Nuclear thyroxine binding in human mononuclear blood cells from hyperthyroid and hypothyroid patients before and after treatment

Nuclear binding of [125I]T4 in human mononuclear blood cells was examined in six hyperthyroid and six hypothyroid patients before and after treatment. In hypothyroid patients the nuclear T4 binding was initially increased and subsequently normalized as the patients became euthyroid suggesting a homeostatic counter-regulation. The thyroid state did not affect the nuclear T4 binding in hyperthyroid patients. Treatment with methimazol however increased the nuclear T4 binding, suggesting either a direct effect of methimazol on the hormone-receptor interaction or that the patients had become slightly hypothyroid during the treatment. The TSH was shown not to affect nuclear T4 binding. The thyroid state of the patients did not significantly affect the nuclear accumulation of the T3 produced intracellularly by deiodination of T4.     

Basal cyclic AMP levels in human blood mononuclear cells

Basal cyclic AMP levels in blood mononuclear cells from healthy human donors has been studied. A great variation in cyclic AMP content ranging from below 2 to 20 pmol 10(-6) cells was seen. Incubation of the cells at 37 degrees C mostly showed a time-dependent decrease in basal cyclic AMP, levelling off after 16 h. When treated synchronously, cells from blood from the first sample of blood sampling contained more cyclic AMP than that of later samples. Presence of adenosine deaminase, theophylline or indomethacin influenced cyclic AMP concentrations in a non-consistent manner. Variations in cyclic AMP content may in some individuals be ascribed to early increase of cyclic AMP, in others to a progressive reduction in basal cyclic AMP during blood sampling, cell preparation and incubation.     

Resveratrol modulates apoptosis and oxidation in human blood mononuclear cells

BACKGROUND: We examined the effect of resveratrol (RS), a nonflavonoid polyphenolic phytoalexin found in grapes and red wine, and RS coincubated with the oxidant 2-deoxy-D-ribose (dR), on apoptosis and on the oxidative metabolic status of normal human peripheral blood mononuclear cells (PBMNCs) isolated ex vivo from healthy donors. MATERIAL AND METHODS: Apoptosis was measured by changes of membrane permeability to propidium iodide (PI), plasma membrane exposure of phosphatidylserine (PS) and intracellular caspase activity. Oxidative status was assessed by recording the intracellular glutathione concentration (GSH), the activities of the enzymes y-glutamyltransferase (y-GT) and glutathione-S-transferase (GST), and intracellular lipid peroxidation (MDA). RESULTS: Neither apoptotic nor oxidative parameters were affected by culturing PBMNCs in medium containing RS up to 20 micro M for 5 days, while the frequency of cells with intermediate permeability to PI (17% +/- 5) increased at 50 micro M of RS. Thus resveratrol was slightly toxic, but there was little apoptosis in these cells. Peripheral blood mononuclear cells were also grown first in medium plus RS for 24 h and then for 96 h in medium containing RS plus 10 mM of dR, an oxidant sugar that is apoptogenic for human lymphocytes. The apoptotic changes triggered by dR were counteracted by the phytoalexin in a dose-dependent manner, but RS activity was absent at the lowest concentration (5 micro M) and significantly reduced at the highest concentration used (50 micro M). In PBMNCs coincubated with 20 micro M of RS and 10 mM of dR the antioxidant effect of RS manifested with a significant reduction of caspases-3, -8, y-GT, GST activities and MDA content. CONCLUSIONS: Peripheral blood mononuclear cells acquire antioxidant capacity when treated with RS. Grape resveratrol may make a useful dietary supplement for minimizing oxidative injury in immune-perturbed states and human chronic degenerative diseases.     

Cis-platinum-induced immunosuppression: relationship to melatonin in human peripheral blood mononuclear cells

Objectives: The purpose of this study was to investigate the immunomodulatory effect of melatonin (MLT) on human peripheral blood mononuclear cells (PBMC) and to address its effects on Cis-platinum (CDDP)-induced cytotoxicity.

Methods and results: The obtained data from this study revealed that treatment of cells with MLT (100 μg/ml) for 24 h enhanced cell viability. When cells were exposed to CDDP (5 μg/ml), cell proliferation in response to phytohaemagglutinin (PHA) stimulation was reduced by 49.63% compared to control cells as detected by ³[H]-thymidine uptake. Furthermore, Cis-platinum significantly depleted intracellular glutathione (GSH) levels by 47% below that of untreated cells and led to apoptotic changes in the target cells as evidenced by DNA fragmentation (45% compared to 5% in control cells as measured by diphenylamine assay). DNA fragmentation was also confirmed by agarose gel electrophoresis. However, MLT enhanced cell proliferation by 8.63% above the control values, and counteracted the antiproliferative effect of CDDP. The GSH levels were significantly increased in response to MLT (71% above control values) and it protected the cells against GSH depletion induced by CDDP. Moreover, DNA fragmentation and laddering produced by CDDP were significantly reduced or even disappeared when the cells were pretreated with MLT or the latter was simultaneously added with CDDP.

Conclusions: The findings from this study indicated that melatonin is a potent immunomodulatory hormone that protects PBMC against cis-platinum–induced immunosuppressive effects. These effects might improve the patients’ response to cis-platinum therapy and, therefore, their survival rates.     

Effect of immunosuppressive drugs on DNA repair in human peripheral blood mononuclear cells

Introduction

Cancer is a major cause of mortality among transplant recipients. Immunosuppressive treatment is a modifiable factor contributing to this phenomenon. Cyclosporine in kidney transplant recipients was associated with reduced UV-induced DNA repair by peripheral blood mononuclear cells (PBMC) and increased cancer rate. H₂O₂ is a common cellular reactive oxygen species (ROS), which induces DNA damage followed by DNA repair.

Aim

To investigate the effect of currently used immunosuppressive drugs on DNA repair.

Methods

H₂O₂-induced DNA repair by human PBMC was tested in vitro in the presence of the calcineurin inhibitors (CNI) cyclosporine and tacrolimus, mycophenolic acid (MPA), and the mammalian target of rapamycin (mTOR) inhibitors sirolimus and everolimus, at low to high non-toxic concentrations. The effect of combination therapy at maintenance levels was also tested.

Results

Cyclosporine and tacrolimus suppressed DNA repair throughout the tested dose range. In contrast, MPA, sirolimus and everolimus did so only at the high doses. Maintenance doses of a combination of tacrolimus and MPA, the most frequent treatment regimen, reduced DNA repair, while MPA with sirolimus or everolimus did not.

Conclusion

In an attempt to reduce the risk of post-transplantation malignancy, treatment protocols may be modified by reducing CNI dose.     

Effect of immunosuppressive drugs on spontaneous DNA repair in human peripheral blood mononuclear cells

Introduction

Immunosuppressive treatment increases the risk of post-transplant cancer. Cyclosporine reduced UV-induced DNA repair by peripheral blood mononuclear cells (PBMC) and increased cancer incidence in kidney transplant recipients. Calcineurin inhibitors (CNI), but not mammalian target of rapamycin (mTOR) inhibitors or mycophenolic acid, suppressed H₂O₂-induced DNA repair in human peripheral blood mononuclear cells (PBMC) in vitro at maintenance drug concentrations. DNA repair, when measured in quiescent cells, is named spontaneous DNA repair, and represents a basal ongoing DNA repair in response to endogenous DNA damage. The effect of immunosuppressive drugs on spontaneous DNA repair has not been investigated.

Aim

To investigate the effect of currently used immunosuppressive drugs on spontaneous DNA repair.

Methods

Spontaneous DNA repair by human PBMC was tested in vitro in the presence of the CNI–cyclosporine and tacrolimus; mycophenolic acid (MPA); and the mTOR inhibitors–sirolimus and everolimus, at low to high nontoxic concentrations.

Results

Cyclosporine and tacrolimus suppressed spontaneous DNA repair throughout the tested dose range. In contrast, MPA, sirolimus and everolimus did so only at the high doses.

Conclusion

A reduction in CNI dosage may lead to a decrease in the occurrence of post-transplant malignancy.     

人外周血单个核细胞miR-UL112水平在HCMV感染中的诊断价值研究

目的探讨单个核细胞中人巨细胞病毒(HCMV)微小RNA(miR)-UL112-3p、miR-UL112-5p水平对HCMV潜伏和激发感染的诊断价值。方法选取非肿瘤患者92例[其中HCMV特异性CD8+T淋巴细胞高水平组(简称CD8+细胞高水平组)57例、HCMV特异性CD8+T淋巴细胞低水平组(简称CD8+细胞低水平组)35例]、肿瘤化疗患者30例,提取外周血单个核细胞,采用实时荧光定量-聚合酶链反应(PCR)检测HCMV DNA、miR-UL112-3p及miR-UL112-5p水平。采用受试者工作特征(ROC)曲线评价miR-UL112-3p和miR-UL112-5p水平对HCMV潜伏感染的诊断价值。结果肿瘤化疗组HCMV DNA、miR-UL112-3p及miR-UL112-5p水平明显高于非肿瘤组(P0.05)。非肿瘤组中CD8+细胞高水平组HCMV DNA、miR-UL112-3p及miR-UL112-5p水平明显高于CD8+细胞低水平组(P均0.01);以CD8+细胞低水平组miR-UL112-3p、miR-UL112-5p水平为标准,CD8+细胞高水平组miR-UL112-3p、miR-UL112-5p阳性率分别为59.65%、64.91%。分别以1.09、1.52作为miRUL112-3p和miR-UL112-5p诊断HCMV较高水平潜伏感染状态的Cut-off值,敏感性分别为59.65%、64.91%,特异性分别为97.1%、97.1%。以非肿瘤组miR-UL112-3p、miR-UL112-5p水平的x珋+s(即5.24、6.63)作为诊断HCMV激发感染的标准,肿瘤化疗组miR-UL112-3p、miR-UL112-5p阳性率分别为50.00%、73.33%。结论 miRUL112-3p及miR-UL112-5p对HCMV潜伏和激发感染可能有一定的诊断价值。     

人脐血单个核细胞和脐带间充质干细胞移植用于大鼠脊髓损伤的实验研究

目的:探讨人脐血单个核细胞和脐带间充质干细胞(MSCs)移植对脊髓损伤功能恢复的影响,寻找一种更适合治疗脊髓损伤的细胞源。方法:采集新鲜人脐带血和脐带,分离培养单个核细胞和MSCs。将脊髓损伤模型随机分成3组:单个核细胞移植组、MSCs移植组和低糖必需培养基(L-DMEM)培养组。采用免疫组化和免疫荧光检测细胞移植后1—4周细胞在脊髓内的存活情况和迁移情况,使用BBB行为学评分评估大鼠脊髓功能恢复情况。结果:L-DMEM培养液组在术后各时间点观察评分无明显差异,而细胞移植组脊髓功能处于逐渐恢复过程,与L-DMEM培养液比较,差异有显著性意义。单个核细胞移植组对损伤脊髓功能的修复作用较MSCs移植组显著,且差异有显著性意义。结论:与MSCs相比较,人脐血单个核细胞更适合作为治疗脊髓损伤的细胞源。     

小檗碱抑制人外周血单核细胞COX-2 mRNA及蛋白的表达:c-JUN末端激酶信号转导途径

背景:当细胞受到各种细胞因子及环境刺激时,c-JUN末端激酶信号转导通路可以通过激活不同的受体,对细胞的发育、分化、凋亡、癌变、炎症和免疫反应起调节作用.目的:观察中药小檗碱足否通过c-JUN末端激酶信号转导途径抑制人外周血单核细胞COX-2 mRNA及蛋白表达.方法:取人外周静脉血分离培养单核细胞,分为5组培养:空白对照组、脂多糖组、脂多精+小檗碱25 μmol/L组、脂多糖+小檗碱50 μmol/L组、脂多糖+小檗碱100 μmol/L组.分别在培养后30 min,6 h,12 h,24 h提取细胞,采用RT-PCR测定COX-2 mRNA水平,采用Western blot测定c-JUN末端激酶及COX-2蛋白水平.同时加入选择性c-JUN末端激酶抑制剂,测定COX-2 mRNA及蛋白水平.结果与结论:与空白对照组相比,脂多糖组COX-2 mRNA及蛋白表达明显增强(P<0.01).加入不同浓度小檗碱后COX-2mRNA及蛋白表达明显被抑制(P<0.05),且随着浓度增加,抑制作用更明显,给药后12 h,抑制作用最强.但c-JUN末端激酶活性水平无明显变化(P>0.05),脂多糖+小檗碱100 μmol/L组c-JUN末端激酶活性水平变化明显(P<0.05).加入c-JUN末端激酶抑制剂后,COX-2 mRNA及蛋白水平降低明显(P<0.05).证实小檗碱能抑制人外周血单核细胞COX-2mRNA及蛋白水平,并望浓度依赖性,高浓度小檗碱对c-JUN末端激酶活性蛋白表达有明显抑制作用,其町能通过c-JUN末端激酶信号转导途径抑制人外周血单核细胞COX-2 mRNA及蛋白表达.     

骨髓基质细胞对多细胞因子介导人脐血单个核细胞体外扩增的影响

背景:体外扩增脐血造血细胞的目的是促进脐血造血细胞的植入能力,细胞因子介导的脐血造血细胞能使细胞数有效扩增,但同时也耗竭了具有分化潜能的造血干细胞.目的:观察骨髓基质细胞对多细胞因子组合介导人脐血单个核细胞体外扩增及扩增后黏附分子和CXCR4表达的影响.方法:将分离的人脐血单个核细胞接种在预先建立的人骨髓基质细胞层上,分组培养:对照组仅含有脐血单个核细胞;多种细胞因子+脐血单个核细胞组;骨髓基质细胞+脐血单个核细胞组;骨髓基质细胞+多种细胞因子+脐血单个核细胞组.培养0,7 d检测有核细胞数, CD34+细胞数及CD34+CXCR4+,CD34+CD62L+,CD34+CD49d+的细胞数.结果与结论:单纯骨髓基质细胞和单用细胞因子组均可有效地扩增脐血造血细胞,并提高造血细胞上CD49d,CD62L及 CXCR4表达.而单用细胞因子组促进脐血造血细胞扩增能力较单纯骨髓基质细胞强,提高造血细胞CD49d,CD62L及 CXCR4表达能力较单纯骨髓基质细胞差.提示骨髓基质细胞虽可支持脐血造血细胞扩增,但难以实现造血细胞的大量扩增,但在骨髓基质细胞层的支持下,多细胞因子可有效地促进脐血造血细胞的扩增,并优于单用细胞因子及骨髓基质细胞,证实骨髓基质细胞可协同多细胞因子有效地促进脐血单个核细胞的扩增,促进造血干细胞的黏附和趋化能力.     

Leptin induces tissue factor expression in human peripheral blood mononuclear cells: a possible link between obesity and cardiovascular risk?

BACKGROUND: Obesity is a major modifiable risk factor for cardiovascular disease. Leptin, the hormone synthesized and released primarily by adipose tissue and found increased in obese individuals, has been implicated in the regulation of inflammation and arterial and venous thrombosis. OBJECTIVE: To investigate the role of tissue factor (TF), the pivotal agonist of the clotting cascade, as a link between obesity and cardiovascular disease. METHODS AND RESULTS: In 15 obese patients, plasma levels of leptin and TF as well as TF expression in resting and endotoxin-stimulated mononuclear leukocytes (MN) were increased when compared with healthy donors. In a selected sample of obese patients, loss of body weight led to decreased circulating leptin levels, accompanied by a reduction in plasma TF as well as in TF expression, both in resting and endotoxin-stimulated MN. In subsequent in vitro experiments, leptin was incubated with MN from healthy subjects. Leptin induced TF activity and antigen in a dose-dependent fashion, as assessed by clotting assay and ELISA, respectively. Increased migration of c-Rel/p65 into the nucleus, as determined by EMSA, and development of TF mRNA in monocytes, as assessed by RT-PCR, were observed. Experiments with mitogen-activated protein kinase (MAPK) inhibitors, indicated the involvement of p38 and ERK1/2 pathways. CONCLUSIONS: The presence of TF-expressing MN in blood from obese subjects and the in vitro induction of TF by pharmacologic concentrations of leptin in MN from healthy subjects suggest that TF expression by leptin-stimulated monocytes may contribute to the cardiovascular risk associated with obesity.     

The Trypanosoma cruzi trans-sialidase, through its COOH-terminal tandem repeat, upregulates interleukin 6 secretion in normal human intestinal microvascular endothelial cells and peripheral blood mononuclear cells

The Trypanosoma cruzi trans-sialidase can sensitize mice to become highly susceptible to T. cruzi invasion, through mechanisms that remain unknown. In pursuing this observation, we found that purified trans-sialidase induces the selective release of biologically active interleukin (IL)-6 in naive human intestinal microvascular endothelial cells (HIMECs), peripheral blood mononuclear cells (PBMCs), and bladder carcinoma cells. The trans-sialidase action was independent of its catalytic activity, as demonstrated with a genetically engineered trans-sialidase mutant, an enzymatically active polypeptide, and cocultures of PBMCs with epimastigotes and trypomastigotes. Instead, the trans-sialidase action was reproduced with a recombinant COOH-terminal tandem repeat and with synthetic peptides modeled on the tandem repeat. Most interesting, HIMECs infected with a trypomastigote population expressing trans-sialidase effectively released IL-6, but did not upon infection with the counterpart trypomastigote population expressing low trans-sialidase levels. IL-6 is a key factor in the regulation and symptom formation of infection caused by several types of viruses, such as HIV and influenza A virus. However, the function of IL-6 in protozoan and other parasitic diseases remains unclear. The unique findings presented here suggest that trans-sialidase is a major inducer of IL-6 secretion in T. cruzi infection, independently of immune cell activation. Such IL-6 secretion might underlie some features of Chagas's disease, such as pyrexia, neuroprotection, and fibrosis, and might result in the undermining of normal acquired immunity against T. cruzi.     

The effect of an oral ginger supplementation on NF-κB concentration in peripheral blood mononuclear cells and anthropomorphic data of patients with type 2 diabetes: A randomized double-blind,placebo-controlled clinical trial

IntroductionThe complications of diabetes are extensive which can be caused by excessive oxidative stress, inflammation and impaired insulin system. Plant-sourced bioactive compounds can reduce inflammation and oxidative stress. The aim of present study was to determine the effect of ginger supplementation on diabetic complications.MethodsThe present study is a randomized double blind clinical trial which is conducted with 48 diabetic patients. The participants were randomly divided into two intervention and placebo groups which were received 2 g ginger powder and 2 g wheat flour respectively for 10 weeks. Nuclear factor kappa B (NF-κB) concentration and anthropometric measurements were evaluated at the baseline and at the end of study.ResultsThe effect of ginger supplementation on hip circumference was marginal and there was no significant effect on BMI and waist circumference. Mean NF-κB p65 concentrations were reduced in ginger supplementation group, however, the amount was not statistically significant.ConclusionGinger supplementation had significant effects on anthropometric evaluations. Ginger supplementation decreased mean NF-κB concentration in comparison with placebo, however the significance level was marginal. In order to achieve reliable information, more researches should be complemented with uptake of other diagnostic tools.     

不同时间经静脉移植人脐血单个核细胞对心肌梗死大鼠心功能及结构重构的影响

目的观察不同时间经静脉移植人脐血单个核细胞(HUCBC)对心肌梗死(MI)大鼠心功能及结构重构的影响。方法入选雄性Wistar大鼠共80只,结扎左前降支,制备心肌梗死模型,于梗塞后1d、5d、10d以及30d经尾静脉注入0.5ml人脐血[含单个核细胞(1～3)×107]或0.5ml磷酸盐缓冲溶液(PBS)。不使用免疫抑制剂。术后4周行超声心动图和血流动力学检查,随后处死大鼠,取出梗死心肌,以甲醛固定,进行苏木素伊红(HE)染色以及转移酶介导的三磷酸脱氧鸟苷-生物素刻痕末端标记(TUNEL)计算凋亡细胞。结果 PBS组大鼠左室明显扩大,梗死部位变薄。移植后瘢痕面积减少,室壁较对照组增厚。5d和10d移植瘢痕面积明显减小。与对照组相比,5d和10d移植大鼠左室射血分数(EF),左室压力最大变化率(±dp/dtmax)明显升高,左室收缩末内径(LVESD)明显减小,左室后壁(LVPW)增厚率更高。而梗死后10d移植EF,±dp/dtmax,以及LVPW增厚率的改善最明显。移植大鼠心肌和对照组相比凋亡细胞没有明显差异。结论大鼠心肌梗死后5d和10d经静脉移植HUCBC可以明显抑制心室重构,改善心功能。在梗死后第10天移植对梗死心肌的结构重塑改善最明显,但对细胞凋亡没有明显作用。     

Quinoline compounds decrease in vitro spontaneous proliferation of peripheral blood mononuclear cells (PBMC) from human T-cell lymphotropic virus (HTLV) type-1-infected patients.

In vitro spontaneous proliferation is the immunological hallmark of peripheral blood mononuclear cells (PBMC) from HTLV-1-infected individuals. Quinoline compounds down regulate in vitro cell proliferation of HTLV-1 transformed cell lines. In the present study we assessed the capacity of quinolines to inhibit spontaneous cell proliferation of PBMC from HTLV-1-infected individuals. Twenty-two quinolines were evaluated. Toxicity was first assessed on PBMC from healthy donors by using both the Trypan blue technique and Tetrazolium Salt (XTT) method and then the antiproliferative effect was measured by a classic lymphoproliferative assay on PBMC from three HTLV-1-infected individuals, in the presence of decreasing concentrations of quinolines (from 100muM to 0.8muM), after 5 days of culture. We found that 14 out of 22 compounds were non-toxic to PBMC from uninfected individuals at 100, 50 and 10muM. Four compounds presented a capacity to inhibit more than 80% of the spontaneous proliferation: 7 at 25muM and 10, 20 and 23 at 100muM. Our results indicate that some quinolines block spontaneous proliferation of PBMC from HTLV-1-infected individuals.