 500 nm fluorescent emission of each cell line at fixed intervals upon excitation at 340/380 nm. RESULTS: U937 and peripheral blood monocyte adhesion was followed in HMC by a slow, progressive rise of [Ca(2+)](i) from basal levels of 96+/-9 nM to 339+/-54 at 60 min and 439+/-44 nM at 3 h. The [Ca(2+)](i) elevation reached a steady state thereafter, while parallel monolayers incubated with control media maintained resting levels throughout the co-culture with stable fluoroprobe retention. Receptor sensitivity to vasoconstrictor agents, including compounds not released by monocytes, such as angiotensin II, was rapidly downregulated in HMC co-cultured with U937 cells. No [Ca(2+)](i) changes could be elicited by the octapeptide or by the TxA(2) analogue, U-46619, as early as 30 min after exposure to U937 cells. No [Ca(2+)](i) changes were observed in U937 cells throughout the co-culture. Conditioned media from monocytes and from co-cultured HMC+U937 cells had no effect on [Ca(2+)](i) of HMC. Ca(2+) entry leading to fura 2 saturation was still inducible by Ca(2+) ionophores, such as ionomycin and 4-Br-A23187, which also inhibited the responses to vasoconstrictors. Ca(2+)-free solutions prevented the [Ca(2+)](i) rise as well as subsequent receptor inactivation, implicating Ca(2+) influx through store-operated Ca(2+) channels (SOC), a major pathway for Ca(2+) entry in these cultured cells. Ca(2+) influx was confirmed by Mn(2+)-quenching of fura 2. CONCLUSIONS: In HMC, early changes in [Ca(2+)](i) signal for monocyte adhesion in a co-culture model of glomerular inflammation. This signalling mechanism may mediate the functional responses elicited in glomerular cells by leukocytes, including downregulation of receptors for vasoactive agents.     

Renal extraction of cystatin C vs 125I-iothalamate in hypertensive patients.

BACKGROUND: Several markers are available to estimate the glomerular filtration rate (GFR) in patients. Cystatin C is a relatively new marker and has been suggested as an alternative for creatinine. Numerous studies have been performed to evaluate the usefulness of cystatin C to estimate GFR. The aim of this study is to compare the renal extraction of cystatin C with that of 125I-iothalamate in hypertensive patients. METHODS: Forty hypertensive patients with unilateral renal artery stenosis, and who used at least two antihypertensive agents, were studied. For the determination of the renal extraction ratio, blood samples were drawn simultaneously from the renal vein and the abdominal aorta. The renal extraction ratio was calculated as ([A]-[V])/[A], in which A is the plasma concentration of the compound from the abdominal aorta, and V is the plasma concentration of the compound from the renal vein. RESULTS: The mean difference between the renal extraction ratio of cystatin C and that of 125I-iothalamate was 0.002. The 95% confidence interval (CI) for the mean difference was -0.036 to 0.032, which was not statistically significant. However, the limits of agreement were large (-0.271 and 0.267). CONCLUSIONS: Despite a lower reported glomerular sieving coefficient of cystatin C, the mean renal extraction of cystatin C was equal to the mean renal extraction of 125I-iothalamate in hypertensive patients, suggesting tubular secretion of cystatin C. Combined with the large variation in the renal extraction of cystatin C, these findings cast doubts on its usefulness as a glomerular filtration marker.