Hyperlipoproteinemic low-density lipoprotein receptor-deficient mice are more susceptible to sepsis than corresponding wild-type mice

High circulating concentrations of lipoproteins have been shown to modify the cytokine response and reduce mortality after endotoxin or live bacterial challenge. Sepsis, however, is more complex than endotoxemia, and it is not clear whether elevated plasma lipoproteins will be protective. Previous studies have shown that the low-density-lipoprotein receptor deficient (LDLR-/-) mice with increased circulating LDL are protected against the lethal effects of endotoxemia and Gram-negative infection. We evaluated whether the LDLR-/- mice would be protected against the effects of sepsis induced by cecal ligation and puncture (CLP). Mortality was greater in LDLR-/-mice than in control C57Bl/6J mice. At 120 h after inducing sepsis, 20% of the control mice survived whereas none of theLDLR-/-mice were alive. Prior to inducing sepsis, serum concentrations of amyloid A protein and lipopolysaccharide binding protein (LBP) were significantly elevated in the LDLR-/-mice in comparison to the C57Bl/6J mice. Protein expression of sCD14 was also greater in the serum from the LDLR-/-mice than the C57Bl/6J mice. The elevated serum concentrations of LBP and CD14 were not associated with increases in the levels of liver CD14 mRNA and LBP mRNA. After inducing sepsis, serum concentration of interleukin (IL)-1beta was also significantly higher in LDLR-/-mice than in the control C57Bl/6J mice. These findings indicate that the LDLR-/-mice were more susceptible to the lethal effects of sepsis induced by CLP. The LDLR-/-mice also had higher serum concentrations of baseline, acute phase response proteins, SAA and LBP, and increased production of IL-1beta in response to CLP.     

Mouse livers with macrosteatosis are more susceptible to normothermic ischemic injury than those with microsteatosis

BACKGROUND/AIMS: Fatty livers are increasingly used for transplantation due to the dramatic organ shortage. While steatosis is an established risk factor for post-operative complications, the impact of macro- vs. microvesicular steatosis on ischemic injury is unclear. METHODS: The effects of ischemia and reperfusion were tested in two different models of steatotic mice: ob/ob as a model disclosing predominantly macrovesicular steatosis and choline deficient diet having mainly microvesicular steatosis. Steatotic and lean livers were subjected to 45 min of ischemia. Serial markers of hepatocellular injury, animal survival were measured. Hepatic tissue blood flow and portal vein perfusion were assessed. RESULTS: Ob/ob mice had a significantly lower tolerance to hepatic ischemia. with increased AST release and decreased survival in comparison to the choline deficient mice. No difference in ATP content was found between both steatosis models, but hepatic perfusion and portal vein flow were significantly lower after reperfusion in the ob/ob mice when compared to the choline deficient animals. Ischemic pre-conditioning significantly improved liver reperfusion and injury in both models of steatosis. CONCLUSIONS: Livers with macrovesicular steatosis have a lower tolerance to ischemic injury than those with microvesicular steatosis. Low intrahepatic and portal vein perfusion in macrovesicular fatty livers is, at least partially, responsible for the poorer outcome.     

血管内皮生长因子增加内皮细胞通透性的基质金属蛋白酶-2机制

目的　研究血管内皮生长因子 (VEGF)增加血管内皮细胞对脂蛋白 (LDL)通透性与基质金属蛋白酶 2 (MMP 2 )的关系。方法　用异硫氰酸荧光素 (FITC)标记LDL ,荧光分光光度法测定FITC LDL的含量 ,在牛主动脉内皮细胞通透性模型上研究VEGF对FITC LDL通透率的影响 ;采用明胶酶谱法测定MMP 2活性。结果　VEGF剂量依赖性地增加血管内皮细胞对FITC LDL的通透性 ,MMP 2特异性抗体显著抑制VEGF的作用 ;VEGF还能剂量依赖性地升高MMP 2的活性。结论VEGF增加血管内皮细胞对FITC LDL的通透性的作用可能由MMP 2介导。     

Openings through endothelial cells associated with increased microvascular permeability

Rapid increases in microvascular permeability are associated with the appearance of openings in microvascular endothelium, which are believed to develop between the endothelial cells of venules. Reconstruction of these openings, from electron micrographs of serial sections of the endothelium reveal that many pass through the periphery of the endothelial cells close to intact intercellular junctions. Transcellular pathways are the principal type of opening induced in microvascular endothelium by the ionophore A23187, by VEGF, and by high transmural pressures. Some mediators induce the fusion of vacuoles with the luminal and abluminal surfaces of the endothelium, and it is suggested that the transcellular openings may develop from vacuolar channels.     

CD4+ T cells from elite suppressors are more susceptible to HIV-1 but produce fewer virions than cells from chronic progressors

Elite suppressors/controllers (ES) are HIV-1-infected individuals who maintain stable CD4(+) T-cell counts and viral loads of <50 copies/mL without antiretroviral therapy. Research has predominantly focused on immune factors contributing to the control of viral replication in these patients. A more fundamental question, however, is whether there are differences in the nature of CD4(+) T-cell infection in ES compared with viremic patients. Here, we compare chronic progressor (CP), ES, and uninfected donors in terms of three aspects of CD4(+) T-cell infection: cellular susceptibility to infection, death of infected cells, and production of virus from infected cells. Using multiple methods of infection and both single-cycle and replication-competent virus, we show that unmanipulated CD4(+) T-cell populations from ES are actually more susceptible to HIV-1 infection than those populations from CP. Depletion of highly susceptible cells in CP may contribute to this difference. Using 7AAD and AnnexinV staining, we show that infected cells die more rapidly than uninfected cells, but the increased death of infected cells from CP and ES is proportional. Finally, using an assay for measuring virus production, we show that virus production by cells from CP is high compared with virus production by cells from ES or uninfected donors. This higher virus production is linked to cellular activation levels. These data identify fundamental differences in chronic infection of ES and CP that likely contribute to differential HIV-1 disease progression.     

Human neural stem cells are more sensitive than astrocytes to ethanol exposure

BACKGROUND: Exposure to ethanol (EtOH) can be deleterious to the developing central nervous system. The mechanisms by which EtOH exposure induces neural pathology in utero remain unclear. However, EtOH-induced increases in protein kinase C (PKC) have been associated with apoptosis in human primary cell cultures. Although the toxic effects of EtOH on differentiated neural cells have been studied in laboratory animal models, the susceptibility of the human neural stem cells (NSCs) that predominate in the central nervous system during embryonic development has not been addressed. METHODS: For this study, fetal human brain cells, which satisfied the criteria for NSCs by being CD133-positive, nestin-positive, and differentiated glial fibrillary acidic protein-positive human astrocytes, were studied. The cytotoxic potential of EtOH in NSC and astrocyte cultures was studied by using morphological and biochemical methods. In addition, membrane and cytosolic fraction PKC activity for each cell type was assessed. RESULTS: NSC showed a dose-dependent increase in EtOH-induced toxicity as estimated by terminal transferase-mediated dUTP nick end labeling (TUNEL) stain and viability assays. TUNEL staining indicating DNA degradation consistent with programmed (apoptotic) cell death was detectable in 90% of NSC 16 hr after 2 hr exposure to 10 mM EtOH. NSC also showed a concentration-dependent increase in membrane, but not cytosol, PKC activity over the same EtOH dose range. By contrast, astrocytes showed no cytotoxic effects at any concentrations of EtOH used (0-10 mM). PKC activity of both the membrane and cytosolic fragments from astrocytes also was unaffected by this range of doses. CONCLUSIONS: This study demonstrates the susceptibility of human NSCs, compared with astrocytes, to EtOH and indicates that alterations in PKC signal transduction in NSC may play a role in EtOH-induced neuropathological processes.     

氧化应激损伤后的内皮细胞对瓣膜间质细胞生长的调控

目的建立过氧化氢介导的瓣膜内皮细胞(ECs)氧化应激(OS)模型,探讨OS作用下ECs和间质细胞(VICs)之间的相互作用关系。方法不同浓度过氧化氢(H_2O_2)处理ECs不同时间后,收集OS-ECs上清;上清作用于VICs,通过检测细胞增殖、凋亡、分化及存活率,验证OS-ECs上清对VICs生长有一定影响。结果随着处理时间的延长,ECs和VICs均有不同程度受损。其中,50μmol/L H_2O_2处理ECs,OS-ECs上清可稳定VICs生长状态。结论 50μmol/L H_2O_2处理ECs并收集上清,作用于VICs是建立OS细胞模型的最佳条件。ECs对VICs有一定程度的保护作用。     

Alcohol-induced endothelial changes are associated with oxidative stress and are rapidly reversed after withdrawal

BACKGROUND: Although heavy alcohol drinkers are at an increased risk of developing cardiovascular events, moderate alcohol intake is associated with reduced incidence of cardiovascular death. This paradox might reflect a dose-related effect of different alcohol intakes on endothelial function and this, in turn, might depend on changes in oxidative stress. METHODS: We tested the effects of alcohol withdrawal in heavy alcohol consumers and compared the plasma levels of endothelin-1, nitric oxide, plasminogen activator inhibitor-1, von Willebrand factor, malondialdehyde, and intracellular glutathione with those of alcoholics that did not modify their alcohol intake and teetotalers. In human endothelial cells that had been cultured for 2 weeks in the presence of different concentrations of ethanol, we assessed the same parameters after withdrawal of ethanol exposure. RESULTS: Alcohol increased the levels of endothelin-1, nitric oxide, and plasminogen activator inhibitor-1 and decreased the levels of von Willebrand factor both in vivo and in vitro. These changes were dose dependent, rapidly reversed after withdrawal of exposure, and associated with the presence of increased oxidative stress as indicated by increased levels of both malondialdehyde and intracellular glutathione. Blockade of oxidative stress by incubation of endothelial cells in the presence of oxidants' scavengers prevented the alcohol-induced functional modifications of the endothelium. CONCLUSIONS: Alcohol affects endothelial function with an effect that is mediated by an activated oxidative stress and is rapidly reversed after withdrawal. Dose-related endothelial responses to different alcohol intakes might translate in either vascular protection or vascular damage.     

Individual parathyroid cells are more sensitive to calcium than a parathyroid cell population

Information on the secretory behavior of individual parathyroid cells within a cell population has not previously been available. We now report a technique for examining quantitative changes in hormone secretion in individual parathyroid cells. We have used a reverse hemolytic plaque assay to measure cumulative PTH release in single isolated cells. Bovine parathyroid cells were dispersed with trypsin and mixed with staphylococcal protein-A-linked ovine erythrocytes. Cells were plated in a monolayer in the presence of PTH antiserum. After stimulation by an agonist, complement was added to the cells. Lysis of ovine erythrocytes formed a plaque around each individual cell that releases PTH. Results indicate that inhibition of PTH release by calcium was not affected by trypsinization. Plaque formation was dependent on all reagents; serial dilution of antiserum reduces plaque formation. Cells had a markedly uniform secretory response to calcium. We compared PTH release in individual cells measured by the reverse hemolytic plaque assay with hormone release in a parathyroid cell population measured by RIA. There was an inverse relationship between extracellular calcium concentrations and plaque area. Individual cells were more sensitive to calcium (ED50 = 0.4 mM Ca2+) than cell populations (ED50 = 0.8 mM Ca2+). We demonstrate that PTH release can be quantitated in single viable parathyroid cells.     

Primary cultures of human endothelial cells are susceptible to low doses of Shiga toxins and undergo apoptosis

Various endothelial cells, with the exception of those from human microvasculatures, have been known to resist Shiga toxins (Stxs) in vitro. However, freshly prepared primary cultures of human endothelial cells from the umbilical vein and artery and the saphenous vein were shown to be killed by a very low dose of Stxs. This cytotoxicity of Stxs involves apoptosis, which seems to be caused by a mechanism distinct from the well-known action of Stxs to inhibit protein synthesis, since the blockade of protein synthesis by cycloheximide could not induce apoptosis or enhance the effect of Stxs. Passaged human endothelial cells have been found to be highly resistant to Stxs, which is consistent with previous reports, and not to show any evidence of apoptosis even when they are killed by a high dose of Stxs.     

Intravenous glutathione prevents renal oxidative stress after coronary angiography more effectively than oral N-acetylcysteine

This study proposes the intravenous administration of glutathione (GSH) as a novel strategy to prevent contrast medium-induced renal oxidative stress. Renal oxidative stress is a critical cause of contrast-induced nephropathy (CIN). Recent reports have described that N-acetylcysteine (NAC) may prevent CIN by scavenging reactive oxygen species in the kidney. Twenty-one patients with reduced renal function who underwent coronary angiography (CAG) were equally assigned to the control, NAC and GSH (100 mg/min for 30 min before CAG) groups. CIN occurred in two patients, one in the control and the other in the NAC group. In the control group, the urinary lipid hydroperoxides (LOOHs) increased to 299.5 ± 94.4% of the baseline at 2 h after CAG (mean ± SE, p < 0.01). The increase in LOOHs was completely abolished in the GSH group (5.5 ± 8.8%, p = ns), but not in the NAC group (196.8 ± 81.3%, p < 0.05). In the control group, the serum GSH level fell by 9.4 ± 2.3% at 2 h after CAG (p < 0.01). The decrease was prevented in the GSH group (−1.8 ± 8.5%, p = ns), but not in the NAC group (−10.0 ± 3.3%, p < 0.05). The renal damage by contrast medium-induced oxidative stress occurs soon after CAG, and intravenous GSH is more effective in preventing the oxidative stress than oral NAC. This advantage may make GSH a potentially more effective therapeutic strategy against CIN.     

Alcohol-induced lung damage and increased oxidative stress

BACKGROUND: Alcohol-induced lung damage may be associated with increased oxidative stress. OBJECTIVE: Our aim was to investigate alcohol-induced changes in the biochemistry and histopathology of the lung. METHODS: Rats were divided into two groups, a control group and an ethanol group. The ethanol group received 2 g/kg ethanol (total: 3 ml) intraperitoneally. The controls were given the same amount of saline via the same route. Three hours later, the rats were sacrificed, and blood and lung tissue samples were obtained. Oxidative stress was assessed by measuring the levels of erythrocyte reduced glutathione (GSH), tissue malondialdehyde (MDA), myeloperoxidase (MPO) and Na(+)-K(+) ATPase. Histopathologic evaluation of the lung tissues was also performed. RESULTS: In the ethanol group, serum and tissue MDA levels and MPO activities were increased (p = 0.007, p = 0.001 and p = 0.000), and lung tissue Na(+)-K(+) ATPase activities and erythrocyte GSH were decreased (p = 0.001 and p = 0.000) compared to the controls. Histopathologic examination demonstrated alveolocapillary thickening, alveolar degeneration, leukocyte infiltration and erythrocyte extravasation in the lungs of the ethanol group (p < 0.05). CONCLUSION: These results suggest that high-dose acute alcohol administration aggravates systemic and local oxidative stress leading to acute lung injury, ranging from mild pulmonary dysfunction to severe lung injury. It should be borne in mind that rapid onset of the acute respiratory distress syndrome (ARDS) may also be due to increased oxidative stress following alcohol abuse, especially when ischemic disturbances, e.g. coronary heart disease, acute ischemia of the extremities and traumatic accidents, are concomitantly present. Therefore, precautions against ARDS may prevent morbidity and mortality in alcohol-induced lung damage in at-risk patients.