Angiotensin II type-1 receptor antagonist attenuates LPS-induced acute lung injury

Angiotensin II is able to trigger inflammatory responses through an angiotensin II type 1 (AT1) receptor. The role of AT1 receptor in acute lung injury (ALI) is poorly understood. Mice were randomly divided into three groups (n = 40 each groups): NS group; LPS group (2 mg/kg LPS intratracheally); and LPS + ZD 7155 group, 10 mg/kg ZD 7155 (an AT1 receptor antagonist) intraperitoneally 30 min prior to LPS exposure. Samples from the lung were isolated and assayed for histopathology analyses or proinflammatory gene expressions, angiotensin II receptors expressions and nuclear factors activities. LPS exposure resulted in severe ALI, elevated levels of TNF-α and IL-1β mRNA expressions, and increased activities of NF-κB and activated protein (AP)-1. Upregulation of AT1 receptor and down-regulation of AT2 receptor were also observed after LPS challenge. Pretreatment with ZD 7155 significantly inhibited the increase of AT1 receptor expression and upregulated AT2 receptor expression. ZD 7155 also reduced the mRNA expression of TNF-α and IL-1β, inhibited the activation of NF-κB and AP-1, and improved lung histopathology. These findings suggest that antagonism of AT1 receptor inhibits the activation of NF-κB and AP-1 in the lung, which may mediate the release of TNF-α and IL-1β and contribute to LPS-induced ALI.     

Vasoactive intestinal peptide (VIP) differentially affects inflammatory immune responses in human monocytes infected with viable Salmonella or stimulated with LPS

We compared the effect of VIP on human blood monocytes infected with Salmonella typhimurium 4/74 or stimulated with LPS. VIP (10⁻⁷ M) increased monocyte viability by 24% and 9% when cultured for 24 h with 4/74 or Salmonella LPS (100 ng/ml), respectively. Significantly increased (P < 0.05) numbers of 4/74 were also recovered from monocytes co-cultured with VIP after 6 h post-infection (pi) and this remained high after 24 h pi. Both 4/74 and LPS increased (P < 0.05) the concentration of TNF-α, IL-1β and IL-6 measured in monocyte supernatants. However, LPS induced this effect more rapidly while, with the exception of IL-6, 4/74 induced higher concentrations (P < 0.05). VIP significantly decreased (P < 0.05) TNF-α and IL-1β production by 4/74-infected monocytes after 6 pi, but only after 24 h in LPS-cultured monocytes. This trend was reversed for IL-6 production. However, TNF-α and IL-1β production by 4/74-infected monocytes, cultured with VIP, still remained higher (P < 0.05) than concentrations measured in supernatants cultured only with LPS. VIP also increased (P < 0.05) production of anti-inflammatory IL-10 in both 4/74 and LPS cultures after 24 h. We also show a differential effect of VIP on the expression of TNFα and IL-6 receptors, since VIP was only able to decreased expression in LPS-stimulated monocytes but not in 4/74-infected monocytes.In conclusion, we show a differential effect of VIP on human monocytes infected with virulent Salmonella or stimulated with LPS. Our study suggests that the use of VIP in bacteraemia and/or sepsis may be limited to an adjunctive therapy to antibiotic treatment.     

Inhibitory effects of dynorphin 3-14 on the lipopolysaccharide-induced toll-like receptor 4 signalling pathway

Dynorphin 1-17 (DYN 1-17) is biotransformed rapidly to a range of fragments in rodent inflamed tissue with dynorphin 3-14 (DYN 3-14) being the most stable and prevalent. DYN 1-17 has been shown previously to be involved in the regulation of inflammatory response following tissue injury, in which the biotransformation fragments of DYN 1-17 may possess similar features. This study investigated the effects of DYN 3-14 on lipopolysaccharide (LPS)-induced nuclear factor-kappaB/p65 (NF-κB/p65) nuclear translocation and the release of pro-inflammatory cytokines interleukin-1beta (IL-1β) and tumor necrosis factor-alpha (TNF-α) in differentiated THP-1 cells. Treatment with DYN 3-14 (10 nM) resulted in 35% inhibition of the LPS-induced nuclear translocation of NF-κB/p65. Furthermore, DYN 3-14 modulated both IL-1β and TNF-α release; inhibiting IL-1β and paradoxically augmenting TNF-α release in a concentration-independent manner. A number of opioids have been implicated in the modulation of the toll-like receptor 4 (TLR4), highlighting the complexity of their immunomodulatory effects. To determine whether DYN 3-14 modulates TLR4, HEK-Blue™⁻hTLR4 cells were stimulated with LPS in the presence of DYN 3-14. DYN 3-14 (10 μM) inhibited TLR4 activation in a concentration-dependent fashion by suppressing the LPS signals around 300-fold lower than LPS-RS, a potent TLR4 antagonist. These findings indicate that DYN 3-14 is a potential TLR4 antagonist that alters cellular signaling in response to LPS and cytokine release, implicating a role for biotransformed endogenous opioid peptides in immunomodulation.     

Microbial cell components induced tolerance to flagellin-stimulated inflammation through Toll-like receptor pathways in intestinal epithelial cells

In the intestine, bacterial components activate innate responses that protect the host. We hypothesize that bacterial components reduce Interleukin-8 (IL-8) production in intestinal epithelial cells stimulated by flagellin via the Toll-like receptor (TLR) signaling pathway. Caco-2 cells were pretreated with various doses of lipopolysaccharide (LPS), lipoteichoic acid (LTA), or low-dose flagellin (LDFL) for 24 h. Cells were then treated with flagellin (FL) 500 ng/ml (HDFL) for another 48 h. IL-8 production was measured in the cell culture medium by ELISA. Eighty-four genes in the TLR pathway were evaluated by RT Profiler PCR Array. Pathway Studio 8.0 software was used for altered pathway analysis. HDFL induced IL-8 production by 19-fold (p < 0.01). Pretreatment with LDFL at 20, 10 or 1 ng/ml reduced HDFL-induced IL-8 production by 61%, 52% and 40%, respectively (p < 0.05). LPS at 50 μg/ml decreased HDFL–induced IL-8 production by 38% (p < 0.05). HDFL up-regulated CXCL10, IL1B, IL-8, IRAK2, NF-κB1 and I-κB (all p < 0.05). Pathway Studio analysis showed that HDFL induced cell processes including inflammation, cell death and apoptosis. Pretreatment with LDFL at 10 ng/ml down-regulated FADD, FOS, MAP4K4, MyD88, TLR2, TLR3 and TNFERSF1A compared to HDFL (all p < 0.05). These down-regulated genes are integral for numerous cell functions including inflammatory response, cell death, apoptosis and infection. These results demonstrate that LPS and LDFL provoke tolerance to HDFL-induced IL-8 production. This tolerance effect was accompanied by a complex interaction of multiple genes related to inflammatory as well as other responses in the TLR pathway rather than a single gene alteration.     

Suppressed cytokine production in whole blood cultures may be related to iron status and hepcidin and is partially corrected following weight reduction in morbidly obese pre-menopausal women

Objective: Assess ex vivo whole-blood (WB) cytokine production and its association with iron status and serum hepcidin in obese versus non-obese women. Determine the change in ex vivo WB cytokine production 6 months after restrictive bariatric surgery in the obese group. Subjects: Seventeen obese (BMI: 46.6 ± 7.9 kg/m²) and 19 non-obese (BMI: 22.5 ± 3.0 kg/m²), pre-menopausal women; frequency matched for hemoglobin, age and race. Measurements: At baseline control and ex vivo stimulated IL-6, IL-10, IL-22, IFNγ, and TNFα from heparinized WB cultures, hemoglobin from finger-stick and transferrin receptor, hepcidin, CRP, IL-6, HOMA-IR from fasted serum samples and anthropometric parameters were assessed in the women. All parameters were reassessed 6-months following restrictive bariatric surgery in the obese women only. Results: Whole blood ex vivo LPS and ZY stimulated production of IL-6, TNFα, and IFNγ was reduced, IL-22 increased, and IL-10 was unaffected in obese compared with the non-obese women. Furthermore, ex vivo stimulated production of IL-6 and TNFα normalized, but IFNγ production remained unchanged with weight loss following restrictive bariatric surgery. In the obese women, serum transferrin receptor (a marker of iron status) and serum hepcidin were correlated with ex vivo stimulated IFNγ production at baseline. Conclusion: Ex vivo LPS and ZY stimulated cytokine production from WB cultures was altered in pre-menopausal women with morbid obesity. Significant weight loss resulted in normalization of some but not all observed alterations. Furthermore, iron status and serum hepcidin were associated with ex vivo LPS and ZY stimulated IFNγ in obesity.     

Antithrombin III,but not C1 esterase inhibitor reduces inflammatory response in lipopolysaccharide-stimulated human monocytes in an ex-vivo whole blood setting

In order to examine the immunomodulatory effects of antithrombin III (AT-III) and C1 esterase inhibitor (C1-INH) in human monocytes, we investigated the intracellular expression of interleukin (IL)-6, IL-8, and tumor necrosis factor (TNF)-α in an ex-vivo laboratory study in a whole blood setting.Heparinized whole blood samples from 23 healthy male and female volunteers (mean age: 27 ± 7 years) were pre-incubated with clinically relevant concentrations of AT-III (n = 11) and C1-INH (n = 12), then stimulated with 0.2 ng/mL lipopolysaccharide (LPS) for 3 h. After phenotyping CD14⁺ monocytes, intracellular expression of IL-6, IL-8, and TNF-α was assessed using flow cytometry. In addition, 12 whole blood samples (AT-III and C1-INH, n = 6 each) were examined using hirudin for anticoagulation; all samples were processed in the same way. To exclude cytotoxicity effects, 7-amino-actinomycin D and Nonidet P40 staining were used to investigate probes.This study is the first to demonstrate the influence of C1-INH and AT-III on the monocytic inflammatory response in a whole blood setting, which mimics the optimal physiological setting. Cells treated with AT-III exhibited significant downregulation of the proportion of gated CD14⁺ monocytes for IL-6 and IL-8, in a dose-dependent manner; downregulation for TNF-α did not reach statistical significance. There were no significant effects on mean fluorescence intensity (MFI). In contrast, C1-INH did not significantly reduce the proportion of gated CD14⁺ monocytes or the MFI regarding IL-6, TNF-α, and IL-8. When using hirudin for anticoagulation, no difference in the anti-inflammatory properties of AT-III and C1-INH in monocytes occurs.Taken together, in contrast to TNF-α, IL-6 and IL-8 were significantly downregulated in monocytes in an ex-vivo setting of human whole blood when treated with AT-III. This finding implicates monocytes as an important point of action regarding the anti-inflammatory properties of AT-III in sepsis. C1-INH was unable to attenuate the monocytic response, which supports the hypothesis that other cellular components in whole blood (e.g., neutrophils) might be responsible for the known effects of C1-INH in inflammation.     

Baicalein inhibits nuclear factor-κB and apoptosis via c-FLIP and MAPK in d-GalN/LPS induced acute liver failure in murine models

The hepatoprotective effects and molecular mechanisms of baicalein on acute liver failure induced by d-galactosamine (d-GalN)/lipopolysaccharides (LPS) were investigated in vivo. Mice were administered with different doses of baicalein (50, 100 or 150 mg/kg, p.o.) 1 h before injection of d-GalN (700 mg/kg)/LPS (10 μg/kg) and then sacrificed 6 h after treatment with d-GalN/LPS. Pretreatment with baicalein prevented d-GalN/LPS-induced liver damage by preventing associated increases of serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) and by reducing serum tumor necrosis factor α (TNF-α), nitric oxide (NO) or inducible nitric oxide synthase (iNOS) expressions. The molecular mechanisms involved in baicalein-induced inhibition of d-GalN/LPS-caused apoptosis were associated with the protection of mitochondria, increasing the Bcl-2/Bax ratio, blocking the release of cytochrome c, and suppressing the phosphorylation of IκBα, ERK and JNK. Moreover, baicalein activated c-FLIP_L, XIAP and cIAP2 proteins, potentially blocking the recruitment of NF-κB signaling molecules. The results support the investigation of baicalein as a therapeutic candidate for acute liver apoptosis or injury and indicate that baicalein might inhibit liver apoptosis by mediating one or more of these pathways.     

Elevation in liver enzymes is associated with increased IL-2 and predicts severe outcomes in clinically apparent dengue virus infection

The objective of the present study was to assess the circulating TNF-α and IL-2 levels in dengue virus (DENV) infected patients and to correlate these with clinical severity of DENV infections. A single analyte quantitative immunoassay was used to detect TNF-α and IL-2 in 24 dengue fever (DF) and 43 dengue haemorrhagic fever (DHF) patients, 15 healthy adults and 6 typhoid patients. The mean TNF-α and IL-2 levels of DENV- infected patients were higher than that of healthy adults and typhoid patients. No significant difference in TNF-α levels was noted between DF and DHF patients (p = 0.5) but a significant increase in IL-2 levels was observed in DHF compared with DF patients (mean of DF = 59.7 pg/mL, mean of DHF = 166.9 pg/mL; p = 0.02). No significant association of TNF-α or IL-2 levels was noted with packed cell volume (>45), thrombocytopenia, leucopenia or the presence of viraemia. The liver function tests measuring AST (aspartate aminotransferase) (p = 0.01) and ALT (alanine aminotransferase) (p = 0.02) levels were significantly elevated in DENV-infected patients. AST:ALT was significantly elevated in DHF/DSS (dengue shock syndrome) compared with DF patients. A significant positive linear correlation was noted between AST and IL-2 (r = 0.31; p = 0.01) and ALT and IL-2 elevations (r = 0.2; p = 0.02). Thus, AST and ALT levels correlate with both disease severity and circulating IL-2 levels. We suggest a role for circulating IL-2 in liver dysfunction and propose that a combined assessment of AST/ALT in conjunction with IL-2 at the early stages of symptomatic DENV infection may be useful to predict the severe forms of dengue.     

Tetracyclines downregulate the production of LPS-induced cytokines and chemokines in THP-1 cells via ERK,p38, and nuclear factor-κB signaling pathways

Recent reports have shown that antibiotics such as macrolide, aminoglycoside, and tetracyclines have immunomodulatory effects in addition to essential antibiotic effects. These agents may have important effects on the regulation of cytokine and chemokine production. However, the precise mechanism is unknown. This time, we used Multi Plex to measure the production of cytokines and chemokines following tetracycline treatment of lipopolysaccharide (LPS)-induced THP-1 cells. The signaling pathways were investigated with Western blotting analysis. Three tetracyclines significantly suppressed the expression of cytokines and chemokines induced by LPS. Minocycline (50 μg/ml), tigecycline (50 μg/ml), or doxycycline (50 μg/ml) were added after treatment with LPS (10 μg/ml). Tumor necrosis factor-α was downregulated to 16%, 14%, and 8%, respectively, after 60 min compared to treatment with LPS without agents. Interleukin-8 was downregulated to 43%, 32%, and 26% at 60 min. Macrophage inflammatory protein (MIP)-1α was downregulated to 23%, 33%, and 16% at 120 min. MIP-1β was downregulated to 21%, 11%, and 2% at 120 min. Concerning about signaling pathways, the mechanisms of the three tetracyclines might not be the same. Although the three tetracyclines showed some differences in the time course, tetracyclines modulated phosphorylation of the NF-κB pathway, p38 and ERK1/2/MAPK pathways, resulting in inhibition of cytokine and chemokine production. In addition, SB203580 (p38 inhibitor) and U0126 (ERK1/2 inhibitor) significantly suppressed the expression of TNF-α and IL-8 in LPS-stimulated THP-1 cells. And further, the NF-κB inhibitor, BAY11-7082, almost completely suppressed LPS-induced these two cytokines production. Thus, more than one signaling pathway may be involved in tetracyclines downregulation of the expression of LPS-induced cytokines and chemokines in THP-1 cells. And among the three signaling pathways, NF-κB pathway might be the dominant pathway on tetracyclines modification the LPS-induced cytokines production in THP-1 cells. In general, minocycline and doxycycline suppressed the production of cytokines and chemokines in LPS-stimulated THP-1 cell lines via mainly the inhibition of phosphorylation of NF-κB pathways. Tigecycline has the same structure as the other tetracyclines, however, it showed the different properties of cytokine modulation in the experimental time course.     

Hepatoprotective effect of cryptotanshinone from Salvia miltiorrhiza in d-galactosamine/lipopolysaccharide-induced fulminant hepatic failure

Cryptotanshinone from Salvia miltiorrhiza Bunge was investigated for hepatoprotective effects in d-galactosamine (GalN)/lipopolysaccharide (LPS)-induced fulminant hepatic failure. Cryptotanshinone (20 or 40 mg/kg) was orally administered 12 and 1 h prior to GalN (700 mg/kg)/LPS (10 μg/kg) injection. The increased mortality and TNF-α levels by GalN/LPS were declined by cryptotanshinone pretreatment. In addition, cryptotanshinone attenuated GalN/LPS-induced apoptosis, characterized by the blockade of caspase-3, -8, and -9 activation, as well as the release of cytochrome c from the mitochondria. In addition, cryptotanshinone significantly suppressed JNK, ERK and p38 phosphorylation induced by GalN/LPS, and phosphorylation of TAK1 as well. Furthermore, cryptotanshinone significantly inhibited the activation of NF-κB and suppressed the production of proinflammatory cytokines. These findings suggested that hepatoprotective effect of cryptotanshinone is likely associated with its anti-apoptotic activity and the down-regulation of MAPKs and NF-κB associated at least in part with suppressing TAK1 phosphorylation.     

Effects of PEGylated porcine glucagon-like peptide-2 therapy in weaning piglets challenged with lipopolysaccharide

This study aims to evaluate the therapeutic effect of polyethylene glycosylated porcine glucagon-like peptide-2 (pGLP-2), a long-acting form of pGLP-2, in lipopolysaccharide (LPS)-challenged piglets. Eighteen 21-day-old weaning piglets were randomly assigned into three groups: control (saline solution), LPS (100 μg/kg LPS), and PEG–pGLP-2 (10 nmol/kg PEG–pGLP-2 + 100 μg/kg LPS). All treatments were administered intraperitoneally. Compared with the control treatment, LPS treatment significantly decreased (P < 0.05) the villus heights of the duodenum and jejunum, as well as the villus height/crypt depth ratio of the jejunum. However, PEG–pGLP-2 therapy reduced these effects (P > 0.05). Specifically, PEG–pGLP-2 infusion significantly increased the villus height/crypt depth ratio of the duodenum (P < 0.05) compared with LPS treatment. Compared with the control treatment, LPS treatment significantly increased (P < 0.05) the mRNA expression levels of interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) in the jejunum. However, PEG–pGLP-2 therapy reduced these effects (P < 0.05). Specifically, PEG–pGLP-2 infusion significantly decreased (P < 0.05) the mRNA expression levels of interleukin (IL)-8 and TNF-α in the duodenum and jejunum, IL-10 in the duodenum, and IFN-γ in the jejunum compared with the LPS treatment. LPS treatment increased the caspase-3 activity of the ileum mucosal (P < 0.05), and this effect was significantly reduced by PEG–pGLP-2 treatment. These results indicate that PEG–pGLP-2 infusion alleviates the severity of intestinal injury in weaning piglets by reducing the secretion of inflammatory cytokines and the caspase-3 activity, and increasing the villus height/crypt depth ratio.     

Fish-oil-derived n-3 polyunsaturated fatty acids reduce NLRP3 inflammasome activity and obesity-related inflammatory cross-talk between adipocytes and CD11b+ macrophages

Adipocyte–macrophage cross-talk propagates immune responses in obese adipose tissue (AT). Long-chain n-3 polyunsaturated fatty acids (LC n-3 PUFA) mitigate inflammation, partly through up-regulation of adiponectin; however, specific mechanisms are unclear. We determined if adipocyte–macrophage cross-talk could be mitigated by dietary LC n-3 PUFA and if this was dependent on adiponectin-mediated signaling. We utilized an in vitro co-culture model mimicking the ratio of adipocytes:macrophages in obese AT, whereby 3T3-L1 adipocytes were co-cultured with splenic CD11b⁺ macrophages from C57BL/6 mice fed high-fat control (HF-CON; 34% w/w fat) or fish oil diets (HF-FO; 34% w/w fat containing 7.6% w/w FO), as well as mice fed low-fat control (LF-CON; 10% w/w fat) or FO diets (LF-FO; 10% w/w fat containing 3% w/w FO). Co-culture conditions tested effects of soluble mediator-driven mechanisms (trans-well system), cell contact and low-dose lipopolysaccharide (LPS) mimicking acute or chronic inflammatory conditions. HF-FO macrophages from acute LPS-stimulated trans-well co-cultures had decreased mRNA expression of Casp1, Il1β and Il18, as well as cellular caspase-1 activity compared to HF-CON macrophages (P ≤ .05). Moreover, adipocytes from acute LPS-stimulated HF-FO co-cultures had decreased caspase-1 activity and decreased IL-1β/IL-18 levels following chronic LPS pretreatment compared to HF-CON co-cultures (P ≤ .05). Additionally, in contact co-cultures with adiponectin-neutralizing antibody, the FO-mediated modulation of NFκB activity and decrease in phosphorylated p65 NFκB, expression of NLRP3 inflammasome genes, M1 macrophage marker genes and inflammatory cytokine/chemokine secretion were controlled partly through adiponectin, while cellular caspase-1 activity and IL-1β/1L-18 levels were decreased independently of adiponectin (P ≤ .05). LC n-3 PUFA may decrease the intensity of adipocyte–macrophage cross-talk to mitigate obesity-associated pathologies.     

Interleukin-10 modulates pro-apoptotic effects of TNF-α in human articular chondrocytes in vitro

The aim of this study is to determine if there is an antagonistic effect between tumour necrosis factor (TNF)-α and the immunoregulatory interleukin (IL)-10 on chondrocytes survival. Serum-starved primary human articular chondrocytes were stimulated with either 10 ng/ml recombinant TNF-α, IL-10 or a combination of both (at 10 ng/ml each). Chondrocyte apoptosis was determined by measuring caspase-3/7, -8 and -9 activities using caspase assays. Mitochondrial apoptotic inducer bax, and the suppressor bcl-2 were evaluated using western blotting at 48 h. Results indicated that TNF-α increased caspase activities and resulted in a significant (p = 0.001) increase in bax/bcl-2 ratio. Stimulation with IL-10 did not alter caspase activities, while co-treatment with IL-10 and TNF-α inhibited TNF-α induced caspase activities and significantly (p > 0.004) impaired bax/bcl-2 ratio. At 24 h, mRNA levels for collagen type II, TNF-α and IL-10 were determined using real-time RT-PCR. Stimulation with TNF-α or TNF-α and IL-10 significantly inhibited collagen type II and increased IL-10 and TNF-α mRNA expression. IL-10 modulated the pro-apoptotic capacity of TNF-α in chondrocytes as shown by the decrease in caspase activities and bax/bcl-2 ratio compared to TNF-α stimulated chondrocytes, suggesting a mostly antagonistic interplay of IL-10 and TNF-α on mitochondrial apoptotic pathways.     

A semi-synthetic derivative of artemisinin,artesunate inhibits prostaglandin E2 production in LPS/IFNγ-activated BV2 microglia

Artesunate is a semi-synthetic derivative of artemisinin used to treat malaria, and has been shown to possess anti-inflammatory activity. In this study, we have investigated the effect of artesunate on PGE₂ production/COX-2 protein expression in LPS + IFNγ-activated BV2 microglia. To further understand the mechanism of action of this compound, we investigated its interference with NF-κB and p38 MAPK signalling pathways. PGE₂ production was determined using EIA, while protein expressions of inflammatory targets like COX-2, mPGES-1, IκB, p38 and MAPKAPK2 were evaluated using western blot. An NF-κB-bearing luciferase reporter gene assay was used to test the effect of artesunate on NF-κB-mediated pro-inflammatory gene expression in HEK293 cells stimulated with TNFα (1 ng/ml). Artesunate (2 and 4 μM), significantly (p <0.01) suppressed PGE₂ production in LPS + IFNγ-activated BV2 microglia. This effect was found to be mediated via reduction in COX-2 and mPGES-1 proteins. Artesunate also produced significant inhibition of TNFα and IL-6 production in activated BV2 microglia. Further investigations showed that artesunate (0.5–4 μM) significantly (p <0.001) reduced NF-κB-driven luciferase expression, and inhibited IκB phosphorylation and degradation, through inhibition of IKK. Artesunate inhibited phosphorylation of p38 MAPK and its substrate MAPKAPK2 following stimulation of microglia with LPS + IFNγ. Taken together, we have shown that artesunate prevents neuroinflammation in BV2 microglia by interfering with NF-κB and p38 MAPK signalling.     

Impact of microbial tolerance in persistent secondary Klebsiella pneumoniae peritonitis

Purpose and methodsMicrobial tolerance represents a diminished pro-inflammatory response following repeated stimulation by a host of pathogen-associated molecular patterns (PAMPs) of varying origins. Toll-like receptors (TLRs) have been centrally implicated in the development of tolerance. The purpose of this study was to investigate the impact of tolerance in a previously described murine model.C57BL/6 mice were pretreated intraperitoneally with phosphate buffered saline (PBS), heat-killed Klebsiella 2 × 10⁸ CFU (hkKlebsiella), LPS 10 mg/kg (LPS 10), or BLP 10 mg/kg (BLP 10). Following pretreatment, peritonitis was induced 24 h later using 10³ intraperitoneal Klebsiella CFU. Peritoneal concentrations of TNF-α, IL-10 and nitric oxide (NO), as well as characteristic cell patterns, were determined. Long-term consequences of microbial tolerance were assessed by measuring survival and weight-loss.ResultsFollowing in vitro stimulation with Klebsiella 10⁵ and 10³ CFU, TNF-α and IL-10 secretion were diminished in macrophages harvested from mice pretreated with hkKlebsiella, LPS 10 and BLP 10. Pretreated animals had significantly lower bacterial counts. Conversely, local NO levels were elevated. Survival was not different between the groups.ConclusionPretreatment with TLR ligands induced microbial tolerance, with reduced peritoneal cytokine concentrations and enhanced early bacterial clearance. However, this did not translate into improved survival.     

Esculentin-2CHa: A host-defense peptide with differential cytotoxicity against bacteria,erythrocytes and tumor cells

The host-defense peptide, esculentin-2CHa (GFSSIFRGVA¹⁰KFASKGLGK D²⁰LAKLGVDLVA³⁰ CKISKQC) shows potent (MIC ≤ 6 μM) growth inhibitory activity against clinical isolates of multidrug-resistant strains of Staphylococcus aureus, Acinetobacter baumannii, and Stenotrophomonas maltophilia and differential cytotoxic activity against human erythrocytes (LC₅₀ = 150 μM) and human non-small cell lung adenocarcinoma A549 cells (LC₅₀ = 10 μM). Esculentin-2CHa significantly (P < 0.01) stimulates the release of the anti-inflammatory cytokine IL-10 by mouse lymphoid cells and elevates its production after stimulation with concanavalin A and significantly (P < 0.05) stimulates TNF-α production by peritoneal macrophages. Effects on IL-6 and IL-1β production were not significant. Removal of the hydrophobic N-terminal hexapeptide (GFSSIF) from esculentin-2CHa results in abolition of growth inhibitory activity against S. aureus and cytotoxic activity against erythrocytes and A549 cells as well as a marked (≥16-fold) reduction in potency against A. baumannii and S. maltophilia. The primary structure of esculentin-2 has been poorly conserved between frog species but evolutionary pressure has acted to maintain the hydrophobic character of this N-terminal hexapeptide sequence. Removal of the cyclic C-terminal domain (CKISKQC) and replacement of the Cys³¹ and Cys³⁷ residues by serine resulted in appreciable decreases in cytotoxicity against all microorganisms and against mammalian cells. The more cationic [D20K, D27K] analog showed a modest increase in potency against all microorganisms (up to 4-fold) but a marked increase in cytotoxicity against erythrocytes (LC₅₀ = 11 μM) and A549 cells (LC₅₀ = 3 μM).     

Greater impact of dietary fat manipulation than apolipoprotein E genotype on ex vivo cytokine production – Insights from the SATgenε study

Apolipoprotein E (APOE) genotype is believed to play an important role in cardiovascular risk. APOE4 carriers have been associated with higher blood lipid levels and a more pro-inflammatory state compared with APOE3/E3 individuals. Although dietary fat composition has been considered to modulate the inflammatory state in humans, very little is known about how APOE genotype can impact on this response. In a follow-up to the main SATgenε study, we aimed to explore the effects of APOE genotype, as well as, dietary fat manipulation on ex vivo cytokine production. Blood samples were collected from a subset of SATgenε participants (n = 52/88), prospectively recruited according to APOE genotype (n = 26 E3/E3 and n = 26 E3/E4) after low-fat (LF), high saturated fat (HSF) and HSF with 3.45 g docosahexaenoic acid (DHA) dietary periods (each diet eight weeks in duration assigned in the same order) for the measurement of ex vivo cytokine production using whole blood culture (WBC). Concentrations of IL-1beta, IL-6, IL-8, IL-10 and TNF-alpha were measured in WBC supernatant samples after stimulation for 24 h with either 0.05 or 1 μg/ml of bacterial lipopolysaccharide (LPS). Cytokine levels were not influenced by genotype, whereas, dietary fat manipulation had a significant impact on TNF-α and IL-10 production; TNF-α concentration was higher after consumption of the HSF diet compared with baseline and the LF diet (P < 0.05), whereas, IL-10 concentration was higher after the LF diet compared with baseline (P < 0.05). In conclusion, our study has revealed the amount and type of dietary fat can significantly modulate the production of TNF-α and IL-10 by ex vivo LPS-stimulated WBC samples obtained from normolipidaemic subjects.     

Acute high-dose sodium selenite administration improves intestinal microcirculation without affecting cytokine release in experimental endotoxemia

We evaluated the effects of acute high-dose sodium selenite (SEL) administration on the intestinal microcirculation and the release of the cytokines TNF-α, IL-1β, IL-6 and IL-10 in experimental endotoxemia (induced by lipopolysaccharide-LPS). Three groups of animals (n=30) were studied: control group, endotoxemic group (15 mg kg^?1 i.v. LPS from E. coli) and SEL-treated LPS group (100 μg kg^?1 SEL i.v.). SEL treatment resulted in a significant reduced number of firmly adhering leukocytes in intestinal submucosal venules and reduced significantly the impairment of the intestinal functional capillary density. Despite of the improvement of microcirculatory parameters, we did not detect any changes in the pattern of cytokine release. In conclusion, administration of high-dose sodium SEL attenuates leukocyte adhesion and improves capillary perfusion within the intestinal microcirculation without affecting release of the cytokines TNF-α, IL-1β, IL-6 and IL-10 in experimental endotoxemia.     

Rapid cloning,expression and purification of a novel high-activity alkaline phosphatase with detoxification of lipopolysaccharide

Lipopolysaccharide (LPS) is a bacterial endotoxin leading to endotoxemia. Its virulence factor ‘diphosphoryl lipid A’ can be abolished by alkaline phosphatase (AP). A novel AP gene (without introns) was cloned from Saccharomyces boulardii ATCC MYA-796 with a GenBank accession number KF471017, and the recombinant AP (rAP) was expressed as a soluble protein in Pichia pastoris X-33 with a yield of 43.66 mg/l at the end of 120 h of induction in a shaker flask. After purification by affinity-column chromatography, the purity of rAP was over 90%. The optimal reaction conditions of rAP were pH 9.6, temperature at 60 °C and 2 mM Mg²⁺ in diethanolamine buffer, and EDTA was a potent inhibitor of rAP activity. The specific activity of rAP was 9912.01 U/mg under the optimal conditions. Furthermore, rAP showed a broad dephosphorylation activity to LPS over a broad pH range (pH 2–10) in vitro and peaked at pH 4 in Tris–HCl buffer. After LPS dephosphorylated by rAP was injected intraperitoneally into mice, the serum level of tumor necrosis factor (TNF)-α was significantly reduced compared to that of the LPS group (p < 0.01). These findings suggest that rAP has great potential to cure diseases caused by LPS.