Poly(I:C) source,molecular weight and endotoxin contamination affect dam and prenatal outcomes,implications for models of maternal immune activation

The viral mimetic polyinosinic:polycytidylic acid (poly(I:C)) is increasingly used to induce maternal immune activation (mIA) to model neurodevelopmental disorders (NDDs). Robust and reproducible phenotypes across studies are essential for the generation of models that will enhance our understanding of NDDs and enable the development of improved therapeutic strategies. However, differences in mIA-induced phenotypes using poly(I:C) have been widely observed, and this has prompted the reporting of useful and much needed methodological guidelines. Here, we perform a detailed investigation of molecular weight and endotoxin variations in poly(I:C) procured from two of the most commonly used suppliers, Sigma and InvivoGen. We demonstrate that endotoxin contamination and molecular weight differences in poly(I:C) composition lead to considerable variability in maternal IL-6 response in rats treated on gestational day (GD)15 and impact on fetal outcomes. Specifically, both endotoxin contamination and molecular weight predicted reductions in litter size on GD21. Further, molecular weight predicted a reduction in placental weight at GD21. While fetal body weight at GD21 was not affected by poly(I:C) treatment, male fetal brain weight was significantly reduced by poly(I:C), dependent on supplier. Our data are in agreement with recent reports of the importance of poly(I:C) molecular weight, and extend this work to demonstrate a key role of endotoxin on relevant phenotypic outcomes. We recommend that the source and batch numbers of poly(I:C) used should always be stated and that molecular weight variability and endotoxin contamination should be minimised for more robust mIA modelling.     

Lipid A as a Drug Target and Therapeutic Molecule

In this review, lipid A, from its discovery to recent findings, is presented as a drug target and therapeutic molecule. First, the biosynthetic pathway for lipid A, the Raetz pathway, serves as a good drug target for antibiotic development. Several assay methods used to screen for inhibitors of lipid A synthesis will be presented, and some of the promising lead compounds will be described. Second, utilization of lipid A biosynthetic pathways by various bacterial species can generate modified lipid A molecules with therapeutic value.     

Men and women differ in inflammatory and neuroendocrine responses to endotoxin but not in the severity of sickness symptoms

Impaired mood and increased anxiety represent core symptoms of sickness behavior that are thought to be mediated by pro-inflammatory cytokines. Moreover, excessive inflammation seems to be implicated in the development of mood/affective disorders. Although women are known to mount stronger pro-inflammatory responses during infections and are at higher risk to develop depressive and anxiety disorders compared to men, experimental studies on sex differences in sickness symptoms are scarce. Thus, the present study aimed at comparing physiological and psychological responses to endotoxin administration between men and women. Twenty-eight healthy volunteers (14 men, 14 women) were intravenously injected with a low dose (0.4 ng/kg) of lipopolysaccharide (LPS) and plasma concentrations of cytokines and neuroendocrine factors as well as negative state emotions were measured before and until six hours after LPS administration. Women exhibited a more profound pro-inflammatory response with significantly higher increases in tumor necrosis factor (TNF)-α and interleukin (IL)-6. In contrast, the LPS-induced increase in anti-inflammatory IL-10 was significantly higher in men. The cytokine alterations were accompanied by changes in neuroendocrine factors known to be involved in inflammation regulation. Endotoxin injection induced a significant increase in noradrenaline, without evidence for sex differences. The LPS-induced increase in cortisol was significantly higher in woman, whereas changes in dehydroepiandrosterone were largely comparable. LPS administration also increased secretion of prolactin, but only in women. Despite these profound sex differences in inflammatory and neuroendocrine responses, men and women did not differ in endotoxin-induced alterations in mood and state anxiety or non-specific sickness symptoms. This suggests that compensatory mechanisms exist that counteract the more pronounced inflammatory response in women, preventing an exaggerated sickness response. Disturbance of these compensatory mechanisms by environmental factors such as stress may promote the development of affective disorders in women.     

酒精性肝病与肠道通透性

近年研究发现乙醇及其代谢衍生物乙醛,通过改变细胞内信号传导通道,进而破坏上皮细胞紧密连接,增加肠道细胞侧壁对大分子物质的通透性,促进内毒素血症与ALD的发生发展。此文就乙醇及乙醛介导的肠道通透性改变与ALD发生发展的关系作一综述。     

Effect of Prostaglandin E Receptor Subtype EP4 Selective Agonist on the Secretion of Tumor Necrosis Factor-α by Macrophages in Acute Ethanol-Loaded Rats

Background: It is suggested that endotoxin and proinflammatory cytokines play an important role in the development and progression of alcoholic liver disease. Recently, a prostaglandin receptor subtype EP4 agonist with cytoprotective effect has been developed. We examined the efficacy of an EP4 agonist ONO-AE1-437 on tumor necrosis factor-α (TNF-α) secretion of Kupffer cells, splenic macrophages, and alveolar macrophages in acute ethanol-loaded rats.
Methods: Kupffer cells, splenic macrophages, and alveolar macrophages were isolated from control and acute ethanol-loaded rats (5 mg/g body weight of ethanol, intraperitoneally). After the preculture in the medium that containing 0, 0.1, 1, 10, or 100 nmol/liter of ONO-AE1-437, TNF-α secretion of these cells stimulated by 100 ng/ml of endotoxin was determined for 3 hr.
Results: The amount of TNF-α secreted from alveolar macrophages was largest in both the control and the acute ethanol-loaded rats. Acute ethanol load enhances TNF-α secretion of splenic macrophages. The addition of ONO-AE1-437 significantly inhibited TNF-α secretion of Kupffer cells and splenic macrophages in both the control and the acute ethanol-loaded rats. Alveolar macrophages were less affected.
Conclusions: An EP4 agonist ONO-AE1-437 suppresses excess TNF-α secretion from macrophages and seems promising for future trial in patients with severe alcoholic hepatitis.     

Tumor Necrosis Factor in Alcohol Enhanced Endotoxin Liver Injury

Endotoxin administration causes liver injury. Patients with alcoholic liver disease frequently have portal vein and systemic endotoxemia, and some investigators have suggested that endotoxin plays an etiologic role in alcoholic liver injury. Many of the metabolic effects of endotoxin are mediated by the cytokine tumor necrosis factor (TNF). It was the purpose of this study to determine whether TNF plays a role in ethanol-enhanced endotoxin liver injury. Rats were fed either a diet in which 36% of the calories were from ethanol or an isocaloric control diet. After 6 weeks, groups of 10 rats were intravenously injected with either saline, 1 mg/kg endotoxin, or 30 micrograms/kg of a prostaglandin E1 (PGE1) analogue + 1 mg/kg endotoxin 24 hr prior to sacrifice. Ethanol/endotoxin-treated rats had significantly higher liver enzyme levels (ALT: 1064 +/- 355 IU/liter, AST: 2024 +/- 515 IU/liter) compared with isocaloric/endotoxin controls (ALT: 237 +/- 54 IU/liter, AST: 602 +/- 80 IU/liter). Ethanol/endotoxin rats also had significantly higher peak serum TNF concentrations (992 +/- 200 units/ml) compared with isocaloric/endotoxin controls (344 +/- 96 units/ml). Pretreatment of ethanol/endotoxin rats with PGE1 caused significant attenuation of liver injury (ALT: 267 +/- 64 IU/liter, AST: 612 +/- 77 IU/liter) and a diminished serum TNF response. In contrast to chronic ethanol administration, acute gavage with 2 mg/kg ethanol (30% w/v) followed by intravenous injection of 2 mg/kg endotoxin produced significantly lower peak serum TNF concentrations (401 +/- 76 units/ml) than gavage with distilled water (1152 +/- 208 units/ml).(ABSTRACT TRUNCATED AT 250 WORDS)     

Cytokine-induced nitric oxide inhibits mitochondrial energy production and induces myocardial dysfunction in endotoxin-treated rat hearts

The mechanism responsible for cardiac depression in septic shock remains unknown. The present study examined whether nitric oxide (NO) overproduced by inducible NO synthase (iNOS) can inhibit aerobic energy metabolism and impair the myocardial function in endotoxin-treated rat hearts. Lipopolysaccharide (LPS) significantly decreased systolic blood pressure (BP) to 44% of control during the 48 h treatment. Hearts from control and LPS-treated rats were perfused in a Langendorff apparatus. After LPS injection, left ventricular (LV) developed pressure (LVDP) was significantly depressed, plasma NO2-/NO3- (NO(x)) concentration was markedly increased, and myocardial adenosine 5'-triphosphate (ATP), creatine phosphate (CrP), and the ratio of ATP/adenosine 5'-diphosphate were progressively decreased with time. Immunological examination showed a significant expression of iNOS protein in the LPS-treated myocytes. Aminoguanidine, an inhibitor of iNOS, significantly attenuated these LPS-induced functional and metabolic changes. Myocardial cyclic guanosine 3',5'-monophosphate (cGMP) content was significantly increased after LPS injection. Methylene blue, an inhibitor of soluble guanylate cyclase, blunted this increase in cGMP and significantly restored the LPS-induced contractile dysfunction 6 h after LPS injection. In addition, there was a significant negative correlation between LVDP and myocardial cGMP levels as well as a significant negative correlation between LVDP and plasma NO(x) levels. In contrast, 48 h after LPS injection, methylene blue no longer affected cardiac performance, and there was a significant positive correlation between LVDP and myocardial ATP content. Furthermore, the normalized activities (as a ratio of the citrate synthase activity) of mitochondrial NADH-CoQ reductase, succinate-CoQ reductase, and ATPase, were significantly inhibited, and the swelling or disruption of mitochondria cristae was seen in the 48 h LPS treatment. These LPS-induced functional and morphological disorders in the mitochondria were significantly improved by aminoguanidine. The findings suggest that sustained production of NO by iNOS leads to contractile dysfunction via cGMP in the early stage, but that it can directly impair the mitochondrial function, lower myocardial energy production, and contribute significantly to the myocardial dysfunction in the later stage of septic shock.     

Lowering of oxidative stress in hemodialysis patients by dialysate cleaning: in relation to arteriosclerosis

The objective of this study was to investigate changes in oxidative stress associated with the cleaning of the dialysate. Thirty-six dialysis patients were studied. Changes in soluble CD-14 (sCD-14), malondialdehyde-low-density lipoprotein (MDA-LDL), and oxidized-LDL (Ox-LDL) were monitored for 1 year before and 1 year after dialysate cleaning. The mean endotoxin (ET) level in the dialysate had previously been confirmed to decrease from 39.0 EU/L to an undetectable level after the cleaning. The mean levels of sCD-14, MDA-LDL, and Ox-LDL decreased significantly after the cleaning (sCD-14, P < 0.0001; MDA-LDL, P < 0.001; Ox-LDL, P < 0.001). One year after the cleaning, six cases still showed high levels of MDA-LDL and Ox-LDL. Cardiovascular events occurred in four of those six cases within 2.8 years after the cleaning. These four patients suffered from strong oxidative stress during dialysis, even after the cleaning. We therefore concluded that high levels of MDA-LDL and Ox-LDL are improved in dialysis patients by cleaning of the dialysate. These results indicate that even a dialysate containing 50 EU/L or less ET may stimulate monocytes and cause oxidative stress. They also suggest that even low levels of ET may aggravate arteriosclerosis in dialysis patients. Thus, in order to prevent cardiovascular events in dialysis patients, it is necessary to purify the dialysate.     

非酒精性脂肪性肝炎发病机制探讨

目的建立大鼠非酒精性脂肪性肝炎（NASH）动物模型,探讨脂质过氧化、一氧化氮（NO）和一氧化氮合酶（NOS）以及肿瘤坏死因子α（TNF-α）、转化生长因子-β（TGF-β）等细胞因子在NASH致病中的作用.方法通过持续16周的高脂肪、高胆固醇饮食,建立大鼠NASH模型,造模结束时检测模型组和正常组血清转氨酶,血清及肝组织丙二醛（MDA）、超氧化物歧化酶（SOD）、NO、NOS、谷胱甘肽（GSH）含量.免疫组化标记α平滑肌肌动蛋白（α-SMA）、KP-1、TNF-α、TGF-β.结果实验结束时,模型组动物体重、肝脏指数比正常组显著增高（P值分别＜0.05和0.01）.血清转氨酶较正常组显著升高（P＜0.01）.除血清NO显著升高外（P＜0.05）,MDA、SOD、NOS、GSH均与正常组差异无统计学意义（P＞0.05）;而肝匀浆中,MDA、NO较正常组显著升高（P值分别＜0.05和＜0.01）,SOD、NOS、GSH则较正常组显著下降（P值分别＜0.05和0.01）.模型组α-SMA、KP-1、TNF-α、TGF-β免疫组化显示阳性细胞较正常组显著增加（P值均＜0.01）.结论脂质过氧化损伤增强,抗氧化能力降低,以及TNF-α、TGF-β、NO等炎性介质活化,共同参与了NASH的发病,并预示NASH可发展为肝纤维化、肝硬化.