Disturbed LDL and scavenger receptor functions in monocytes from chronic haemodialysed patients.

BACKGROUND: The most frequent complication in patients with end-stage renal failure on chronic haemodialysis (HD) treatment is atherosclerosis, i.e. the different forms of heart and vascular diseases. The complete disorder of serum lipid and lipoprotein patterns is well demonstrated, whereas our knowledge about the low-density lipoprotein (LDL) and scavenger receptor expression and function are poorly understood. METHODS: In our current work, LDL and scavenger receptor expression and functions were simultaneously studied in monocytes obtained from 15 healthy male control subjects and from 11 chronic HD male patients applied with (125)I-labelled LDL, isolated from healthy volunteers. To study the scavenger LDL receptors, labelled acetylated LDL (acLDL) was used. RESULTS: LDL binding to the monocytes of the HD-group was found to be decreased in comparison to that of the controls. As a result, the 50 microg LDL protein-induced inhibition of endogenous cholesterol synthesis was also diminished. In contrast, acLDL binding was greatly increased, though it could trigger only a low apoE synthesis. Consequently the number of cholesterol inclusions in monocytes was increased. CONCLUSIONS: The disturbed expression and function of LDL and scavenger receptors both may play significant roles in pathogenesis of cardiovascular complications in chronic HD patients. Based on our present results, it can be assumed that dysfunction of scavenger receptors is at the centre of cardiovascular complications of HD patients with renal failure.     

Comparison of blood biocompatibility during haemodialysis with cuprophane and polyacrylonitrile membranes

Clotting within the dialyser is one of the most significant clinical parameters of biocompatibility. A study was designed to evaluate the biocompatibility of two different dialysis membranes (cuprophane and polyacrylonitrile) during therapy with conventional heparin. Transient leukopenia during cuprophane but not during polyacrylonitrile haemodialysis was observed, and elastase release using polyacryonitrile membranes was reduced (P less than 0.001). An elevation in F VIII:C activity during cuprophane haemodialysis has to be taken as an indication of endothelial disturbances. There was a significant (P less than 0.001) platelet activation (beta-thromboglobulin) and combined thrombin/plasmin generation using cuprophane membranes. This new synthetic polyacrylonitrile membrane inactivates the clotting in an extracorporeal system to a sufficient degree and allows a reduction in dosages of heparin. Platelet activation, platelet turnover, disturbances of endothelium, fibrinolysis activation, and granulocyte activation are reproducible parameters of a described interaction model. They also permit a comparison of different haemodialysis membranes.     

右美托咪啶对脂多糖诱导大鼠外周血单核细胞Toll样受体4mRNA表达的影响

目的 探讨右美托咪啶对脂多糖(LPS)诱导大鼠外周血单核细胞Toll样受体4(TLR4)mRNA表达的影响.方法 健康雄性Wistar大鼠40只,取外周血分离培养单核细胞,采用随机数字表法,将其随机分为5组(n=8),A组:阴性对照;B组:单核细胞中加入LPS(终浓度为1μg/ml);C组:单核细胞中加入LPS(终浓度为1μg/ml)+右美托咪啶(终浓度为0.5 ng/ml);D组:单核细胞中加入LPS(终浓度为1μg/ml)+右美托咪啶(终浓度为5.0 ng/ml);E组:单核细胞中加入LPS(终浓度为1μg/ml)+右美托咪啶(终浓度为50.0 ng/ml).孵育24 h后,收集上清液,采用ELISA法测定TNF-α、IL-1β和IL-6的浓度,采用RT-PCR法测定TLR4 mRNA的表达.结果 与A组比较,B组TNF-α、IL-1p、IL-6的浓度升高,TLR4 mRNA表达上调(P＜0.01);与B组比较,C组、D组和E组TNF-α、IL-1β、IL-6的浓度降低,TLR4 mRNA表达下调(P＜0.05或0.01);与C组比较,D组和E组TNF-α、IL-1β、IL-6的浓度降低(P＜0.01),TLR4 mRNA表达差异无统计学意义(P＞0.05);D组和E组各指标比较差异无统计学意义(P＞0.05).结论 右美托咪啶可通过下调TLR4 mRNA表达,抑制TLR4的合成,从而抑制LPS诱导大鼠外周血单核细胞TNF-α、IL-1β和IL-6的生成与释放.

Abstract：

Objective To investigate the effects of different concentrations of dexmedetomidine on the expression of Toll-like receptor 4 (TLR4) mRNA in rat peripheral blood monocytes exposed to lipopolysaccharide ( LPS ). Methods Peripheral blood monocytes isolated from male Wistar rats were seeded in 24-well plate in RPMI 1640 liquid culture medium in CO2 incubator at 37 ℃ and 5% CO2 for 2 h, and were randomly divided into 5 groups ( n = 8 each): group A negative control; group B was exposed to LPS 1 μg/ml and C, D and E groups were exposed to LPS 1 μg/ml + dexmetomidine 0.5, 5.0 and 50.0 ng/ml respectively. The monocytes were then incubated for 24 h. The concentrations of TNF-α, IL-1β and IL-6 in the supernatant of the cultured monocytes were detected by ELISA. The expression of TLR4 mRNA in the monocytes was detected by RT-PCR.Results Exposure to LPS significantly increased the expression of TLR4 mRNA and the concentrations of TNF-α, IL-1β and IL -6 in group B as compared with group A ( P ＜ 0.01 ). Dexmedetomidine attenuated the LPS-induced increase in the expression of TLR 4 mRNA and the concentrations of TNF-α, IL-1β and IL-6 in a dose-dependent manner ( P ＜0.05or 0.01 ). Conclusion Dexmedetomidine can inhibit the synthesis of TLR4 and inhibit the secretion and dilivery of TNF-α, IL-1β and IL-6 by down-regulating the gene expression of TLR4 in rat peripheral blood monocytes exposed to LPS.     

HLA-DR antigen expression on peripheral blood monocytes correlates with surgical infection

Monocyte human leukocyte antigen-DR (HLA-DR) expression has correlated closely with clinical outcome in severely injured patients at high risk for infection. Monocytes from 77 asymptomatic volunteers expressed HLA-DR antigen with minimal variability in respect to age, gender, race, time of day or year, or serum alcohol level. Patients who developed infection after elective laparotomy had a significantly lower mean percentage of monocytes expressing HLA-DR antigen and a lower mean fluorescent intensity than uninfected patients (p less than 0.05). Severely infected nonsurgical patients had significantly lower values than normal volunteers (p less than 0.01), and the mean fluorescent intensity of those who died from infection was significantly lower than that of those who survived (p less than 0.05). Patients on immunosuppressive regimens after renal transplantation had levels of HLA-DR expression similar to those of the volunteers. Monocyte HLA-DR expression was found to be a reliable marker of clinical infection and showed remarkable reproducibility within the normal uninfected study population.     

Cytokine secretion by peripheral blood monocytes from patients with acute poststreptococcal glomerulonephritis

We have measured the release of interleukin-1β (IL-1) and tumour necrosis factor-α (TNF) by unstimulated monocytes and monocytes stimulated with lipopolysaccharide (LPS) isolated from the peripheral blood of two patients with acute poststreptococcal glomerulonephritis (AGN) and 16 healthy controls. We have demonstrated that spontaneous and LPS-induced cytokine release correlated with disease activity in the AGN patients. We speculate thatin vivo streptococcal infection itself may alter peripheral blood monocyte cytokine secretion.     

Tumour necrosis factor does not increase during routine cuprophane haemodialysis in healthy well-nourished patients

Serum tumour necrosis factor (TNF) was measured by both bioassay and immunoassay (ELISA) during routine cuprophane acetate haemodialysis in 17 asymptomatic patients. In 14 (82%) there was no change in the serum values during haemodialysis. TNF was found to increase, using both assays, in three patients, the responders. These patients differed from the others in terms of body mass index, mean index 16.8 kg/m2 (range 14.8-18.2), compared to the non-responders, mean 24.1 kg/m2 (range 19.6-33.1), P less than 0.05, and had an increased serum calcium, mean 2.9 mmol/l (range 2.6-3.2) compared to the non-responders, mean 2.4 mmol/l (range 1.7-2.8), P less than 0.05. Two of the TNF responders subsequently died of cachexia and respiratory infection. The third underwent a successful parathyroidectomy, and when retested after an increase in body-weight with a normal serum calcium concentration no longer showed an increase in TNF during haemodialysis.     

Expression profile of Toll-like receptors in monocytes of peripheral blood from osteoarthritic patients undergoing arthroplasty

To investigate the difference in the expression of TLRs in monocytes from patients with/without implants. Blood samples from 21 osteoarthritic patients undergoing joint replacement (11 cases) or arthroscopy (10 cases) were tested. Blood samples were obtained within 24 h before surgery and the third postoperative day to measure cell-associated TLRs by FCM. The results showed that in the absence of clinical infection, implantation of joint prosthesis seems to be associated with a significant decrease in circulating TLR2- and TLR7-positive cells, whereas the decrease in TLR4- and TLR9-positive cells was not statistically significant. A small intervention in the form of knee arthroscopy was without effect. TLRs seem to be affected by the implantation of joint prosthesis, perhaps by migration to and entrapment in peri-implant tissues. Due to their role in innate and adaptive immunity, TLRs may have consequences for the postoperative inflammation and infection.     

Negative effect of uraemia and cuprophane haemodialysis on natural killer cells

The evidence indicating the important role of natural killer (NK) cells in immune surveillance against tumours and certain infections is accumulating. Uraemic and dialysed patients are known to be at greater risk of infections and malignant diseases. NK cells were analysed in patients with advanced uraemia, and in patients treated with different dialysis techniques. Number of NK cells was morphologically identified as large granular lymphocytes in blood smears. NK activity was determined as mononuclear cell cytotoxicity against K562 cells. In a group of uraemic patients, large granular lymphocyte number was reduced to 39%, and NK activity to 41%-52% of control values. Large granular lymphocyte number and NK activity in patients haemodialysed on cuprophane membranes was significantly reduced, compared to corresponding values in controls and uraemic patients, declining to 17% and 8%-16% of respective control values. In a group of patients treated by CAPD, and in a group haemodialysed on polyacrylonitrile membranes, NK activity was close to values in the uraemic group, but significantly greater than those of cuprophane-haemodialysed patients. Haemodialysis on cuprophane membranes has an additional negative effect on NK cells, which are already seriously depressed by the uraemic state.     

米非司酮对药物流产患者外周血中差异基因表达研究

目的研究米非司酮对药物流产患者外周血中Th1/Th2型细胞因子干扰素-γ(IFN-γ)、IL-2、IL-5、IL-6、IL-10以及细胞因子EDN1、EDNRA、EDNRB、NOS1、NOS2、NOS3、VEGF在药物流产过程中的表达变化。方法收集2014年4~7月就诊于天津医科大学总医院妇产科月经延迟7d以内要求药物流产的孕妇,随机分为两组。A组(5例):第1天口服米非司酮100mg,于2d后口服600μg米索前列醇;B组(6例):第1天口服米非司酮200mg,于2d后口服200μg米索前列醇。两组均分别在服米非司酮前和服米非司酮48h后收集受试者的外周血,采用qRT-PCR方法检测各基因在全血中的mRNA表达水平。结果与同组服用米非司酮前相比较,A组服用米非司酮后EDN1水平显著上调(P0.05),上调至服药前的(2.19±1.20)倍,而B组服用米非司酮后EDN1水平显著下调(P0.05),下调至服药前的(0.49±0.15);A组服用米非司酮后VEGF水平显著上调至服药前的(1.22±0.43)倍(P0.05),而B组服用米非司酮后VEGF水平显著下调(P0.05),下调至服药前的(0.72±0.14);A组和B组中,服用米非司酮前后IFN-γ水平差异无统计学意义(P0.05)。结论在药物流产过程中不同剂量的米非司酮可能存在不同的作用机制;EDN1基因和VEGF基因可能在米非司酮药物流产过程中起着重要的作用。     

人急性胰腺炎早期外周血单核细胞Toll样受体4及CD14表达变化

目的探讨人急性胰腺炎早期外周血单核细胞（peripheral blood monocytes，PBMCs）表面Tbll样受体4（toll like receptor4，TLR4）、CD14表达变化。方法收集发病24h内入院的早期急性胰腺炎病人36例，采集入院当日及第3、第7日外周血，分离单核细胞。用流式细胞术检测单核细胞表面TLR4、CD14表达变化情况。同时检测血清肿瘤坏死因子（tumor necrosis factor alpha，TNF-α）、白细胞介素6（interleukin6，IL-6），研究它们之间的相关性。结果急性胰腺炎病人外周血单核细胞表面TLR4发病后表达上调，在轻症病人渐下降，1周左右恢复正常。TNF-α的变化一致。结论人急性胰腺炎早期可能通过先天性免疫门户蛋白TLR4激活单核巨噬细胞系统导致TNF-α等促炎细胞因子的产生和释放。     

IL-10 synthesis and secretion by peripheral blood mononuclear cells in haemodialysis patients

Purpose of study: IL-10 may explain the paradox between immunodeficiency and oversecretion of cytokines in chronic haemodialysis (HD) patients. We analysed the secretion of IL-10 by PBMC and the expression of IL-10 mRNA in 10 long-term HD patients (108-276 months), 10 short-term HD patients (3-18 months), and 10 healthy controls. Results: Spontaneous IL-10 secretion was higher in HD patients than in controls (15 pg/mol vs 2 pg/ml), P=0.001). It was detected in 13 of 20 patients and in 1 of 10 controls (P=0.01). IL-10 mRNA expression was also higher in HD patients than in controls. Spontaneous secretions of IL-10 and IL-6 were positively correlated in patients. Il-10 secretion in response to LPS was higher than the upper limit of control range in 4 of 10 long-term HD patients and in no short-term HD patients (P=0.04). IL-10 mRNA expression was also higher in long-term than in short-term HD patients. Conclusions: This study demonstrates that IL-10 is spontaneously synthesized and secreted in HD patients, supporting an immunomodulating role in this setting. The greater IL-10-producing capacity in long-term HD patients indicates a chronic effect of haemodialysis on PBMC responsiveness.     

卵巢摘除大鼠外周血单核细胞ER-mRNA表达与骨小梁退变的相关研究

目的探讨外周血单核细胞及其雌激素受体（ＥＲ）ｍＲＮＡ表达水平，在绝经后骨质疏松发生机理中所超的作用。方法成年雌性ＳＤ大鼠３０只，随机分３组，去势组（ＯＶＸ组），假手术组（Ｓｈａｍ组），ＯＶＸ＋Ｐｒｅｍａｒｉｎ组（ＥＲＴ组）。术后６周后采各大鼠心脏血，分别以①用放免法测定血清雌二醇；②外周血单核细胞计数以及分离培养纯化单核细胞后运用ＲＴ－ＰＣＲ法检测ＥＲ－ｍＲＮＡ；③各组行骨组织形态计量学测定。结果     

Interleukin-6 gene expression in peripheral blood mononuclear cells from patients undergoing hemodialysis or continuous ambulatory peritoneal dialysis

To compare the interleukin-6 (IL-6) gene expression in the peripheral blood mononuclear cells (PBMCs) and plasma IL-6 levels in patients undergoing continuous ambulatory peritoneal dialysis (CAPD) with those in patients undergoing hemodialysis. Eleven hemodialysis patients, 10 CAPD patients, 15 non-dialyzed patients with end-stage kidney disease (ESKD), and 7 healthy controls were included in this study. PBMCs were collected by differential centrifugation. Plasma IL-6 concentration was measured by enzyme immunoassay. Plasma IL-6 levels were significantly increased in the hemodialysis and CAPD patients as compared with non-dialyzed ESKD patients and normal subjects (p < 0.01). Following hemodialysis, plasma IL-6 levels exceeded those before hemodialysis. No significant difference was found in plasma IL-6 levels in CAPD patients and in hemodialysis patients when blood was drawn before hemodialysis. Low but steady-state levels of IL-6 mRNA expression were observed in the non-dialyzed ESKD patients. The expression of IL-6 mRNA in PBMCs was significantly increased in the patients undergoing hemodialysis or CAPD as compared with the non-dialyzed ESKD patients. The PBMC IL-6 mRNA was significantly lower in CAPD patients than in hemodialysis patients (p < 0.01). A significant correlation was found between the plasma concentration of IL-6 and the expression of IL-6 mRNA in PBMCs from patients undergoing hemodialysis or CAPD (p < 0.01). The hemodialysis or CAPD procedure contributed to the increase in PBMC IL-6 mRNA expression and plasma IL-6 concentration. CAPD treatment stimulated the production of IL-6 to a lesser extent than hemodialysis treatment.     

Abnormal regulation of insulin-like growth factor gene expression in peripheral blood mononuclear cells from patients with IgA nephropathy.

We investigated insulin-like growth factor (IGF)-I and -II mRNA expression in peripheral blood mononuclear cells (PBMC) and T cells obtained from 31 patients with IgA nephropathy (IgAN), 43 patients with other types of glomerulonephritis and 16 health age-matched controls. The majority of patients with IgAN showed elevated IGF-I and -II mRNA expression in PBMC, while no IGF-I and -II mRNA expression was detected in PBMC obtained from patients with other types of glomerulonephritis or normal controls. In T cells obtained from IgAN, other types of glomerulonephritis and normal controls, however, IGF-I and -II mRNA expression was not detected. A positive correlation was noted between IGF-I and -II mRNA levels and urinary protein excretion. IGF-I and -II mRNA expression also correlated with the histopathological findings in the renal tissue of patients with IgAN. Sixty-nine percent of patients with more than 1.0 g/day proteinuria showed strong [more than (++)] IGF-I and -II mRNA expression in their PBMC. Eighty-one and 76% of patients with grade III and IV histopathological findings, respectively, showed strong IGF-I and -II gene expression in their PBMC. We also studied the clinical course of 11 patients with IgAN during hospitalization. The IGF-I and -II mRNA levels in these patients decreased gradually, as did proteinuria, after treatment. These studies suggest that abnormal regulation of IGF-I and -II gene expression in PBMC may be associated with the progression of IgAN and may be useful as an indicator of disease activity.     

Resting T cells negatively regulate osteoclast generation from peripheral blood monocytes

There is accumulating evidence that T cells may be involved in osteoclastogenesis in a variety of murine systems. However, the precise role of human T cells in the regulation of osteoclast generation is still unclear. To address this issue, we investigated the effect of resting peripheral T cells on receptor activator of NF-kappaB ligand (RANKL)-induced osteoclast generation from human peripheral monocytes. Although osteoclasts were not generated in the culture of human peripheral blood mononuclear cells (PBMC) in the presence of RANKL and macrophage colony-stimulating factor (M-CSF), the addition of cyclosporine A (CsA), a potent inhibitor of T-cell function, resulted in the formation of an increasing number of lacunae resorption on dentine, suggesting T cells may inhibit osteoclast formation. In a coculture of T cells and monocytes, which were isolated from PBMC, T cells inhibited the osteoclast generation from monocytes, as determined by tartrate-resistant acid phosphatase (TRAP) staining and a pit assay using dentine. This inhibition of osteoclast generation by T cells was also observed in a culture of the parathyroid hormone-stimulated SaOS4/3 osteoblast cell line and monocytes. The culture in Transwell plates revealed that the cell-to-cell interaction was not required for the inhibition, suggesting that T-cell cytokines may be responsible for the inhibition. Among inhibitory T-cell cytokines on osteoclastogenesis, granulocyte-macrophage colony-stimulating factor (GM-CSF) and interferon-gamma (IFN-gamma) were actively produced by CD4 T cells but not CD8 T cells in the coculture of T cells with monocytes, and the neutralizing antibodies to these cytokines partially rescued the T-cell-induced inhibition of osteoclast formation. Although CsA did not affect RANKL-induced osteoclast generation in the culture of monocytes alone, it completely rescued the T-cell-induced inhibition of osteoclast formation and strongly inhibited the production of GM-CSF and IFN-gamma. Thus, we demonstrate that resting T cells negatively regulate the osteoclast generation via production of GM-CSF and IFN-gamma by CD4 T cells and that CsA stimulates the osteoclast generation through the inhibition of the production of these cytokines. These findings provide new insight into therapeutic strategies for immunosuppression-induced bone loss in transplant and other diseases.     

Characterization of osteoclasts derived from CD14+ monocytes isolated from peripheral blood

Bone resorption is solely mediated by osteoclasts. Therefore, a pure osteoclast population is of high interest for the investigation of biological aspects of the osteoclasts, such as the direct effect of growth factors and hormones, as well as for testing and characterizing inhibitors of bone resorption. We have established a pure, stable, and reproducible system for purification of human osteoclasts from peripheral blood. We isolated CD14-positive (CD14+) monocytes using anti-CD14-coated beads. After isolation, the monocytes are differentiated into mature osteoclasts by stimulation with macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor κB ligand (RANKL). Osteoclast formation was only observed in the CD14+ population, not in the CD14− population, and only in the presence of both M-CSF and RANKL, confirming that the CD14+ system is a pure population of osteoclast precursors. No expression of osteoclast markers was observed in the absence of RANKL, whereas RANKL dose-dependently induced the expression of cathepsin K, tartrate-resistant acid phosphatase (TRACP), and matrix metallo proteinase (MMP)-9. Furthermore, morphological characterization of the cells demonstrated that actin rings were only formed in the presence of RANKL. Moreover, the osteoclasts were capable of forming acidic resorption lacunae, and inhibitors of lysosomal acidification attenuated this process. Finally, we measured the response to known bone resorption inhibitors, and found that the osteoclasts were sensitive to these and thereby provided a robust and valid method for interpretation of the effect of antiresorptive compounds. In conclusion, we have established a robust assay for developing osteoclasts that can be used to study several biological aspects of the osteoclasts and which in combination with the resorption marker CTX-I provides a useful tool for evaluating osteoclast function in vitro.     

Enhanced expression of intracellular heme oxygenase-1 in deactivated monocytes from patients with severe systemic inflammatory response syndrome

BACKGROUND: Monocyte deactivation is an important contributor to infectious susceptibility in critically ill patients. However, the mechanism of monocyte deactivation has not been fully elucidated. Recently, intracellular heme oxygenese-1 (HO-1), an anti-inflammatory heat-shock protein, was reported to be activated by Toll-like receptors (TLRs), and to inhibit inflammatory cytokine production such as that of TNF-alpha. In the present study, we evaluated the expression of intracellular HO-1 and TLRs in monocytes from patients with severe systemic inflammatory response syndrome (SIRS) and examined the role of HO-1 in monocyte deactivation. PATIENTS: Twenty-seven patients who fulfilled the criteria for severe SIRS and had a serum C-reactive protein (CRP) level >10 mg/dL were included in this study. The cause of SIRS was sepsis in 16 patients, trauma in 7, and other in 4. Expression of intracellular HO-1, surface TLR2 and TLR4, and intracellular cytokines (TNF-alpha, Interleukin-6) stimulated via TLR activation were measured in circulating monocytes by flow cytometry. Intracellular HO-1 expression was evaluated in normal monocytes stimulated with patient serum. Serum cytokine levels were also measured. Patient data were compared with data from healthy volunteers (n = 16). RESULTS: Cytoplasmic HO-1 was clearly detected by fluorescence microscopy. Expression of HO-1, TLR2, and TLR4 in monocytes was significantly enhanced in patients with severe SIRS compared with that in healthy volunteers, whereas intracellular TNF-alpha expression with peptidoglycan was significantly decreased (p < 0.05) in patients compared with that in healthy volunteers. HO-1 expression was significantly enhanced in normal monocytes stimulated with patient serum. Intracellular HO-1 levels were positively related to serum TNF-alpha levels in patients (r = 0.46). CONCLUSIONS: Expression of intracellular HO-1 and of TLRs was enhanced in deactivated monocytes from patients with SIRS. Increased production of intracellular HO-1 in response to serum factors may play a role in monocyte deactivation after systemic inflammation.