Mediators of pulmonary injury induced by inhalation of bacterial endotoxin

The purpose of this study has been to further define the pathophysiologic aspects of lung injury caused by the inhalation of endotoxin (LPS) using the morphometric approach to identify mediators that influence distal lung structure and function. Hamsters were divided into 3 groups 24 h prior to low dose LPS inhalation exposure (4 micrograms/m3 for 5 h): (1) pretreated with cobra venom factor to deplete complement in vivo, (2) pretreated with indomethacin to block prostaglandin production, and (3) untreated control group. Both pretreatments abolished LPS-induced decreases in lung volume as well as increases in capillary PMN and platelets seen in untreated control animals. Neither pretreatment had any effect on the LPS-induced decreases of other capillary leukocytes. Similarly, both methods of pretreatment failed to block increases in cellular interstitium of distal capillary septa induced with LPS alone. LPS provoked changes in capillary endothelium, especially seen as an increase in numerical density of endothelial pinocytotic vesicles. Decomplementation failed to alter this increase, but indomethacin pretreatment blocked the effect. Neither treatment had any effect on their size. Low dose LPS inhalation also altered pulmonary capillary permeability to a 125I-BSA probe, which was found in significantly greater amounts in LPS-exposed lungs than in those of saline aerosol control lungs, but was not present in the air space as evidenced by negligible counts in bronchoalveolar lavages. It is evident that endotoxin on the epithelial side of the air-blood barrier leads to changes on the other side of that barrier.(ABSTRACT TRUNCATED AT 250 WORDS)     

Application of thrombelastography in liver injury induced by endotoxin in rat

Liver injury developing in patients with sepsis may lead to an increased risk of mortality. Thrombelastography (TEG) is generally applied to evaluate hemostatic disturbance in patients undergoing liver transplantation or cardiopulmonary bypass. The aim of this study was to investigate the development of liver injury and coagulopathy in a lipopolysaccharide (LPS)-induced animal model and to assess the relationship between TEG variables and liver injury. Male Wistar rats received LPS (30 mg/kg over a 4-h intravenous infusion) to induce experimental liver injury or isotonic saline as a control. Variables of hemodynamics and liver biochemistry were measured during the subsequent 6?h after the start of infusion. TEG variables (R-time, K-time, α-angle and maximal amplitude), thrombin-antithrombin complex and plasminogen activator inhibitor-1 were also measured. After LPS infusion, liver injury [examined by biochemical variables (e.g. alanine aminotransferase, ALT) and histological studies] was developed and inflammatory cytokines (tumor necrosis factor-α and interleukin-6) were raised. At the initial period of LPS infusion, R-time was shortened and α-angle was increased. Thereafter, α-angle and maximal amplitude were decreased progressively, demonstrating that endotoxin induced coagulation disturbances. Furthermore, there were strong positive correlation between K-time and natural log (Ln)(ALT) (r?=?0.823, P?=?0.001); also, there were strong negative correlations between α-angle and Ln(ALT) (r?=?-0.762, P?=?0.002) as well as maximal amplitude and Ln(ALT) (r?=?-0.732, P?=?0.004) at 6?h after LPS infusion. These results demonstrated that TEG could be a potential tool to evaluate the development of liver injury in endotoxemia.     

Hepatocellular injury with distinctive mitochondrial changes induced by lergotrile mesylate: a dopaminergic ergot derivative.

Increased serum activities of the enzymes alanine aminotransferase (ALT) and aspartate aminotransferase (AST) occurred in 12 out of 19 patients with idiopathic parkinsonism when they were treated with the ergot derivative lergotrile at an oral dose varying from 50 to 150 mg daily. Hepatocellular injury was confirmed by microscopic examination of liver biopsies obtained from 3 of these patients when the serum activities of ALT and AST were appreciably elevated. Light microscopy revealed features of mild acute hepatocellular injury, and electron microscopy showed proliferation of the smooth endoplasmic reticulum and apparently unique mitochondrial changes in hepatocytes. This is the first report of pathological changes in the liver associated with the therapeutic use of an ergot derivative. The presence of a potentially reactive cyanide group in the lergotrile molecule could be causally related to the observed hepatocellular injury. It is suggested that serum ALT and AST activities should be monitored carefully when the therapeutic potential of any new ergot derivative is assessed.     

Blood group does not correlate with disease severity in patients with Fabry disease (alpha-galactosidase A deficiency)

Blood groups B and P1 are substrates for the lysosomal enzyme alpha-galactosidase A. Therefore, patients with alpha-Gal A deficiency and blood groups B or P1 may exhibit more severe disease. In 48 Fabry patients distribution of blood group was not different from that in the Dutch population. No patient had blood group B. Clinical symptoms did not differ between bloodgroup P1 or P2 patients. We conclude that blood groups B and P1 are not overrepresented in Dutch Fabry patients. Blood group P1 is not correlated with more severe disease and cannot be considered a significant risk factor.     

Dynamic changes in apoptotic and necrotic cell death correlate with severity of ischemia-reperfusion injury in lung transplantation

Ischemia-reperfusion (IR) injury is a major cause of organ dysfunction following lung transplantation. We have recently described increased apoptosis in transplanted human lungs after graft reperfusion. However, a direct correlation between ischemic time, cell death, and posttransplant lung function has not yet been demonstrated. We hypothesized that an increased ischemic period would lead to an increase in cell death, and that the degree and type of cell death would correlate with lung function. To investigate this, we preserved rat lungs at 4 degrees C for 20 min and 6, 12, 18, and 24 h, and then transplanted the lungs and reperfused them for 2 h. Cell viability was determined with a triple staining technique combining trypan blue, terminal deoxynucleotidyl transferase-uridine nucleotide end-labeling, and propidium iodide nuclear staining. Percentages of apoptotic and necrotic cells were calculated from total cell numbers. Following 20 min and 6 and 12 h of cold preservation, less than 2% of graft cells were dead, whereas after 18 and 24 h of cold preservation, 11% and 27% of cells were dead (p < 0.05), the majority of which were necrotic. After transplantation and reperfusion, the mode of cell death changed significantly. In the 6- and 12-h groups, approximately 30% of cells were apoptotic and < 2% were necrotic, whereas in the 18- and 24-h groups, 21% and 29% of cells, respectively, were necrotic and less than 1% were apoptotic. Lung function (Pa(O(2))) decreased significantly (p < 0.05) with increasing preservation time. The percentage of necrotic cells was inversely correlated with posttransplant graft function (p < 0.0001). The study demonstrates a significant association among cold preservation time, extent and mode of cell death, and posttransplant lung function, and suggests new potential strategies to prevent and treat IR injury.     

Exhaled H(2)O(2) in steady-state bronchiectasis: relationship with cellular composition in induced sputum, spirometry, and extent and severity of disease.

STUDY OBJECTIVES: To determine the concentration of exhaled H(2)O(2) in patients with bronchiectasis, and to study the relationship between levels of exhaled H(2)O(2), extent of disease, symptoms score, spirometry, and cellular composition obtained from induced sputum; furthermore, to account for possible confounding effects of inhaled corticosteroids (ICS) usage, long-term oral antibiotic treatment, and chronic colonization with Pseudomonas aeruginosa. DESIGN: Cross-sectional study. PATIENTS: Thirty patients with steady-state bronchiectasis. RESULTS: Mean (95% confidence interval [CI]) exhaled H(2)O(2) levels were significantly elevated in patients with bronchiectasis compared to normal subjects: 1.1 (0.87 to 1.29) microM vs 0.3 (0.19 to 0.36) microM, respectively (p < 0.0001). Patients treated with ICS had similar values as steroid-na?ve patients. The group of patients with P aeruginosa colonization showed a significantly increased concentration of H(2)O(2) compared to the group without P aeruginosa colonization. Patients receiving long-term oral antibiotic treatment had significantly higher values of H(2)O(2) compared to those not receiving antibiotics. There was a significant positive correlation between H(2)O(2) and either the percentage of neutrophils in induced sputum or the extent of the disease as defined by high-resolution CT. A significant negative correlation was found between H(2)O(2) and FEV(1) percent predicted. Finally, there was a significant positive correlation between H(2)O(2) and the symptoms score. CONCLUSIONS: Patients with bronchiectasis in stable condition showed increased levels of exhaled H(2)O(2). The above-mentioned levels were not decreased either by ICS or long-term oral antibiotic treatment, but were significantly affected by chronic colonization with P aeruginosa. H(2)O(2) levels could be an indirect index of neutrophilic inflammation, impairment of lung function, and extension and severity of the disease.     

Melatonin protection against lethal myocyte injury induced by doxorubicin as reflected by effects on mitochondrial membrane potential.

Melatonin (MLT) is highly protective against cardiotoxicity caused by doxorubicin (DOX). DOX induces cardiac damage via production of reactive oxygen species. This study tests the hypothesis that oxygen radicals generated by DOX disrupt mitochondrial membrane potential (Delta(psi)(m)) prior to severe cell injury. Myocytes were incubated with 20 micromol/l DOX for 24 h. Myocyte damage was estimated by lactate dehydrogenase (LDH) release. Mitochondrial membrane potential was determined by staining myocytes with 5, 5', 6, 6'-tetrachloro-1, 1', 3, 3'-tetraethylbenzimidazolcarbocyanine iodide (JC-1) using confocal microscope. A significant amount of LDH was observed after 24 h of treatment with DOX. Mitochondria in DOX-treated myocytes exhibited a collapse of Delta(psi)(m). Pretreatment with melatonin (1 mmol/l) for one hour prevented the release of LDH and restored Delta(psi)(m). The data support the hypothesis that DOX induces damage to mitochondria through radicals, and this is reflected in depolarization of Delta(psi)(m), which was prevented by melatonin.     

清除循环肿瘤坏死因子对急性肺损伤的影响

目的研究亲和免疫吸附清除循环肿瘤坏死因子（ＴＮＦ）对内毒素性急性肺损伤动物的保护作用。方法以６１只新西兰白兔为实验动物，建立内毒素性急性肺损伤模型。随机分为三组：（１）免疫吸附组（２７只），颈静脉注射自制脂多糖（ＬＰＳ）１００×１０８ｃｆｕ·ｋｇ－１后１小时开始血液灌流，通过含重组肿瘤坏死因子α（ｒＨｕＴＮＦα）单克隆抗体的吸附柱，灌流时间２小时；（２）致伤组（２７只），颈静脉注射ＬＰＳ，无吸附柱，仅有相同血量的体外循环；（３）对照组（７只），颈静脉注射生理盐水２ｍｌ，无吸附柱，仅有体外循环。通过亲和免疫吸附清除循环ＴＮＦ，观察了肺系数、肺通透指数、肺泡灌洗液（ＢＡＬＦ）中ＴＮＦ和白细胞介素８（ＩＬ８）水平、白细胞数的变化以及对存活率的影响。结果免疫吸附组能有效清除循环ＴＮＦ、ＢＡＬＦ中ＴＮＦ及ＩＬ８水平较致伤组显著降低（Ｐ＜０．０１），肺毛细血管通透性等指标明显改善（Ｐ＜０．０１），存活率提高（Ｐ＜０．０１）。结论免疫吸附清除循环ＴＮＦ，能有效地保护内毒素所致的急性肺损伤。     

Sputum cysteinyl-leukotriene levels correlate with the severity of pulmonary disease in children with cystic fibrosis.

Sputum samples from 13 children with cystic fibrosis (CF) were analyzed for leukotrienes (LTs) LTB4, LTC4, LTD4, and LTE4. Distribution of LTB4 appeared to be normal, and of cysteinyl-LTs log normal. Total cysteinyl-LT levels, of which on average 75% was LTE4, were nearly 10 times higher than in earlier studies. Log LTE4 and total cysteinyl-LT levels correlated with the overall severity of pulmonary disease assessed by Chrispin-Norman chest radiograph score (Log LTE4: r = 0.701, r2 = 49.1%, P = 0.008. Log total cysteinyl-LTs: r = 0.715, r2 = 51.1%, P = 0.006). There was no apparent relationship between LTB4 levels and Chrispin-Norman chest radiograph score, nor between the level of any of the LTs and age or organism cultured from sputum. These findings suggest that the cysteinyl-LTs may be involved in the pathophysiology of pulmonary disease in CF.     

Cellular alterations of parotid gland of rats with acute pancreatitis induced by cerulein.

To explore the cellular and subcellular alterations of the parotid gland during acute pancreatitis, we examined the redistribution of lysosomal enzyme, cathepsin B, along with the discharge of the LDH and cathepsin B from parotid acini of rats with acute pancreatitis induced by a supramaximal dose of cerulein (5 micrograms/kg/h for 3.5 h). Both the serum amylase level and parotid-gland amylase content were increased significantly in rats with acute pancreatitis. The dry-/wet-wt ratio ratio was significantly lower than in the control. In vitro studies showed that discharge of LDH from the parotid acini and leakage of cathepsin B from lysosomes in the acini were significantly higher than in the control. In addition, there was redistribution of the cathepsin B (shifting from the lysosomal pellet to the zymogen pellet) in the parotid gland. These results indicate that in acute pancreatitis there is edema and accumulation of amylase in the parotid glands, along with increased cellular and lysosomal fragility. Thus, there seems to be a close relationship between the exocrine pancreas and the parotid glands. Gut hormones, such as cerulein, also appear to play an important role in the pathophysiology of the parotid glands.     

Early mitochondrial damage in the induction of haemorrhagic necrosis in the Crocker sarcoma (S 180) by endotoxin

Summary Disturbances in the functional properties of tumor mitochondria have been studied during the course of induction of haemorrhage brought about by endotoxin in the murine Crocker sarcoma (S 180). Extensive impairment of function was already present in mitochondria isolated from control tumors, as shown by low respiratory control ratios. The existing mitochondrial damage intensified promptly in response to injection of endotoxin long before the onset of haemorrhage at 4 h. The nature of the additional damage took two forms, depending on the duration of exposure to endotoxin; first, at 30 min, a true uncoupling of oxidative phosphorylation was seen, largely reversible in vitro by pre-treatment of the isolated organelles with bovine serum albumin (BSA). Second, at 1 h and later, oxygen utilisation in the presence of succinate, ADP and inorganic phosphate (P _i) was depressed. The pre-addition of BSA consistently lowered respiration rates with succinate andP _i in all preparations. The extent of endogenous inhibition of the adenine nucleotide translocase appeared unaltered by endotoxin in vivo.     

Serum adiponectin concentrations correlate with severity of rheumatoid arthritis evaluated by extent of joint destruction

Adiponectin is a hormone released by adipose tissue with antidiabetic, antiatherogenic, and anti-inflammatory properties. The present observational study focused on the relation between serum adiponectin level and the disease severity of established rheumatoid arthritis (RA). Ninety patients with more than 5-year diagnosis of RA and 42 age- and BMI-matched control were enrolled. The severity of RA was evaluated according to the number of destructed joints of overall 68 joints on plain radiographs (37 patients had mild RA and 53 had severe RA). Serum adiponectin level was significantly higher in the severe RA group (17.7 ± 6.7 μg/ml) than in the control (9.1 ± 3.8 μg/ml) and mild RA groups (13.9 ± 6.5 μg/ml) (control vs. mild RA group, P < 0.001; mild RA vs. severe RA group, P < 0.01). These results suggest that increased number of joint destruction is associated with hyperadiponectinemia in established RA patients.     

JAK2/STAT3 activation by melatonin attenuates the mitochondrial oxidative damage induced by myocardial ischemia/reperfusion injury

Ischemia/reperfusion injury (IRI) is harmful to the cardiovascular system and causes mitochondrial oxidative stress. Numerous data indicate that the JAK2/STAT3 signaling pathway is specifically involved in preventing myocardial IRI. Melatonin has potent activity against IRI and may regulate JAK2/STAT3 signaling. This study investigated the protective effect of melatonin pretreatment on myocardial IRI and elucidated its potential mechanism. Perfused isolated rat hearts and cultured neonatal rat cardiomyocytes were exposed to melatonin in the absence or presence of the JAK2/STAT3 inhibitor AG490 or JAK2 siRNA and then subjected to IR. Melatonin conferred a cardio‐protective effect, as shown by improved postischemic cardiac function, decreased infarct size, reduced apoptotic index, diminished lactate dehydrogenase release, up‐regulation of the anti‐apoptotic protein Bcl2, and down‐regulation of the pro‐apoptotic protein Bax. AG490 or JAK2 siRNA blocked melatonin‐mediated cardio‐protection by inhibiting JAK2/STAT3 signaling. Melatonin exposure also resulted in a well‐preserved mitochondrial redox potential, significantly elevated mitochondrial superoxide dismutase (SOD) activity, and decreased formation of mitochondrial hydrogen peroxide (H₂O₂) and malondialdehyde (MDA), which indicates that the IR‐induced mitochondrial oxidative damage was significantly attenuated. However, this melatonin‐induced effect on mitochondrial function was reversed by AG490 or JAK2 siRNA treatment. In summary, our results demonstrate that melatonin pretreatment can attenuate IRI by reducing IR‐induced mitochondrial oxidative damage via the activation of the JAK2/STAT3 signaling pathway.     

Intravenous (−)-epicatechin reduces myocardial ischemic injury by protecting mitochondrial function

Background

Targeting the mitochondria during ischemia/reperfusion (IR) can confer cardioprotection leading to improved clinical outcomes. The cardioprotective potential of (−)-epicatechin (EPI) during IR via modulation of mitochondrial function was evaluated.

Methods and results

Ischemia was induced in rats via a 45 min occlusion of the left anterior descending coronary artery followed by 1 h, 48 h, or 3 week reperfusion. EPI (10 mg/kg) was administered IV 15 min prior to reperfusion for the single dose group and again 12 h later for the double dose group. Controls received water. Experiments also utilized cultured neonatal rat ventricular myocytes (NRVM) and myoblasts. A single dose of EPI reduced infarct size by 27% at 48 h and 28% at 3 week. Double dose treatment further decreased infarct size by 80% at 48 h, and 52% by 3 weeks. The protective effect of EPI on mitochondrial function was evident after 1 h of reperfusion when mitochondria demonstrated less respiratory inhibition, lower mitochondrial Ca^2 + load, and a preserved pool of NADH that correlated with higher tissue ATP levels. Mechanistic studies in NRVM revealed that EPI acutely stimulated maximal rates of respiration, an effect that was blocked by inhibitors of the mitochondrial pyruvate carrier, nitric oxide synthase, or soluble guanylyl cyclase. In myoblasts, knockdown of components of the mitochondrial pyruvate carrier blocked EPI-induced respiratory stimulation.

Conclusions

IV EPI confers cardioprotection via preservation of mitochondrial function potentially through enhanced substrate provision. These provocative results document a novel mechanism of a natural product with potential clinical utility.     

Delayed neutrophil elastase inhibition prevents subsequent progression of acute lung injury induced by endotoxin inhalation in hamsters

To define the role of neutrophil elastase (NE) in the progression of acute lung injury (ALI), we examined the effects of post-treatment with a specific NE inhibitor, sivelestat sodium hydrate (sivelestat), on ALI induced by endotoxin (ET) inhalation in hamsters. Inhalation of ET (300 microg/ml, 30 min) in conscious hamsters increased inflammatory cell count, protein concentration, and hemorrhage in bronchoalveolar lavage fluid (BALF) that peaked 24 h after ET inhalation. These changes were significant 2 h after ET inhalation and paralleled the increase in NE activity in BALF. When intravenously infused from 2 to 24 h post-ET inhalation, sivelestat (0.03 to 3 mg/kg/h) dose-dependently attenuated changes in these BALF parameters at 24 h post-ET inhalation in a manner dependent on the inhibition of NE activity in BALF. Histopathological analysis also indicated that sivelestat prevented the progression of lung inflammation such as alveolar neutrophil infiltration and hemorrhage. In contrast, dexamethasone (3 mg/kg, intravenously) was not effective in this model when administered 2 h after ET inhalation, although it was highly effective when applied before ET. We conclude that delayed inhibition of NE activity with sivelestat prevents subsequent progression of ALI in hamsters after ET inhalation. Thus NE may play an important role in the progression of ALI.     

Canine granulopoiesis: alterations induced by suppression of gram-negative flora.

We investigated alterations in canine granulopoiesis following suppression of bacterial flora by antibiotic treatment. Beagles were decontaminated in sterile isolation with laminar air flow, by an antifungal and antibiotic regimen. Skin and fecal specimen cultures were usually negative after 4 days. The reduction of gram-negative bacteria resulted in a significant decrease in plasma colony-stimulating factor (CSF) levels, marrow CFU-c concentration, and the cytopoietic activity of marrow-derived diffusion chamber (DC) progenitor cells. The period of conventionalization was characterized by a significant although delayed increase in plasma CSF as well as marked increases in marrow CFU-c concentration and cytopoietic activity of marrow DC progenitors. There was also mobilization of marrow DC progenitors into the circulation. All parameters returned to control values within 7 days of conventionalization. These data supported the hypothesis that the gram-negative bacteria of the gut play a significant role in the regulation of normal canine granulopoiesis.