Detection of cardiac allograft rejection by real-time PCR analysis of circulating mononuclear cells

BACKGROUND: Detection of cardiac allograft rejection is based on the histological examination of endomyocardial biopsies (EMB). We have explored the possibility of whether graft rejection could be detected by characteristic gene expression patterns in peripheral blood mononuclear cells (PBMC) of heart-transplant recipients. METHODS: The study included 58 blood samples of 44 patients. On the day of EMB, mononuclear cells were isolated from peripheral blood, and gene expression was measured by quantitative real-time PCR. Thirty-nine parameters, including cytokine and chemokine genes were analyzed. Gene expression results were correlated with histological assessment of concomitant evaluated EMB according to International Society for Heart and Lung Transplantation (ISHLT) nomenclature. RESULTS: Gene expression of perforin, CD95 ligand, granzyme B, RANTES, CXCR3, COX2, ENA 78 and TGF-beta1 was significantly different in PBMC of patients with mild to moderate degrees of allograft rejection (> or =grade 2) compared with patients exhibiting no or minor forms of rejection ( or =grade 2 vs. 相似文献   

Radiation and androgen withdrawal alter expression of apoptosis pathway genes of prostate cancer cells

Aim: To investigate the altered expression of apoptosis pathway genes of prostate cancer cells treated by radiation and androgen withdrawal and whether the combined treatment may induce additive apoptosis. Methods: Androgen sensitive prostate cancer cell line LNCaP was cultured and treated by radiation, androgen withdrawal and combination of the two. Apoptosis was determined using apoptotic cells staining and mononuclear cell direct cytotox-icity assay. The total RNA were extracted and harvested. cDNA probes were prepared and labeled with biotin-16-dUTP and then hybridized to commercially available cDNA arrays, including apoptosis pathway-specific genes. The expression of important gene was further determined using RT-PCR. Results: Radiation induced additive apoptosis of prostate cancer cells; androgen withdrawal exhibited synergetic action. TNFRSF8 variant 2, DFFA, LTbR, mdm2, Myd88, TNFRSF14 and TNFSF4 mRNA were up regulated by radiation, while Survivin and Bar mRNA were down regulated. Mcl-1, TNFRSF14     

CD4+ cells play a major role in xenogeneic human anti-pig cytotoxicity through the Fas/Fas ligand lytic pathway

BACKGROUND: In this study, the role of cell-mediated cytotoxicity by human leukocytes against pig endothelial cells was examined in vitro. The aim was to determine which cell subsets were responsible for this phenomenon and which pathways were involved in cell lysis. METHODS: Primed human peripheral blood mononuclear cells (PBMC) or purified CD4+ or CD8+ T cells were used in a cell-mediated cytotoxicity assay in which cytotoxicity of an SV40 transformed porcine endothelial cell (EC) line (SVAP) was determined by Annexin V binding. RESULTS: Human PBMC demonstrated specific lysis of porcine EC that was proportional to the effector: target ratio. CD4+ T cells accounted for >60% of this lysis, whereas CD8+ T cells accounted for <20%. CD4+ T cell-mediated lysis depended on direct recognition of porcine major histocompatibility complex class II molecules as inhibition of swine leukocyte antigen class II on porcine EC-inhibited CD4+ T cell cytotoxicity. This lysis was mediated through the Fas/FasL pathway as addition of anti-Fas and/or anti-FasL antibody profoundly inhibited antiporcine lysis. In addition, FasL gene expression was detected in primed PBMC and CD4+ T cells by RT-PCR, whereas granzyme B gene expression was not. Primed CD4+ T cells demonstrated high level FasL protein by Western blotting and two-color FACS analysis, whereas NK cells and CD8+ T cells did not. Finally, recombinant human FasL induced apoptosis in Fas expressing porcine EC cells, demonstrating that human FasL interacted with and activated Fas on porcine EC cells. CONCLUSIONS: In conclusion, human to pig cell-mediated cytotoxicity was mediated predominantly by CD4+ T cells through the Fas/FasL pathway of apoptosis. These results suggest that direct cytotoxicity by xenoreactive CD4+ T cells may be one of several effector mechanisms involved in cellular xenograft rejection.     

人单核细胞共刺激分子在异种免疫反应中的表达及其作用机制

目的 揭示人单核细胞共刺激分子在异种免疫反应中的表达及其作用机制.方法 从猪的主动脉分离血管内皮细胞(PEC)并培养扩增;从人单个核细胞(PBMC)中纯化CD4+T淋巴细胞和单核细胞.建立PEC和人PBMC混合培养体系,培养后收集细胞,然后加入荧光标记的单克隆抗体,通过流式细胞术检测CD14+单核细胞表面共刺激分子表达情况.为了检测淋巴细胞增殖反应以及阻断共刺激分子对PEC免疫反应的作用,在PEC和人PBMC混合培养体系中分别加入抗CD154、CD80和CD86单克隆抗体.在培养的最后24 h加入同位素,于培养结束后收集细胞并经同位素计数仪进行检测.纯化的单核细胞经PEC刺激后与CD4+T淋巴细胞共培养来研究这些单核细胞诱导CD4+T淋巴细胞的增殖以及阻断共刺激分子的作用.结果 PEC和人PBMC混合培养后可检测到PBMC对异种PEC的高度免疫增殖反应;流式细胞术检测到PBMC中的CD14+单核细胞表面无CD40和CD80的表达,但表达CD86,经PEC刺激后,CD14+单核细胞膜表面显著上调CD40和CD80蛋白分子的表达,CD86表达上调.与未经刺激的单核细胞相比较,经PEC刺激后的单核细胞和CD4+T淋巴细胞共培养后可诱导CD4+T淋巴细胞明显增殖,抗人CD154、CD80、CD86单克隆抗体可以阻断CD4+T淋巴细胞对PEC的增殖反应.结论 人CD14+单核细胞在异种免疫反应过程的间接抗原提呈和共刺激信号传导中发挥重要作用,通过上调其共刺激分子的表达与CD4+T淋巴细胞共刺激分子CD154和CD28相互作用形成第二信号,并诱导CD4+T淋巴细胞对PEC的增殖反应;阻断共刺激分子可抑制异种细胞免疫反应.     

Human allogeneic vascular rejection after arterial transplantation and peripheral lymphoid reconstitution in severe combined immunodeficient mice

BACKGROUND: Interspecies differences create important shortcomings in existing animal models used to describe in vivo events responsible for allograft rejection. Alloimmune destruction of human dermal microvessels, histologically consistent with rejection, has been demonstrated in human skin-grafted severe combined immunodeficient (SCID) mice receiving allogeneic human peripheral blood mononuclear cells (PBMC). We have now documented human alloimmune injury in a vascularized, SCID-human arterial transplantation model. METHODS: Fresh human artery was used to replace the CB.17 SCID/beige mouse infrarenal aorta. Seven days later, 3x10(8) human PBMC were administered intraperitoneally, and lymphocyte engraftment was considered successful when circulating human CD3+ cells were later identified in peripheral blood. RESULTS: Forty-six of 49 (94%) mice undergoing transplantation survived, including 14 controls with arterial grafts receiving no PBMC. Twenty-eight of 32 mice demonstrated circulating human CD3+ cells, 14 days after PBMC administration. Animals were killed at 14, 21, or 28 days after receiving allogeneic PBMC, and arteries were recovered for histology and immunohistology. All 14 control mice had patent transplanted grafts with normal vascular histology and no lymphoid infiltration. Damage to transplanted arteries among lymphocyte-engrafted mice was apparent by 14 and 21 days in some animals, whereas 16 of 22 exhibited moderate to severe intimal, medial, and/or adventitial lymphocytic infiltration with intimal expansion by day 28. The infiltrate consisted of HLA-A, -B, -C+, and -DR+, human CD3+ cells, approximately equally distributed as CD4+ and CD8+ subsets. Some infiltrating lymphocytes were cytolytic cells as demonstrated by perforin staining. The endothelium of transplanted human arteries exhibited endothelialitis, and the endothelial cells stained intensely with anti-HLA-A, -B, -C and anti-HLA-DR antibodies. The expanded intima was predominantly smooth muscle cells, staining positively for smooth muscle alpha-actin, HLA-A, -B, -C and HLA-DR. Medial necrosis was not observed. CONCLUSION: The results provide evidence of alloimmune-mediated vascular rejection in this human arterial transplantation model.     

Adrenomedullin gene transcription is decreased in peripheral blood mononuclear cells of patients with IgA nephropathy

We measured mRNA levels of adrenomedullin (AM), C-type natriuretic peptide (CNP), vascular endothelial growth factor (VEGF), interleukin 1beta (IL-1beta) and interleukin 6 (IL-6) in peripheral blood mononuclear cells (PBMC) of patients with IgA nephropathy. To evaluate these mRNA levels, we employed a real-time quantitative PCR method which was performed using a hybridization probe labeled with two fluorescence dyes. This strategy was found to afford the standard curves with a high correlation, suggesting that this method is useful for evaluations of mRNA levels. By this method, levels of AM, CNP, VEGF, IL-1beta and IL-6 mRNA in PBMC of 49 IgA nephropathy patients and 35 healthy volunteers were evaluated. Among the mRNAs examined, AM mRNA levels were significantly lower in severe-grade than in mild-grade IgA nephropathy patients. Furthermore, AM mRNA levels correlated with CNP mRNA levels in PBMC of patients with IgA nephropathy, and each peptide generated from these mRNAs has antiproliferative effects on mesangial cells. These data indicate that gene expression of AM in PBMC is regulated according to the pathophysiological states of IgA nephropathy and that decreased AM production may contribute to the progression of IgA nephropathy.     

腹腔感染脓毒症时肺内主要模式识别受体表达变化及其与肺损伤的关系

目的观察腹腔感染脓毒症时肺组织内病原菌相关模式分子(内毒素、脂蛋白、DNA)的主要模式识别受体的表达变化及其意义。方法采用盲肠结扎穿孔(CLP)术建立30只昆明种小鼠腹腔感染脓毒症模型,将模型小鼠随机分为CLP组和假手术组,每组各15只鼠,分别于术后8,12和24h取5只鼠处死取右肺组织,采用逆转录聚合酶链反应(RT PCR)法检测肺组织内毒素受体[清道夫受体(SR)、白细胞分化抗原14(CD14)、TLR4]及细菌脂蛋白受体(TLR2)、细菌DNA受体(TLR9)mRNA的表达,酶联免疫吸附实验(ELISA)法检测肺组织内肿瘤坏死因子α(TNFα)含量,采用分光光度计法测定肺组织髓过氧化物酶(MPO)活性。结果与假手术组比较,术后8hCLP组肺组织内CD14mRNA(1.143±0.139,t=0.022,P<0.05),TLR2mRNA(0.418±0.102,t=0.021,P<0.05),TLR4mRNA(0.595±0.052,t=0.0001,P<0.01)和TLR9mRNA(0.743±0.178,t=0.0023,P<0.01)表达上调,其中TLR9mRNA呈持续上调变化,SRmRNA(0.659±0.159,t=0.029,P<0.05)呈持续下调改变;肺组织CD14、TLR4、SR、TLR2和TLR9mRNA的表达变化分别与MPO和TNFα变化呈明显的相关关系(P<0.05或P<0.01)。结论脓毒症发病过程中,肺组织内主要模式识别受体表达变化与肺损伤相关,除LPS外,其他细菌成分也可能参与了肺损伤过程。     

显性调控相关早反应基因的大规模扫描和分析

目的：动态观察白细胞介素10（IL－10）作用后，淋巴细胞早反应基因表达谱变化，为下一步评价患者免疫状态和诱导治疗提供理论依据。方法：应用DNA微矩阵技术，分离正常人外周血单个核细胞（PBMC），予以抗CD3McAb及CD3McAb IL-10刺激，抽提24h PBMC mRNA，反转录后与芯片杂交，通过生物信息学方法比较分析，结果：培养24h后，抗CD3McAb IL-10组与抗CD3McAb组比较有15个基因发生改变，5个基因表达明显下调，10个基因上调，主要功能在于促进基因编码区连接，影响 细胞骨架和运动，不同部位的磷酸化，去磷酸化，细胞识别，会促进细胞生长作用，细胞凋亡，信号传递的基因则重点涉及钙离子－钙调蛋白，G－蛋白信号通路。结论：在IL－10作用，细胞凋亡，信号传递的基因则重点涉及钙离子－钙调蛋白，G－蛋白信号通路。结论：在IL－10作用下，一系列基因转录活化，其中许多先前与免疫细胞无明确功能的相关基因参与了T细胞活化与调控，是否该群基因即代表IL－10作用下淋巴细胞的基因表达标签，尚须进一步证实；要完全明确T细胞调控网络，还应全面分析T淋巴细胞基因表达谱，从中筛选差异有显著意义的基因，进行功能研究。     

Expression of Toll-like receptors 2 and 4 is downregulated after operation

Background

Toll-like receptors (TLR) that recognize microbial pathogens play a critical role in innate immunity; however, their expression and function after surgery remain unknown. The aim of this study was to examine TLR2 and TLR4 expression on monocytes and their responses to each agonist after surgical insults.

Methods

Blood samples were obtained from 83 patients who underwent gastrointestinal surgery. TLR2, TLR4, and inducible nitric oxide synthase expressions on peripheral blood mononuclear cells (PBMCs) were analyzed by flow cytometry. Macrophage-activating lipopeptide-2 or lipopolysaccharide-induced tumor necrosis factor-α and interleukin-6 production was measured by enzyme-linked immunosorbent assay.

Results

TLR2 and TLR4 decreased and showed the lowest values on the postoperative days 3 and 1, respectively. Macrophage-activating lipopeptide-2-stimulated tumor necrosis factor-α and interleukin-6 production was decreased immediately after the operation (P<.05), increased to a maximum value on postoperative day 1, and then decreased gradually. Lipopolysaccharide-stimulated tumor necrosis factor-α production was also suppressed immediately (P<.05) after operation then showed a gradual increase to maximum values on postoperative day 3. Inducible nitric oxide synthase in cultured PBMC that was obtained immediately after operation was upregulated (P<.05).

Conclusion

Expressions of TLR2 and TLR4 were downregulated by operation, and agonist-induced cytokine production was suppressed transiently and soon increased through the activation of PBMC. The present study may offer new insights for postoperative modulation of innate immunity under surgical stress.     

Toll-Like Receptor 4 and CD14 Gene Polymorphisms in Tunisian Kidney Transplantation

BackgroundAcute and chronic rejections remain an important cause of graft loss after renal transplantation. Currently, activation of innate immune responses through Toll-like receptors (TLRs) is suspected to be implied in the loss of the transplant tolerance.ObjectivesWe investigated functional single nucleotide polymorphisms (SNPs) of TLR4 and its coreceptor CD14 in kidney transplantation and looked for any potential role in acute rejection (AR) and chronic allograft nephropathy (CAN) and impact on graft survival.Patients and MethodsTLR4 (Asp299Gly) and CD14 (C/T -159) SNPs were detected using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) in 209 kidney transplant recipients (KTRs) including 132 treated with mycophenolate mofetil (MMF+). AR occurred in 59 patients and 24 were identified as having CAN by biopsy and scored according to the Banff criteria.ResultsThere were no significant associations between TLR4 and CD14 genotypes and alleles and the occurrence of both AR episodes and CAN. Moreover, TLR4 and CD14 SNPs did not seem to influence kidney graft survival. Analysis according to human leukocyte antigen (HLA) compatibility status, positivity of anti-HLA antibodies, and immunosuppression by MMF confirmed the absence of correlation of the investigated SNPs with the graft outcome. In addition, incidence of post-transplantation infections, including cytomegalovirus (CMV) infections, was not influenced by both TLR4 and CD14 SNPs.ConclusionThese results suggest that TLR4 (Asp299Gly) and CD14 (C/T -159) functional SNPs do not play a major role in AR, CAN, and kidney graft survival. Therefore, intragraft monitoring of TLR4/CD14 genes expression by messenger RNA (mRNA) would provide clarity on the exact role of these receptors in graft injuries.     

Low incidence of acute rejection after living-donor liver transplantation: immunologic analyses by mixed lymphocyte reaction using a carboxyfluorescein diacetate succinimidyl ester labeling technique

BACKGROUND: To monitor antidonor alloreactivity for accurate diagnosis of acute rejection after living-donor liver transplantation (LDLT), we used a mixed lymphocyte reaction (MLR) assay using an intracellular fluorescent dye carboxyfluorescein diacetate succimidyl ester (CFSE)-labeling technique (CFSE-MLR) in 29 consecutive patients who underwent adult-to-adult LDLT. METHODS: For patients who developed moderate or severe disorders in liver function, CFSE-MLR was performed together with needle biopsy of the liver allografts immediately after liver dysfunction had occurred. CFSE-labeled peripheral blood mononuclear cells (PBMC) from recipients and irradiated autologous, donor, or third-party PBMC were cultured, and then proliferation and CD25 expression in each of the CD4+ and CD8+ T cell subsets were analyzed by flow cytometry. RESULTS: Twelve (41.4%) of the 29 patients developed moderate or severe disorders in liver function within 6 months after LDLT. Eight of the 12 patients (overall incidence of 27.6%) suffering from liver function disorder were diagnosed on the basis of liver biopsy results as having mild or moderate acute rejection. However, only 4 of the 12 patients (overall incidence of 13.8%) showed remarkable proliferation of CD8+ T cells in association with CD25 expression on antidonor CFSE-MLR. The other eight patients were eventually diagnosed as having recurrence of original hepatitis, drug-induced hepatotoxicity, or congestion of the anterior segment of the liver allograft by further extensive examinations or in retrospect. CONCLUSIONS: The results of CFSE-MLR assays, which could be used for rigorously monitoring rejection, provided evidence of low incidence of acute rejection after LDLT.     

Propofol has anti‐inflammatory effects on alveolar type II epithelial cells

Background: We investigated whether lipopolysaccharide (LPS) induced inflammation in alveolar epithelial type II (ATII) cells is through cluster of differentiation 14 (CD14) and Toll‐like receptor 4 (TLR4) and the effect of different dosages of propofol on the inflammation in primary cultured rat ATII cells. Methods: Cultured ATII cells were randomly assigned to one of the following five groups: Group C: untreated group (control) cultured in the absence of propofol and LPS; Group LPS: treated with 1 μg/ml LPS; Group P1: treated with 1 μg/ml LPS and 25 μM propofol; Group P2: treated with 1 μg/ml LPS and 50 μM propofol; Group P3: treated with 1 μg/ml LPS and 100 μM propofol. ATII cells in all groups were cultured at 37 °C for 3 h. CD14 and TLR4 mRNA was detected using real‐time polymerase chain reaction. Western blot was used to detect CD14 and TLR4 protein expression. CD14 and TLR4 expression on the ATII cells was imaged using immunofluorescence. Tumor necrosis factor‐α (TNF‐α) production was determined using an ELISA kit. Results: LPS stimulation resulted in an increased CD14 and TLR4 expression and increased TNF‐α production in ATII cells. Propofol, at concentrations ≥50 μM, significantly (P<0.05) and dose‐dependently decreased CD14 and TLR4 mRNA expression and protein expression in ATII cells. This was accompanied by a decrease in TNF‐α production (P<0.05). Conclusion: These results suggest that propofol, at clinically relevant concentrations, can reduce inflammatory responses in LPS‐induced ATII cells injury through downregulation of CD14 and TLR4 expression.     

Increased Toll-like receptor 4 expression on T cells may be a mechanism for enhanced T cell response late after burn injury

BACKGROUND: Burn injury is associated with a dynamic T cell response. We have previously reported an enhanced functional T cell response 14 days after burn injury. Toll-like receptors (TLR), primarily expressed on innate immune cells, have recently been identified on certain T cell subsets, including activated and memory T cells. Our hypothesis is that increased TLR4 expression on memory T cells may be a mechanism for enhanced T cell response 14 days after burn injury. METHODS: Splenocytes from wild-type C57Bl/6 mice were harvested 14 days after a 20% total body surface area (TBSA) scald burn or sham injury. Splenocytes ex vivo were surface stained either with monoclonal anti-CD3, anti-CD4, anti-CD8, or anti-CD44 antibodies or a two-step biotin-TLR4 monoclonal antibody-streptavidin-FITC surface stain and results analyzed by flow cytometry. RESULTS: TLR4 expression is successfully detected on CD4 and CD8 T cells. TLR4 expression is significantly (p < 0.05) increased on CD4 T cells and CD8 T cells 14 days after burn injury. There is a significant (p < 0.05) increase in CD44 (memory) CD4 and CD44 (memory) CD8 T cells 14 days after burn injury and this is associated with a significant (p < 0.05) increase of TLR4 expression in both T cell populations. CONCLUSIONS: This study demonstrates for the first time the potential role of TLR4 expression on memory T cells generated late after burn injury. Although further analysis is required, these data reiterate the importance of adaptive immunity and the complexity of the immune response to burn injury.