急性呼吸窘迫综合征患者肺组织细胞凋亡及Fas／FasL表达？…

目的 探讨细胞凋亡在急性呼吸窘迫综合征（ＡＲＤＳ）发病中的作用机制。方法 ＡＲＤＳ组９例,对照组５例,采用ＴＵＮＥＬ法以及免疫组织化学技术观察ＡＢＤＳ患肺组织细胞凋亡及Ｆａｓ／ＦａｓＬ系统表达的变化,结果 ＡＲＤＳ急性期患肺组织细胞凋亡率明显高于对照组,且主要表现为肺泡上皮和肺血管内皮细胞凋亡增加;Ｆａｓ抗原,ＦａｓＬ在ＡＲＤＳ急性期患肺组织中的表达与对照组比较明显下调,并且与肺组织细胞凋亡     

地塞米松对急性损伤大鼠肺组织细胞凋亡及Fas基因与Fas基 …

目的 探讨细胞凋亡与急性肺损伤（ＡＬＩ）发生、发展的关系以及地塞米松在全身炎症反应及急性肺损伤中的作用机制及意义。方法 通过复制内毒素性大鼠ＡＬＩ模型,采用ＴＵＮＥＬ法、原位杂交、ＳｑＲＴ－ＰＣＲ以及免疫组化等技术观察ＡＬＩ肺组织细胞凋亡变化、Ｆａｓ基因与Ｆａｓ基因配体系统ｍＲＮＡ和蛋白质表达的改变以及地塞米松对细胞凋亡及Ｆａｓ、ＦａｓＬ表达的影响。结果 ＡＬＩ早期肺组织细胞凋亡明显增加;Ｆdispla     

Alveolar granulocyte colony-stimulating factor and alpha-chemokines in relation to serum levels, pulmonary neutrophilia, and severity of lung injury in ARDS

OBJECTIVES: To determine granulocyte colony-stimulating factor (G-CSF), epithelial neutrophil-activating peptide (ENA)-78, and interleukin (IL)-8 in BAL fluid (BALF), epithelial lining fluid (ELF), and serum for establishing the concentration gradient of G-CSF, ENA-78, and IL-8 between the blood and the alveolar space in ARDS and acute lung injury (ALI); and to evaluate the relationship of G-CSF, IL-8, and ENA-78 to pulmonary neutrophilia and severity of lung injury. DESIGN: Prospective study. SETTING: An adult trauma/surgical ICU. PATIENTS: Nineteen patients with ARDS and 10 patients with ALI. INTERVENTIONS: None. Measurements and main results: BAL and blood sampling simultaneously within 12 h and 24 h after onset of ARDS/ALI; G-CSF was detected in BALF in 18 of 19 patients with ARDS, in 7 of 10 patients with ALI, and in all serum samples. G-CSF in BALF and serum was significantly higher in ARDS than in ALI. ENA-78 was detected in BALF in 14 of 19 patients with ARDS, in 8 of 10 patients with ALI, and in serum of all patients. Levels in BALF and serum were not different between ARDS and ALI. IL-8 was detected in all patients; concentrations in BALF in ARDS were significantly higher than in ALI. Concentrations of G-CSF, ENA-78, and IL-8 in ELF were significantly higher than in serum. G-CSF in BALF and serum and IL-8 in BALF correlated positively with pulmonary neutrophilia. G-CSF in serum and IL-8 in BALF correlated negatively with PaO(2)/fraction of inspired oxygen (FIO(2)) ratio. However, ENA-78 did not show a correlation with neutrophil count or with PaO(2)/FIO(2) ratio. CONCLUSIONS: G-CSF may be pathophysiologically important for accumulation and activation of neutrophils in ARDS. Local G-CSF production is the likely driving force for neutrophils rather than elevation of circulating levels. In comparison to ENA-78, IL-8 seems to be the predominant neutrophil chemoattractant in the early phase of ARDS.     

Fibroproliferation occurs early in the acute respiratory distress syndrome and impacts on outcome

The fibroproliferative phase of acute respiratory distress syndrome (ARDS) has traditionally been regarded as a late event but recent studies that suggest increased lung collagen turnover within 24 h of diagnosis challenge this view. We hypothesized that fibroproliferation is initiated early in ARDS, characterized by the presence of fibroblast growth factor activity in the lung and would relate to clinical outcome. Patients fulfilling American/European Consensus Committee criteria for ARDS and control patients ventilated for non-ARDS respiratory failure underwent bronchoalveolar lavage (BAL) and serum sampling within 24 h of diagnosis and again at 7 d. The ability of BAL fluid (BALF) to stimulate human lung fibroblast proliferation in vitro was examined in relation to concentrations of N-terminal peptide for type III procollagen (N-PCP-III) in BALF/serum and clinical indices. At 24 h, ARDS lavage fluid demonstrated potent mitogenic activity with a median value equivalent to 70% (range 31-164) of the response to serum, and was significantly higher than control lavage (32% of serum response, range 11-42; p < 0.05). At 24 h, serum N-PCP-III concentrations were elevated in the ARDS group compared with control patients (2.8 U/ml; range 0.6-14.8 versus 1.1 U/ml; range 0.4-3.7, p < 0.0001) as were BALF N-PCP-III concentrations (2.9 U/ml; range 0. 6-11.4 versus 0.46 U/ ml; range 0.00-1.63, p < 0.01). In addition, BALF N-PCP-III concentrations at 24 h were significantly elevated in nonsurvivors of ARDS compared with survivors (p < 0.05). At 7 d, the mitogenic activity remained elevated in the ARDS group compared with control (p < 0.05) and was also significantly higher in ARDS nonsurvivors compared with survivors (67%; range 45-120 versus 31%; range 16-64, p < 0.05). These data are consistent with the hypothesis that fibroproliferation is an early response to lung injury and an important therapeutic target.     

Perforin and granzyme B of cytotoxic T lymphocyte mediate apoptosis irrespective of Helicobacter pylori infection: possible act as a trigger of peptic ulcer formation

BACKGROUND/AIMS: The perforin/granzyme and Fas/Fas ligand pathways are two known major pathways of cytotoxic T lymphocyte-mediated apoptosis. We designed a clinical study to examine whether cytotoxic T lymphocyte-mediated apoptosis associated with peptic ulcer formation may occur via either or both of these two pathways. METHODOLOGY: Mucosal biopsy specimens were obtained endoscopically from the marginal zone of active stage gastric and duodenal ulcers in patients with or without Helicobacter pylori infection. RT-PCR, immunoblotting and immunohistochemistry were used to study the samples for the expression of apoptotic cells, perforin, granzyme B, Fas, Fas ligand and caspase 3. RESULTS: Apoptotic cells (Tunnel positive cells) appeared in the marginal zone of gastric and duodenal ulcers with and without H. pylori infection. Perforin/granzyme B and caspase 3 were expressed consistently, however Fas ligand was not. Furthermore, the immunohistochemical findings demonstrated apoptotic changes of target cells caused by perforin/granzyme B. CONCLUSIONS: These results suggest that the main pathway of cytotoxic T lymphocyte-mediated apoptosis in peptic ulcer formation is the perforin/granzyme pathway irrespective of H. pylori infection.     

Defining the roles of perforin,Fas/FasL,and tumour necrosis factor alpha in T cell induced mucosal damage in the mouse intestine

BACKGROUND AND AIMS: Mucosal flattening and epithelial cell apoptosis are typical features of T cell induced inflammatory diseases of the bowel, such as coeliac disease and graft versus host disease. Mice injected with a T cell activating anti-CD3 antibody develop a severe diarrhoeal illness. We describe the histological features of this enteropathy and define the effector mechanisms involved in T cell induced mucosal injury in this in vivo model. METHODS: Wild-type and genetically modified mice were injected with the anti-CD3 antibody 3C11 (50 microg). Changes in the murine intestine were characterised by light microscopy analysis and terminal uridine nick-end labelling (TUNEL) assay. The role of perforin, Fas/Fas ligand (FasL), tumour necrosis factor alpha (TNF-alpha), and interferon gamma (IFN-gamma) in T cell induced mucosal damage was assessed using selected immunodeficient mouse strains. RESULTS: T cell activation caused severe damage, including small intestinal mucosal flattening and apoptosis of crypt epithelial cells. Mucosal damage was unaltered in anti-CD3 treated mice lacking IFN-gamma, Fas, or TNF-alpha receptors. In mice lacking TNF-alpha receptors and Fas (TNF-R1xR2 lpr/lpr strain), enterocyte apoptosis was diminished but there was no significant reduction in tissue damage. Apoptosis and mucosal injury were significantly reduced in perforin knockout mice. Abrogation of both FasL and perforin (perforin KOxgld mice) further significantly reduced tissue damage and apoptotic bodies. CONCLUSIONS: T cell induced mucosal injury is mediated by the combined effect of multiple pathways but predominantly by perforin. The redundancy of the mechanisms of tissue damage will have significant impact on therapeutic strategies aimed at specific and targeted inhibition of inflammatory processes.     

Fas- and tumor necrosis factor receptor 1-dependent but not perforin-dependent pathways cause injury in livers infected with an adenovirus construct in mice

Intravenous injection of type 5 adenovirus, deleted in the E1 and E3 regions, is shown to result in expression of viral antigens in the liver, initiating lymphocyte infiltration and liver injury. Following this infection, induction of Fas ligand (FasL), tumor necrosis factor alpha (TNF-alpha), and perforin mRNA are all demonstrable in the liver, pointing to a role of respective pathways in liver injury. Making use of mice in which the genes coding for Fas, FasL, TNF receptors (TNFRs), and perforin are inactivated, as well as recombinant proteins that inhibit Fas- and TNF-alpha-mediated apoptosis, it is shown that a functional perforin-mediated mechanism is not obligatory for cellular infiltration and progression of liver injury. In contrast functional Fas- and TNF-alpha-mediated mechanisms were found to be essential for liver injury to occur. Results are presented demonstrating that signaling through TNFR1, but not TNFR2, is involved in TNF-alpha-mediated liver damage. The conclusion is drawn that although perforin mRNA is induced in the virus-infected liver, Fas- and TNF-alpha-mediated mechanisms constitute the principal pathways by which the cell-mediated immune system causes acute liver injury.     

Fas ligand down-regulates cytokine-induced Fas receptor expression on insulinoma (NIT-1), but not islet cells, from autoimmune nonobese diabetic mice

In the pathogenesis of autoimmune type 1 diabetes, the apoptosis receptor Fas appears de novo on the surface of insulin-producing beta-cells. Fas expression is thought to be induced by proinflammatory cytokines, such as IL-1beta, interferon-gamma (IFNgamma), and TNFalpha, released by islet-infiltrating mononuclear cells. To determine whether beta-cells can modulate their sensitivity to apoptosis at the level of Fas, we investigated the effect of Fas ligand (FasL) on surface expression of Fas in NIT-1 insulinoma cells from nonobese diabetic (NOD) mice prone to autoimmune diabetes and islet cells from NOD and nonautoimmune BALB/c mice. In NIT-1 insulinoma cells, Fas expression induced by the cytokine combination IL-1beta and IFNgamma was reduced in the presence of FasL, whereas in islet cells Fas expression was unaffected by FasL. The effect of FasL on NIT-1 cells was evident during and after the induction of Fas expression by IL-1beta and IFNgamma. Thus, FasL down-regulates cytokine-induced Fas expression in NOD mouse-derived NIT-1 cells, but not in NOD or BALB/c mouse islets. The ability of NIT-1 cells to down-regulate Fas receptor in response to ligation is similar to that of a variety of tumor cells, which may use this mechanism to escape destruction by cytotoxic T cells. Islets apparently cannot protect themselves against FasL-induced apoptosis by down-regulating the Fas receptor. Understanding how NIT-1 insulinoma cells down-regulate Fas receptor in response to ligation by FasL has therapeutic implications for protecting normal beta-cells in autoimmune type 1 diabetes.     

Expression of perforin,granzyme A and Fas ligand mRNA in caecal tissues upon Eimeria tenella infection of naïve and immune chickens

Cytotoxic cells of the immune system may kill infected or transformed host cells via the perforin/granzyme or the Fas ligand (FasL) pathways. The purpose of this study was to determine mRNA expression of perforin, granzyme A and FasL in Eimeria tenella‐infected tissues at primary infection and infection of immune chickens as an indirect measure of cytotoxic cell activity. Chickens were rendered immune by repeated E. tenella infections, which were manifested as an absence of clinical signs or pathological lesions and significantly reduced oocyst production upon challenge infection. During primary E. tenella infection, perforin, granzyme A and FasL mRNA expression in caecal tissue was significantly increased at 10 days after infection, compared to uninfected birds. In contrast, at infection of immune birds, perforin and granzyme A mRNA expression in caecal tissue was significantly increased during the early stages of E. tenella challenge infection, days 1–4, which coincided with a substantial reduction of parasite replication in these birds. These results indicate the activation of cytotoxic pathways in immune birds and support a role for cytotoxic T cells in the protection against Eimeria infections.     

Granzyme B as a novel factor involved in cardiovascular diseases

Apoptosis plays an important role in cardiovascular diseases such as atherosclerosis, ischemic heart disease, and congestive heart failure. Previous studies have demonstrated that oxidative stress, physiological stress, and inflammatory cytokines such as tumor necrosis factor and Fas ligand are involved in apoptosis of cardiovascular system. We demonstrate that another apoptosis-related pathway, i.e. granzyme B/perforin system is involved in cardiovascular diseases. Expression of granzyme B, a member of serine protease family is increased in acute coronary syndrome, coronary artery disease with end-stage renal disease, and subacute stage of acute myocardial infarction. Although granzyme B is extensively researched in immunological disorders, the role of granzyme B/perforin system was not clear in the cardiovascular field. In addition, little is known regarding the inhibition of granzyme B system in the clinical situation. In this review we demonstrate recent findings of granzyme B in cardiovascular diseases and possible therapeutic applications of inhibiting the granzyme B/perforin system.     

A mouse CD8 T cell-mediated acute autoimmune diabetes independent of the perforin and Fas cytotoxic pathways: possible role of membrane TNF

Double transgenic mice [rat insulin promoter (RIP)-tumor necrosis factor (TNF) and RIP-CD80] whose pancreatic beta cells release TNF and bear CD80 all develop an acute early (6 wk) and lethal diabetes mediated by CD8 T cells. The first ultrastructural changes observed in beta cells, so far unreported, are focal lesions of endoplasmic reticulum swelling at the points of contact with islet-infiltrating lymphoblasts, followed by cytoplasmic, but not nuclear, apoptosis. Such double transgenic mice were made defective in either the perforin, Fas, or TNF pathways. Remarkably, diabetes was found to be totally independent of perforin and Fas. Mice lacking TNF receptor (TNFR) II had no or late diabetes, but only a minority had severe insulitis. Mice lacking the TNF-lymphotoxin (LTalpha) locus (whose sole source of TNF are the beta cells) all had insulitis comparable to that of nondefective mice, but no diabetes or a retarded and milder form, with lesions suggesting different mechanisms of injury. Because both TNFR II and TNF-LTalpha mutations have complex effects on the immune system, these data do not formally incriminate membrane TNF as the major T cell mediator of this acute autoimmune diabetes; nevertheless, in the absence of involvement of the perforin or Fas cytotoxic pathways, membrane TNF appears to be the likeliest candidate.     

肝素结合蛋白对脓毒症相关急性呼吸窘迫综合征的预测价值研究

目的探讨肝素结合蛋白(heparin binding protein,HBP)对脓毒症相关急性呼吸窘迫综合征(acute respiratory distress syndrome,ARDS)的预测价值。方法选择2016年6月—2017年6月在我院ICU诊疗并发生ARDS的30例脓毒症患者为ARDS组,30例未发生ARDS的脓毒症患者为非ARDS组。另选取30例健康志愿者为对照组。观察各组HBP、超敏C反应蛋白(hypersensitive C reactive protein,hs-CRP)、IL-6水平的差异,测算脓毒症患者入院48 h内HBP、hs-CRP、IL-6水平对发生ARDS的预测价值。结果 HBP、hs-CRP及IL-6在3组之间差异具有统计学意义(P均0.05);ARDS组和非ARDS组均明显高于对照组(P均0.05),且ARDS组均明显高于非ARDS组(P均0.05)。入院48 h,患者HBP对脓毒症相关ARDS预测价值最高,其最佳界值为28.56 ng/ml,ROC曲线下面积为0.834,诊断敏感度及特异度分别为86.2%和79.3%,约登指数为0.655。结论在脓毒症相关ARDS的预测中,HBP具有较高的价值。     

玄参中苯丙素苷对大鼠肝损伤细胞凋亡的影响

目的:观察玄参中苯丙素苷对大鼠肝损伤细胞凋亡的影响.方法:采用D-氨基半乳糖(D-Gal)造成大鼠急性肝损伤模型,观察玄参中苯丙素苷对此模型肝细胞凋亡及相关基因表达的影响.结果:玄参中苯丙素苷明显抑制模型肝细胞凋亡,上调bcl-2蛋白表达,下调Fas/FasL的表达.结论:玄参中苯丙素苷抗肝损伤细胞凋亡可能与其调控肝细胞凋亡相关基因有关.     

肺缺血再灌注损伤中氧化应激与Fas/FasL系统的关系

目的探讨肺缺血再灌注损伤中氧化应激与Fas/FasL系统的关系。方法采用在体兔单侧肺缺血再灌注损伤模型,日本大耳白兔40只,随机分为对照组,缺血再灌注1h、3h和5h组,每组10只。对比观察各组血浆超氧化物歧化酶活性、丙二醛含量和一氧化氮水平,以及肺组织细胞凋亡指数及Fas/FasL mRNA定位表达。结果缺血再灌注组肺组织凋亡指数明显高于对照组(P<0.01);再灌注后Fas、FasL mRNA在肺血管内皮细胞、肺泡上皮细胞及支气管上皮细胞等呈阳性表达,明显强于对照组(P<0.01);丙二醛含量随再灌注时间延长逐渐增加(P<0.01),而超氧化物歧化酶活性与一氧化氮则随再灌注时间延长有所降低(P<0.01)。结论肺缺血再灌注损伤期间,氧化应激参与Fas/FasL系统的激活。     

High bronchoalveolar levels of tumor necrosis factor and its inhibitors, interleukin-1, interferon, and elastase, in patients with adult respiratory distress syndrome after trauma, shock, or sepsis.

Intrapulmonary activation of leukocytes and release of cellular mediators and enzymes are involved in the pathophysiology of the adult respiratory distress syndrome (ARDS). To investigate a possible role of local cytokines, we measured bronchoalveolar fluid (BALF) and plasma levels of tumor necrosis factor alpha (TNF-alpha) and its soluble inhibitors (sTNF-RI + RII), interleukin-1 beta (IL-1 beta), interferon-alpha (IFN-alpha), and granulocyte elastase in 14 patients at risk for ARDS and in 35 patients developing ARDS after trauma, sepsis, or shock. During clinical development of severe ARDS, BALF cytokines increased markedly: TNF-alpha from 116 +/- 36 to 10,731 +/- 5,048 pg/ml (mean +/- SEM), p = 0.001; sTNF-RI + RII from 3.7 +/- 1.4 to 24.6 +/- 2.6 ng/ml, p less than 0.05; and IL-1 beta from 7,746 +/- 5,551 to 42,255 +/- 19,176 pg/ml, p = 0.01. Plasma cytokines were not increased in most patients, nor were they correlated with the development or severity of ARDS. BALF elastase was higher in patients developing ARDS than in those at risk but not going into pulmonary failure (0.97 +/- 0.26 versus 0.28 +/- 0.13 U/ml, p = 0.026), and the highest values were observed in the early stages of severe ARDS (1.85 +/- 0.39 U/ml). BALF elastase levels correlated with IFN-alpha (r = 0.72, p less than 0.001). In conclusion, local release of TNF-alpha and IL-1 beta, possibly by pulmonary macrophages or other cells, and/or accumulation in the lung is associated with the development of ARDS.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evaluation of bronchoalveolar lavage fluid from ARDS patients with regard to apoptosis

BACKGROUND: Apoptosis is thought to play an important role in the development of acute respiratory distress syndrome (ARDS). We evaluated the bronchoalveolar lavage (BAL) fluid from ARDS patients focusing on apoptosis. METHODS: The study enrolled 31 ARDS patients and 20 healthy controls. BAL fluid levels of caspase-cleaved cytokeratin-18 (CK-18) and soluble mediators such as interleukin-8 (IL-8), soluble Fas (sFas), soluble Fas ligand (sFasL), growth-related oncogene-alpha (GRO-alpha), granulocyte colony-stimulating factor (G-CSF), and tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) were measured using enzyme-linked immunosorbent assay (ELISA). RESULTS: The BAL fluid caspase-cleaved CK-18 levels in ARDS patients were higher than those in controls, reflecting increased epithelial apoptosis, and were correlated with lung injury scores (rs=0.49). The BAL fluid levels of all mediators were significantly higher in ARDS patients than in controls. In ARDS patients, the BAL fluid IL-8 level was positively correlated with the levels of sFas (rs=0.57), GRO-alpha (rs=0.47), and TRAIL (rs=0.45). The BAL fluid IL-8 (rs=0.61), sFas (rs=0.57), G-CSF (rs=0.44), and TRAIL (rs=0.33) levels were correlated with the BAL fluid neutrophil count. The G-CSF levels were significantly higher in non-surviving than in surviving ARDS patients [median 183.4 pg/mL (interquartile range 76.7-315.9) vs. 63.8 pg/mL (36.2-137.2); p<0.05]. The sFas levels were positively correlated with the PaO2/FiO2 ratio (rs=0.40), and the TRAIL levels were negatively correlated with the multiple organ dysfunction scores (rs=-0.37). CONCLUSIONS: Among the mediators in BAL fluid from ARDS patients, G-CSF had the most significant prognostic implications, and the sFas and TRAIL levels were correlated with clinical severity.