Effect of linezolid on cytokine production capacity and plasma endotoxin levels in response to lipopolysaccharide stimulation of whole blood

The purpose of this study was to assess lipopolysaccharide (LPS)-stimulated cytokine production in the presence of linezolid (LZD) in comparison with the drug effect on the plasma endotoxin level. Peripheral venous whole-blood samples collected from five healthy subjects were stimulated with 10 μg/ml of LPS. LZD was then added to the LPS-stimulated blood samples at concentrations of 0, 2, 4, and 15 μg/ml, followed by incubation for 24 h at 37°C in a 5% CO₂–95% air atmosphere. Supernatants of the resultant cultures were assayed to determine the levels of tumor necrosis factor (TNF)-α, interferon (IFN)-γ, interleukin (IL)-10, monocyte chemoattractant protein (MCP)-1, and endotoxin. Significant decreases in the levels of TNF-α and IFN-γ were observed in the LZD 2, 4, and 15 μg/ml groups as compared with that in the 0 μg/ml group (Dunnett’s procedure; P < 0.05). The level of IL-10 tended to increase irrespective of the LZD concentration; however, no significant intergroup differences were observed [analysis of variance (ANOVA); P = 0.68]. No significant decrease of the endotoxin level was observed in the LZD 2, 4, or 15 μg/ml groups as compared with that in the 0 μg/ml group, with no significant intergroup differences (ANOVA; P = 0.83). No change in the MCP-1 levels was observed irrespective of the LZD concentration (ANOVA; P = 0.82). To conclude: (1) it appears possible that LZD inhibits the production of INF-γ and TNF-α to a limited extent; (2) LZD did not exert any inhibitory effect on endotoxin production by bacteria, while suppressing cytokine production. The results indicate that LZD may have a significant role in saving the lives of patients with sepsis.     

Counterregulatory effects of interferon-gamma and endotoxin on expression of the human C4 genes

Susceptibility to autoimmune disease is associated with null alleles at one of the two genetic loci encoding complement protein C4. These two genetic loci, C4A and C4B, are highly homologous in primary structure but encode proteins with different functional activities. Expression of C4A and C4B genes is regulated by IFN-gamma in human hepatoma cells and in murine fibroblasts transformed with the respective genes. In these cell lines, IFN-gamma has a significantly greater and longer-lasting effect on expression of C4A than that of C4B. In this study we examined synthesis and regulation of C4A and C4B in peripheral blood monocytes from normal, C4A-null, and C4B-null individuals. Synthesis of C4 in human peripheral blood monocytes decreases during time in culture. IFN-gamma mediates a concentration- and time-dependent increase in steady-state levels of C4 mRNA and a corresponding increase in synthesis of C4 in normal human monocytes. LPS decreases monocyte C4 expression and completely abrogates the effect of IFN-gamma on the expression of this gene. In contrast, LPS and IFN-gamma have a synergistic effect in upregulating expression of another class III MHC gene product, complement protein factor B. The effect of LPS on constitutive and IFN-gamma-regulated C4 synthesis is probably not mediated via release of endogenous monokines IL-1 beta, TNF-alpha, or IL-6. Synthesis of C4, and regulation of its synthesis by IFN-gamma and LPS, are similar in normal, C4A-, and C4B-null individuals. These results demonstrate the synthesis of C4 at extrahepatic sites and tissue-specific regulation of C4 gene expression.     

Immunomodulatory effects of fosfomycin in an endotoxin model in human blood

OBJECTIVES: Although a wide range of therapeutic strategies have been developed to improve the outcome of severe sepsis, a convincing reduction in mortality is lacking. Recently, increasing attention has been paid to immunomodulatory effects of antimicrobials. This study set out to explore the immunomodulatory effects of fosfomycin, a broad-spectrum antibiotic frequently used in septic patients, at the protein and molecular levels in vitro. METHODS: Whole blood from 11 healthy volunteers was incubated with 50 pg/mL endotoxin and 100 microg/mL fosfomycin or physiological sodium chloride for 4 h. Real-time RT-PCR was performed for various pro- and anti-inflammatory cytokines. Concentrations of tumour necrosis factor (TNF)-alpha and interleukin (IL)-6 in the supernatant were measured using a commercially available ELISA. RESULTS: Incubation of human leucocytes with endotoxin increased messenger RNA (mRNA) levels of cytokines several thousand fold compared with baseline. The addition of fosfomycin significantly inhibited mRNA levels of pro-inflammatory cytokines such as IL-1-alpha, IL-6 and TNF-alpha after 2 h (P < 0.01), while no significant reduction was observed for the anti-inflammatory cytokines IL-4, IL-10 and IL-13 (P = 0.26). At the protein level, the concentrations of IL-6 and TNF-alpha increased approximately 3000- and 600-fold after 4 h of incubation with lipopolysaccharide as compared with baseline, respectively. Addition of fosfomycin significantly reduced cytokine levels by 56% and 73% for IL-6 and TNF-alpha, respectively. CONCLUSIONS: Fosfomycin extensively decreased mRNA levels and release of pro-inflammatory cytokines in human blood. The broad antimicrobial coverage of fosfomycin and its immunosuppressive effects could be clinically useful in patients with sepsis.     

Special collection and storage tubes for blood endotoxin and cytokine measurements.

Commercially available blood-collection tubes may be contaminated with endotoxin (315 +/- 95 pg/tube) and could therefore be unsuitable for blood collection for endotoxin measurement. Plasma separation and storage are a potential source of contamination. To avoid contamination and error, we have developed new blood collection tubes that contain heparin free of endotoxin (LPS) and a gel to separate plasma and blood cells. The LPS content is less than 4 pg/tube. Samples can be stored and frozen without plasma withdrawal to preclude contamination. LPS recovery experiments have shown that the new blood-collection tubes do not bind LPS to the separation gel or vial wall. With these tubes, in vitro formation of tumor necrosis factor (404 +/- 163 ng/L in standard tubes vs less than 40 ng/L in special collection tubes) is minimized.     

Anti-aggregatory effects of physiological concentrations of adenosine in human whole blood as assessed by filtragometry.

1. The anti-aggregatory effect of adenosine (0.3-10 mumol/l), alone or in combination with the adenosine-uptake inhibitor dipyridamole (2 mumol/l), was studied in vitro in whole blood from 11 healthy subjects by filtragometry. 2. ADP (0.05-0.1 mumol/l) was used to reduce the filter occlusion time (tA, a measure of platelet aggregate formation in blood) from approximately 600 s to 71-101 s in the absence of other agents. 3. Adenosine was infused into the tubing system of the filtragometer, yielding a contact time of approximately 25 s with the blood before the filter. Adenosine did not influence the aggregatory response to ADP significantly at 0.3 mumol/l in plasma, whereas tA was prolonged by 19 +/- 6% (P less than 0.02) at 1 mumol/l adenosine and by 259 +/- 78% (P less than 0.02) at 3 mumol/l adenosine. 4. When the rapid elimination of adenosine from plasma was prevented by 2 mumol/l dipyridamole, adenosine caused marked prolongation of ADP-induced tA, with significant effects at 0.3 mumol/l (+143 +/- 72%, P less than 0.05). Dipyridamole per se did not affect tA values. 5. The present results suggest that adenosine has a transient anti-aggregatory effect in whole blood at about 0.3 mumol/l, as this is the highest possible calculated concentration of adenosine at the filter of the apparatus when 1 mumol/l adenosine is infused in the absence of dipyridamole or when 0.3 mumol/l adenosine is infused in its presence. 6. It is concluded that adenosine has anti-aggregatory effects at submicromolar (physiological) concentrations in human whole blood.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of echinocandins on cytokine/chemokine production by human monocytes activated by infection with Candida glabrata or by lipopolysaccharide

Serious Candida glabrata infections, which can be difficult to treat, are often treated with echinocandins. We compared in vitro the effects of high and low concentrations of 3 echinocandins (micafungin [MCF], caspofungin [CAS], and anidulafungin [ANF]), voriconazole (VRC), and amphotericin B (AmB), singly and VRC in combination with MCF, CAS, and ANF, on the production of cytokines/chemokines by human monocyte-derived macrophages (MDM). MDM were activated by infection with C. glabrata or lipopolysaccharide (LPS). Luminex multi-analyte microsphere technology was used for cytokine/chemokine analysis. Concentrations of cytokines/chemokines were significantly elevated following activation by infection or LPS. Treatment with high concentrations of echinocandins, singly or in combination with VRC, was most effective in lowering the elevated cytokine/chemokine levels. This effect occurred only with MDM activated by infection with C. glabrata and not with LPS. Treatment with VRC or AmB alone had little or no effect on cytokine/chemokine levels. In severe C. glabrata infection associated with very high concentrations of dysregulated cytokines/chemokines, echinocandins, singly or in combination with VRC, may decrease cytokine/chemokine concentrations and thus may improve host survival.     

Citrus pectin affects cytokine production by human peripheral blood mononuclear cells

Dietary fibers, including pectin, have been shown to exert a favorable effect on a wide spectrum of pathological conditions. Their positive influence on human health is explained by their anti-oxidative, hypocholesterolemic and anti-cancerous effects. However, little has been reported about their activity on the immune system. Therefore, the present study was undertaken to examine the effect of citrus pectin (CP) on cytokine production by human peripheral blood cells (PBMC). PBMC were incubated without or with CP at different degrees of esterification (DE) (∼30, ∼60 and ∼90% esterified pectin, assigned as DE30, DE60 and DE90, respectively) for detection of IL-1β, IL-1ra, TNFα, IL-6 and IL-10 secretion. Incubation with DE60 and DE90 induced a dose-dependent inhibition of the pro-inflammatory cytokine IL-1β secretion, whereas D30 did not affect this function. However, CP at all three esterification degrees did not alter the secretion of the additional pro-inflammatory cytokines examined, i.e. TNFα and IL-6. Conversely, CP at DE60 and DE90 caused a dose-dependent increased secretion of the anti-inflammatory cytokines IL-1ra and IL-10, whereas D30 did not affect the production of IL-1ra and decreased that of IL-10. The findings indicate that CP possesses the capacity to exert an immunomodulatory response in human PBMC which may have a favorable effect on human health.     

Interleukin 10 inhibits lipopolysaccharide-induced survival and cytokine production by human peripheral blood eosinophils

In this study we have investigated the effects of interleukin 10 (IL- 10) on human peripheral blood eosinophils stimulated with granulocyte/macrophage colony stimulating factor (GM-CSF) and lipopolysaccharide (LPS). We show that LPS was able to enhance eosinophil survival in a dose-dependent manner, as well as release of the cytokines GM-CSF, tumor necrosis factor alpha, and IL-8. LPS- induced eosinophil survival was largely inhibited by an anti-GM-CSF neutralizing antibody and completely blocked by polymyxin B, suggesting GM-CSF involvement in the survival enhancing mechanism and LPS specificity, respectively. IL-10 significantly inhibited survival of, and cytokine production from, eosinophils induced by LPS, but did not inhibit the survival induced by GM-CSF. These observations suggest a novel activation mechanism of eosinophils and, also, that IL-10 may participate in the regulation of diseases characterized by eosinophil infiltration.     

Interleukin 7 induces cytokine secretion and tumoricidal activity by human peripheral blood monocytes

Peripheral blood monocytes can be induced by stimuli such as bacterial lipopolysaccharide (LPS) to secrete an array of cytokines. We have studied the effects of interleukin 7 (IL-7) on human peripheral blood mononuclear cells (PBMC) and found that IL-7 is a relatively potent inducer of IL-6 secretion IL-6 protein levels were determined either by the B9 hybridoma growth factor assay or by enzyme-linked immunosorbent assay, and mRNA for IL-6 was analyzed by Northern hybridization. Detailed examination revealed that, among PBMC, monocytes, rather than lymphocytes, were secreting IL-6 in response to IL-7. In contrast to the low concentrations of IL-7 required to stimulate T cell growth and differentiation (as low as 0.1 ng/ml), relatively high concentrations of IL-7 were necessary to induce IL-6 secretion by monocytes (at least 10 ng/ml). An optimal concentration of IL-7 (100 ng/ml) induced monocytes to secrete 10-fold more IL-6 than an optimal concentration of IL-1 beta (10 ng/ml), and almost as much as LPS. However, significantly more IL-7 than IL-1 beta was required to induce detectable levels of IL-6. The kinetics of IL-6 secretion by monocytes were identical in response to IL-7, IL-1 beta, or LPS, with IL-6 protein detectable in culture supernatants as early as 2 h after the initiation of culture. IL-4 was found to markedly inhibit the ability of IL-7 or LPS to induce IL-6 mRNA and IL-6 secretion. In addition to promoting IL-6 production, IL-7 induced the secretion of immunoreactive IL-1 alpha, IL-1 beta, and tumor necrosis factor alpha (TNF-alpha) by monocytes. IL-7 also induced monocyte/macrophage tumoricidal activity against a human melanoma cell target, an activity that may be related to the secretion of IL-1 alpha, IL-1 beta, and TNF-alpha. Finally, we used a whole blood culture system as a bridge to in vivo analysis to demonstrate that IL-7 induces cytokine secretion in the absence of culture medium, fetal calf serum, and adherence to plastic. Our data suggest that IL-7, in addition to regulating lymphocyte growth and differentiation, has potent effects on cells of the monocytic lineage. Thus, IL-7 may be an important mediator in inflammation and in the macrophage immune response to tumors.     

Inhibition of chemiluminescence by carvedilol in the cell-free system, whole human blood and blood cells

Carvedilol inhibits luminol-enhanced chemiluminescence of reactive oxygen metabolites in vitro. In this study it was found that, in the cell-free system, carvedilol dose-dependently decreased chemiluminescence in the following ranking order of radicals: hydroxyl radical > hydrogen peroxide > superoxide radical. The inhibition of myeloperoxidase was significant with carvedilol concentrations of 10 and 100 micromol/l and manifested in the concentration-dependent shift of chemiluminescence peaks to the right. In whole blood, carvedilol in concentrations of 10 and 100 micromol/l significantly inhibited chemiluminescence induced by both receptor-bypassing stimuli (A23187, PMA) and receptor-operating stimuli (fMLP, OpZ). Carvedilol dose-dependently inhibited chemiluminescence of isolated human polymorphonuclear leucocytes in the ranking order of stimuli: A23187 > OpZ > fMLP. In the presence of blood platelets, carvedilol did not substantially change chemiluminescence induced by fMLP and OpZ, while it was much more effective on chemiluminescence stimulated with calcium ionophore A23187. This could be the result of the supportive effect of serotonin liberated from platelets by A23187.     

Ghrelin inhibits leptin- and activation-induced proinflammatory cytokine expression by human monocytes and T cells

Ghrelin, a recently described endogenous ligand for the growth hormone secretagogue receptor (GHS-R), is produced by stomach cells and is a potent circulating orexigen, controlling energy expenditure, adiposity, and growth hormone secretion. However, the functional role of ghrelin in regulation of immune responses remains undefined. Here we report that GHS-R and ghrelin are expressed in human T lymphocytes and monocytes, where ghrelin acts via GHS-R to specifically inhibit the expression of proinflammatory anorectic cytokines such as IL-1beta, IL-6, and TNF-alpha. Ghrelin led to a dose-dependent inhibition of leptin-induced cytokine expression, while leptin upregulated GHS-R expression on human T lymphocytes. These data suggest the existence of a reciprocal regulatory network by which ghrelin and leptin control immune cell activation and inflammation. Moreover, ghrelin also exerts potent anti-inflammatory effects and attenuates endotoxin-induced anorexia in a murine endotoxemia model. We believe this to be the first report demonstrating that ghrelin functions as a key signal, coupling the metabolic axis to the immune system, and supporting the potential use of ghrelin and GHS-R agonists in the management of disease-associated cachexia.     

血液内部成分对全血细胞介电特性的影响

目的 分析血液内部成分对成人全血细胞介电谱特性的影响.方法 在0.01-110MHz频率范围,利用4294A阻抗分析仪测量了50个正常健康人血液样本的交流阻抗,通过介电谱、Cole-Cole图、介电损耗频谱、电导率虚部频谱和损耗角正切频谱的数据分析.建立正常健康人全血细胞电生理的频率特性;同时检测红细胞比容、血沉、血糖、血浆纤维蛋白原及D-二聚体指标,用多元回归统计学分析方法分析上述实验指标对全血细胞介电谱特性的影响.结果 直线相关分析显示血沉与电导率低频极限值呈正相关(r=0.412,P=0.02),与最大电导率虚部负相关(r=-0.410,P=0.048),与电导率高频极限值正相关(r=0.435,P=0.02);红细胞比容是影响全血细胞介电性能的最主要因素,介电常数低频极限值、电导率松弛强度、介电损耗因子、电导率虚部及损耗角正切与红细胞比容呈正相关,介电常数高频极限值、电导率低频极限值、电导率高频极限值与红细胞比容负相关,血糖与介电常数高频极限值负相关而与电导率松弛强度正相关.结论 血液内部成分对全血细胞介电特性有影响,其中红细胞比容是影响全血介电特性最主要的因素,血糖对全血介电参数亦有一定的影响;血沉与部分介电参数呈线性关联.