Antimicrobial penetration into polymorphonuclear leukocytes and alveolar macrophages.

Infections caused by intracellular organisms often involve the lung and may be implicated in chronic disease. These intracellular bacteria may be protected from otherwise lethal concentrations of extracellular antimicrobials. Knowledge of the intracellular concentration of usual antimicrobials used to treat pneumonia may allow physicians to refine their initial choice of therapy. Lipid-insoluble antimicrobials, such as penicillin, cephalosporins, aminoglycosides, trimethoprim, and imipenem penetrate poorly into cells, if they penetrate at all. Isoniazid, tetracycline, and lincomycin have intermediate intracellular penetration, and chloramphenicol, rifampin, ethambutol, quinolones, and lincosamides, plus macrolides, are avidly concentrated. Nonetheless, to date it has been difficult to correlate intracellular concentrations of antimicrobials with cellular killing or clinical outcome. Information derived from a more standardized approach to the evaluation of antimicrobial agent intracellular penetration will be useful in improving the direct application of in vitro study results to the clinical care of patients with pneumonia.     

吸烟致冠状动脉慢血流现象研究

目的:评估吸烟对冠状动脉造影正常者血流速度的影响。方法:连续性选择冠状动脉造影正常的男性患者123例,依据吸烟情况,分为吸烟组(57例)和非吸烟组(66例)。计算左前降支、左回旋支和右冠状动脉的心肌梗死溶栓(TIMI)血流帧数及全冠状动脉的平均TIMI血流帧数,分析缓慢血流及其相关危险因素。结果:与非吸烟组比较,吸烟组的TIMI血流帧数(左前降支41.43±15.29比36.5±11.75,左回旋支29.48±9.95比23.43±12.54,右冠状动脉25.33±10.92比20.63±11.28,均P﹤0.05)和全冠状动脉平均TIMI血流帧数(31.23±13.11比27.12±9.68,P﹤0.05)均显著增高。吸烟、肥胖、糖尿病和高脂血症为冠状动脉缓慢血流的独立危险因素。结论:吸烟者的冠状动脉尽管形态正常,但是其血流速度已受损。     

Cigarette smoking decreases interleukin-8 secretion by human alveolar macrophages

Cigarette smoking can impair pulmonary immune function, and hence influences the development of lung diseases. Interleukin-8 (IL-8) is a proinflammatory peptide and a potent chemotactic factor for neutrophils, and is produced by both immune and non-immune cells including monocytes and alveolar macrophages (AM). We investigated the effect of cigarette smoking on the secretion of IL-8 by human AM. The IL-8 concentration in bronchoalveolar lavage fluid (BALF) was much higher in smokers than in non-smokers (18.4 +/- 3.9 vs 4.1 +/- 1.0 pg ml-1; P < 0.005). However, spontaneous IL-8 secretion by cultured AM was lower in smokers than in non-smokers (46.8 +/- 12.7 vs 124.1 +/- 24.0 ng ml-1; P < 0.01). When stimulated with lipopolysaccharide (LPS), AM from smokers secreted significantly less IL-8 than those from non-smokers at all tested concentrations of LPS. In contrast, the amount of IL-8 secreted by peripheral blood monocytes with or without LPS stimulation was comparable in smokers and non-smokers. These observations indicate that smoking decreases IL-8 secretion by AM, which may modify or decrease the inflammatory response in the lung.     

Activated polymorphonuclear leukocytes inhibit phosphatidylcholine synthesis in cultured type II alveolar cells.

Activated human neutrophils (PMNs) were demonstrated to inhibit total de novo phosphatidylcholine (PC) synthesis in monolayered rat alveolar type II cells (T2C). Non-activated PMNs had no effect on PC synthesis in this system. The magnitude of inhibition T2C PC synthesis by phorbol myristate acetate-activated PMNs in six experiments averaged 59.0 +/- 13%. Exogenous chelated iron (ferric pyrophosphate) did not appear to augment the PMN-mediated inhibition of T2C PC production in this model. Alpha-1-antiprotease usually provided no protection relative to the PMN insult towards the T2C. However, superoxide dismutase and catalase alone or in combination generally provided a significant, protective effect. Although activated PMNs consistently decreased T2C PC synthesis, this effect did not appear to involve generalized T2C cytotoxicity, as assessed by lack of release of cytosolic lactate dehydrogenase. These results indicate that PMNs can inhibit T2C PC synthesis in vitro, probably via oxyradical injury. This type of pulmonary host autoinjury may be operative in a variety of acute lung injury syndromes involving pulmonary sequestration of activated PMNs.     

Antibiotic proteins of polymorphonuclear leukocytes

Abstract: The polymorphonuclear leukocyte (PMN) plays an essential role in the innate defense of the mammalian host against bacterial invaders. Responding chemotactically, the PMN delivers a complex antibiotic arsenal to sites of infection. Among these cytotoxic systems is an array of antimicrobial proteins and peptides that the PMN directs at microorganisms both before (i.e. extracellularly) and after sequestration into a phagocytic vacuole. In addition to their microbicidal capacity, several of these proteins bind to and neutralize the endotoxic activity of Gram-negative bacterial lipopolysaccharides (LPS). In this review the principle features of these antibiotic proteins are briefly summarized with emphasis on their possible actions in biological settings. In many instances, additional functions independent of cytotoxicity have been described raising the possibility that some of these proteins subserve multiple roles in inflammation.     

Cytolytically inactive terminal complement complex causes transendothelial migration of polymorphonuclear leukocytes in vitro and in vivo.

Intravital microscopy was used to monitor leukocyte traffic across rat mesenteric postcapillary venules induced by the inactive terminal complement (C) complex (iTCC) topically applied to ileal mesentery. Leukocytes started rolling within 15 minutes from the administration of iTCC, and by 1 hour they adhered almost completely to the endothelium emigrating from the vessels in the next 3 hours. C5a caused a similar, though less marked, effect, whereas boiled iTCC was inactive, excluding the contribution of contaminating lipopolysaccharide. The complex stimulated the migration of polymorphonuclear neutrophils (PMNs) across endothelial cells (ECs) in a transwell system after a 4-hour incubation of ECs with iTCC added to the lower chamber of the transwell, whereas a 30-minute incubation was sufficient for C5a and interleukin (IL)-8 to induce the passage of PMNs. C5a was not responsible for the effect of iTCC because this complex had no chemotactic activity and contained too small an amount of C5a to account for the transendothelial migration of PMNs. Similarly, the effect of iTCC was not mediated by IL-8 released by stimulated ECs because anti-IL-8 failed to inhibit the migration of PMNs induced by the complex. Unlike tumor necrosis factor-alpha, iTCC did not cause the redistribution of platelet-endothelial cell adhesion molecule-1 (PECAM-1), and PMN mobilization was partially blocked by anti-PECAM-1 antibodies.     

Cigarette smoking in social interaction

Studied behavior patterns by smokers and others in social interaction correlated with various behaviors comprising cigarette smoking. Field observation methods were used in observing 91 smokers in 117 social interaction settings. Settings were chosen to vary in degree of intimacy between participants in social interaction, and in degree of physical activity. Reliability of observations was established using independent field observers and raters of a videotape of one interaction. Findings generally were that smoking behaviors occur at points in the interaction when the smoker is relatively uninvolved and passive in the context of the social interaction. It is speculated that smoking is a form of self-involvement which is incompatible, on a momentary basis, with active social involvement.     

Cigarette smoking in the HIV-infected population

As mortality due to AIDS-related causes has decreased with the use of antiretroviral therapy, there has been a rise in deaths related to non-AIDS-defining illnesses. Given the exceedingly high prevalence of cigarette smoking among individuals living with HIV infection, tobacco has been implicated as a major contributor to this paradigm shift. Evidence suggests that smoking-related illnesses, such as cardiovascular disease, respiratory illnesses, and certain malignancies, contribute substantially to morbidity and mortality among HIV-infected persons. In this review, we summarize the adverse health consequences of smoking relevant to HIV-infected individuals and discuss smoking cessation in this unique population, including a discussion of barriers to quitting and a review of studies that have examined smoking cessation interventions.     

Effects of serine protease inhibitors on accumulation of polymorphonuclear leukocytes in the lung induced by acute pancreatitis in rats

The administration of a high-dose of a serine protease inhibitor is recommended in patients complicated by multiple organ failure (MOF), including adult respiratory distress syndrome (ARDS), induced by acute pancreatitis. The accumulation of polymorphonuclear leukocytes (PMN) in affected organs is considered to be one of the causative factors of MOF. Adhesion to endothelial cells (EC), via adhesion molecules, and the transendothelial migration of PMN is closely associated with the accumulation of PMN. We examined the effects of two serine protease inhibitors, ulinastatin (UT) and gabexate mesilate (GM), on EC-PMN adhesion and transendothelial migration in human umbilical vein EC and⁵¹Cr-labeled PMN in vitro. EC-PMN adhesion, and the expression of intercellular adhesion molecule-1 (ICAM-1) and endothelial cell adhesion molecule-1 (ELAM-1) on EC induced by IL-1β and TNFα, were reduced by the pretreatment of EC with these inhibitors. The transendothelial migration of PMN stimulated by IL-8 was also inhibited by pretreating PMN with UT or GM. We also examined whether these inhibitors reduced PMN accumulation in the lung in rats with acute pancreatitis induced by a closed duodenal loop. The myeloperoxidase activity in and histological findings of the lung suggested that UT and GM reduced PMN accumulation. In conclusion, serine protease inhibitors may inhibit PMN accumulation in ARDS due to acute pancreatitis.     

Characteristics of light and heavy polymorphonuclear leukocytes

Whole blood gravity sedimentation technique can be modified for studying leukocyte sedimentation properties. Previously, we demonstrated that the displacement rate of leukocytes was associated with activation of leukocytes during traditional gravity sedimentation of the whole blood. The plasma flow as well as the difference between the specific gravity of leukocytes and plasma propel the leukocytes upward in the sedimentation tube while the erythrocyte aggregates are descending. The leukocyte ascension rate can be described as the increment of leukocyte concentration in the upper half section of the blood column after one-hour sedimentation. The aim of the present study was to characterize the ascending and non-ascending leukocytes using a flow cytometric technique. Venous blood samples were taken from 8 healthy controls and 8 septic patients after major thoracic or abdominal surgical procedures. The upper and lower halves sections of venous blood column were separately removed from the sedimentation tube after one hour gravity sedimentation. Using flow cytometry, the leukocyte subsets were identified by their CD45 density and side scatter parameters followed by characterization of their cellular size and cytoplasmic granularity. The size indices of septic patients' ascending polymorphonuclear leukocytes (PMNs) were significantly lower than that of the non-ascending ones (253 +/- 22 versus 387 +/- 12 (SEM), p < 0.002) or the ascending PMN fraction taken from healthy individuals (382 +/- 28, p < 0.005). Septic patients' ascending PMNs presented significantly lower cytoplasmic granularity indices compared to non-ascending (447 +/- 23 versus 538 +/- 18, p < 0.05) or healthy ascending PMNs (539 +/- 20, p < 0.05). The cellular size and cytoplasmic granularity indices of heavy and light monocytes as well as lymphocytes were similar in both groups. It can be assumed that venous blood samples of septic patients contain significantly smaller PMNs with less cytoplasmic granularity than healthy control cells.     

Antibiotic proteins of human polymorphonuclear leukocytes.

Nine polypeptide peaks with antibiotic activity were resolved from human polymorphonuclear leukocyte azurophil granule membranes. All but 1 of the 12 constituent polypeptides were identified by N-terminal sequence analysis. Near quantitative recovery of protein and activity permitted an assessment of the contribution of each species to the overall respiratory-burst-independent antimicrobial capacity of the cell. Three uncharacterized polypeptides were discovered, including two broad-spectrum antibiotics. One of these, a defensin that we have designated human neutrophil antimicrobial peptide 4, was more potent than previously described defensins but represented less than 1% of the total protein. The other, named azurocidin, was abundant and comparable to bactericidal permeability-increasing factor in its contribution to the killing of Escherichia coli.     

Cigarette smoking extract causes hypermethylation and inactivation of WWOX gene in T-24 human bladder cancer cells

Genomic, epigenetic and expression alterations of WW domain containing oxidoreductase (WWOX) have been implicated in multiple tumor types. The current study was designed to examine the expression of WWOX in tumor tissues of human bladder transitional cell carcinoma (BTCC) and the influence of cigarette smoke extract (CSE) on WWOX expression and methylation status in T-24 bladder cancer cells. WWOX protein expression was evaluated by immunohistochemistry staining in a series of tumor samples from 78 patients with BTCC and 26 normal bladder tissues. The expression level and methylation status of WWOX in CSE-treated cells were examined by using quantitative Real-Time RT-PCR and methylation specific PCR, respectively. The expression levels of DNA methyltransferases (DNMTs) 1, 3A and 3B were also detected. We found that WWOX expression was absent or reduced in 79.5% (62/78) of BTCC tissues, but only in 19.2% (5/26) of normal bladder tissues. Loss of WWOX expression was correlated with tumor grade (P=0.019) and cigarette smoking (P=0.031), but was not associated with age, gender, tumor size and tumor number. Hypermethylation of WWOX promoter and exon 1 was specifically induced by CSE with a kinetics concurrent to the suppression of WWOX mRNA in T-24 cells. Furthermore, CSE treatment induced a significant time-dependent increase in the level of DNMT1, but has no effects on DNMT3A and DNMT3B. Taken together, these novel findings suggest that hypermethylation of WWOX induced by cigarette smoking may represent one underlying mechanism for the loss expression of WWOX in bladder cancer.     

急性肺损伤大鼠多形核白细胞L选择素变化及其在肺内扣押中的作用

目的 探讨急性肺损伤(ALI)大鼠循环血多形核白细胞(PMN)L选择素表达的变化及其于肺内扣押中的作用.方法 通过静脉注射内毒素(5 mg/kg)复制大鼠ALI模型,56只大鼠随机分为7组,每组8只.分别为(1)内毒素5 min组;(2)内毒素15 min组;(3)内毒素30 min组;(4)内毒素60 min组;(5)fucodin干预5 min组;(6)Fucodin干预15 min组静脉注射fucodin 5 mg/kg后,立即静脉注射内毒素5 mg/kg;(7)正常对照组静脉注射等量生理盐水替代内毒素.用免疫荧光间接法和流式细胞仪检测大鼠ALI过程中PMN L选择素蛋白表达的变化;用髓过氧化酶(MPO)分析法及组织学检查定量ALI过程中PMN于肺内的扣押量.结果 (1)PMN L 选择素的表达于静脉注射内毒素后5 min为7.8±1.6,与对照组(10.5±2.1)比较差异有显著性(P<0.05),静脉注射内毒素后15 min(2.9±0.5)、30 min(3.5±0.7)和60 min(4.9±0.7)与对照组比较差异更具显著性(P<0.01).(2)大鼠肺组织MPO活力于静脉注射内毒素后5 min [(0.359±0.074) U/mg肺组织]、15 min [(0.490±0.069) U/mg肺组织]、30 min [(0.565±0.111 ) U/mg肺组织]、60 min [(0.710±0.112) U/mg肺组织]与对照组[(0.069±0.011) U/mg肺组织]比较差异有显著性(P<0.01);fucodin干预5 min组[(0.391±0.071)U/mg肺组织]和对应时相点的内毒素组[(0.359±0.074) U/mg肺组织]比较差异无显著性,fucodin干预15 min组[(0.396±0.061) U/mg肺组织]和对应时相点的内毒素组[(0.490±0.069) U/mg肺组织]比较差异有显著性(P<0.05).结论 (1)正常状态下L选择素在PMN表面呈结构性表达,内毒素致伤后PMN L选择素表达迅速减少,伤后15 min时最低,其后呈回升趋势.(2)内毒素性ALI大鼠PMN肺内的早期扣押可能是L选择素非依赖性的,但L选择素对维持肺PMN的持续扣押仍然是重要的.