MicroRNA 181a-2-3p Alleviates the Apoptosis of Renal Tubular Epithelial Cells via Targeting GJB2 in Sepsis-Induced Acute Kidney Injury.

Acute kidney injury (AKI) is the most common complication of sepsis. MicroRNAs (miRNAs) play important roles in the sepsis-induced AKI. This paper aimed to explore the role of miRNA 181a-2-3p (miR-181a-2-3p) in the sepsis-induced AKI and the underlying mechanism. Our results revealed that miR-181a-2-3p showed low expression levels in patients with sepsis and mouse models undergoing cecal ligation and puncture (CLP). The addition of miR-181a-2-3p antagonists aggravated the sepsis-induced kidney injuries and inflammatory response in CLP mouse models, as suggested by hematoxylin and eosin (H&E) staining and quantitative real-time PCR (qRT-PCR). In addition, miR-181a-2-3p mimic alleviated the lipopolysaccharide (LPS)-induced inflammatory response, along with apoptosis of TCMK-1. Moreover, results from the GSE46955 data set indicated that GJB2 was highly expressed in septic patients but lowly expressed after recovery. Further, the dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay were carried out, which confirmed that GJB2 was a target of miR-181a-2-3p, and overexpression of GJB2 reversed the anti-inflammatory and antiapoptotic effects of miR-181a-2-3p mimic on the LPS-induced sepsis cell models. In conclusion, miR-181a-2-3p alleviates the inflammatory response and cell apoptosis of septic patients and animal models by upregulating GJB2 expression, which may provide a new therapeutic strategy for sepsis.

Anti-inflammatory potential of invasive sun corals (Scleractinia: Tubastraea spp.) from Brazil: alternative use for management?

OBJECTIVES: The objective was to analyse the anti-inflammatory potential of the invasive coral species Tubastraea coccinea and Tubastraea tagusensis. METHODS: Methanolic extracts, fractions and synthesized compounds were evaluated for their anti-inflammatory ability, and their composition was elucidated through chemical analysis. KEY FINDINGS: The genus Tubastraea (Order Scleractinia, Family Dendrophylliidae) (known as sun corals) presents compounds with pharmacological value. The introduction of these azooxanthellate hard corals into Brazil, initially in Rio de Janeiro state, occurred through their fouling of oil and gas platforms from the Campos oil Basin. The two invasive species have successfully expanded along the Brazilian coast and threaten endemic species and biodiversity. The HPLC-MS and GC-MS data suggest the presence of aplysinopsin analogues (alkaloids). Anti-inflammatory activity was observed in all samples tested in in-vivo assays, especially in T. coccinea. The ethyl acetate fraction from this sample was more effective in in-vitro assays for anti-inflammatory activity. Depending on the concentration, this fraction showed cytotoxic responses. CONCLUSIONS: These species have potential pharmacological use, and considering their invasive nature, this study presents a potential alternative use, which may enhance the management of this biological invasion.

Toll-Like Receptor 4 Signaling Licenses the Cytosolic Transport of Lipopolysaccharide From Bacterial Outer Membrane Vesicles.

Outer membrane vesicles (OMVs), released by variety of bacteria, are membrane-enclosed entities enriched in microbial components, toxins, and virulent factors. OMVs could deliver lipopolysaccharide (LPS) into the cytosol of host cells and subsequently activate caspase-11, which critically orchestrates immune responses and mediates septic shock. Although it is known that caspase-11 is activated by intracellular LPS, how OMVs deliver LPS into the cytosol remains largely unknown. Here we show that the activation of toll-like receptor 4 (TLR4), a LPS receptor on the cytoplasmic membrane, licenses macrophages to transport LPS from OMVs into the cytosol through TIR domain-containing adaptor-inducing interferon-ß (TRIF). TRIF-mediated cytosolic delivery of LPS from OMVs depends on the production of type 1 interferon and the expression of guanylate-binding proteins (GBPs). Deletion of TRIF or GBPs prevents pyroptosis and lethality induced by OMVs or OMVs-releasing Escherichia coli. Together, these findings provide novel insight into how host coordinates extracellular and intracellular LPS sensing to orchestrate immune responses during gram-negative bacterial infection.

Prolyl hydroxylases positively regulated LPS-induced inflammation in human gingival fibroblasts via TLR4/MyD88-mediated AKT/NF-κB and MAPK pathways.

OBJECTIVES: Prolyl hydroxylases (PHDs) play essential roles in oxygen-sensing system, whereas the effects of PHDs on inflammation have not been totally uncovered. Our study aimed to investigate the role of PHDs in lipopolysaccharide (LPS)-induced inflammation of human gingival fibroblasts (HGFs) and clarify the potential mechanisms. MATERIALS AND METHODS: A pan hydroxylase inhibitor, dimethyloxallyl glycine (DMOG), and RNA interference were used to explore the role of PHDs in inflammation. Cytotoxic effect of DMOG was determined by cell-counting kit-8 and flow cytometry respectively. The secretion levels of IL-6 and IL-8 were assessed by ELISA. The mRNA levels of inflammatory cytokines, Toll-like receptor (TLR) 4 and MyD88 were evaluated by quantitative real-time PCR. The activation of NF-κB, mitogen-activated protein kinase (MAPK) and PI3K/AKT pathways were detected by western blot and the nuclear translocation of NF-κB p65 was examined by immunofluorescence. Downregulation of PHD1 and PHD2 was performed with siRNA transfection. RESULTS: Dimethyloxallyl glycine inhibited LPS-induced inflammatory cytokine, TLR4 and MyD88 expression in gene level and the elevated secretion of IL-6 and IL-8 was also downregulated. Additionally, LPS-induced activation of NF-κB, MAPK and AKT pathways was abolished by DMOG treatment. Importantly, LPS-induced inflammatory cytokine expression was merely suppressed by PHD2 knockdown. CONCLUSIONS: Prolyl hydroxylases acted as a positive regulator in LPS-induced inflammation of HGFs via TLR4/MyD88-mediated NF-κB, MAPK and AKT signalling pathways and PHD2 among three isoforms was principally responsible for the effects.

Distinct pattern of immune tolerance in dendritic cells treated with lipopolysaccharide or lipoteichoic acid.

Cytokine induction is often critical for the host defense during acute immune responses while, if not tightly regulated, it may cause an immunological pathology coincident with tissue damage. Despite the fact that gram-positive bacterial infection has become increasingly prevalent, immune modulation induced by lipoteichoic acid (LTA), the major cell wall component of gram-positive bacteria has not been studied thoroughly at the cellular level. In the current study, tolerance induction in mouse bone marrow-derived dendritic cells (BMDCs) treated with single or repeated stimulation of Staphylococcus aureus LTA was compared with those of Escherichia coli lipopolysaccharide (LPS). The results showed that repeated LTA stimulation significantly suppressed pro-inflammatory cytokine (TNF-α and IL-6) production in BMDCs, comparable to that of LPS, but with less extent, down-regulated IL-10 and enhanced the inhibitory molecule, LAG-3-associated protein (LAP). Furthermore, we observed a sustained expression of unique negative regulators, Toll interacting protein (TOLLIP) and Indoleamine 2,3-dioxygenase (IDO), in BMDCs treated with LTA. A transient hyporesponsiveness period appeared when DCs were treated repeatedly with LTA or LPS showing a distinctive pattern. Intriguingly, LPS exposure induced cross tolerance to LTA while LTA exposure did not to LPS, implicating that a distinct signaling components are involved in response to LTA. Collectively, a distinct immune regulation appeared to be responsible for the LPS- and LTA-induced tolerance on cytokine production, expression of surface markers and intracellular proteins.

Retinoic acid-mediated anti-inflammatory responses in equine immune cells stimulated by LPS and allogeneic mesenchymal stem cells.

Retinoic acid (RA), an active metabolite of vitamin A, has shown potential therapeutic immunomodulatory properties. Allogeneic mesenchymal stem cells (MSCs)-based therapy is an effective approach to induce tissue healing and regeneration in many equine orthopedic conditions. However, MSCs-based therapies induced inflammatory responses in vivo. This study aimed to: 1. Determine the effect of RA cell culture treatment on inflammatory responses of lipopolysaccharides (LPS)- and allogeneic MSCs-stimulated peripheral blood mononuclear cells (PBMCs). 2. Determine the effect of RA on stimulated MSCs viability and morphology. Allogeneic MSCs-stimulated PBMCs had significant decreases in the anti-inflammatory cytokines (IL-10, IL-1ra, TGF-ß1), increases in the pro-inflammatory mediators (IL-1ß, IL-6, TNF-α, SAA), and increases of CD14 and MHC II percent positive cells compared to LPS- and non-stimulated PBMCs. Retinoic acid treatment of LPS- and allogeneic MSCs-stimulated PBMCs counterbalanced the induced inflammatory responses. Moreover, RA significantly improved the viability and morphology of stimulated MSCs. These findings highlighted the potential complications of equine allogeneic MSCs-based therapies and the immuno-modulatory effect of RA on equine stimulated cells. In conclusion, the use of RA to ameliorate allogeneic MSCs therapy associated inflammation may offer advantages that would require further investigations.

The cell wall component lipoteichoic acid of Staphylococcus aureus induces chemokine gene expression in bovine mammary epithelial cells.

Staphylococcus aureus (SA) is a major cause of bovine mastitis, but its pathogenic mechanism remains poorly understood. To evaluate the role of lipoteichoic acid (LTA) in the immune or inflammatory response of SA mastitis, we investigated the gene expression profile in bovine mammary epithelial cells stimulated with LTA alone or with formalin-killed SA (FKSA) using cap analysis of gene expression. Seven common differentially expressed genes related to immune or inflammatory mediators were up-regulated under both LTA and FKSA stimulations. Three of these genes encode chemokines (IL-8, CXCL6 and CCL2) functioning as chemoattractant molecules for neutrophils and macrophages. These results suggest that the initial inflammatory response of SA infection in mammary gland may be related with LTA induced chemokine genes.

Characteristics of Lung Injury in Rats after Inhalation of Ultradisperse Bacterial LPS in Lipophilic and Hydrophilic Phases.

We compared cellular and tissue reactions of the lungs in Wistar rats after inhalation of LPS from E. coli 0128: B12 in the form of ultradispersion in water-salt phase and lipid phase. The estimated dose of the toxin was 200 mg/kg in both series. The content of LPS and TNF-α in lung homogenates, bronchoalveolar fluid, and blood serum was determined 3, 8, and 24 h after inhalation, morphological changes in the lungs were analyzed. Inhalation of LPS in the lipid phase was accompanied by its less intensive accumulation in the lung tissue and bloodstream, and more pronounced accumulation in the bronchoalveolar fluid as well as less intense release of TNF-α in comparison with the effects of LPS in water-salt phase. Macrophage reaction, leukocyte infiltration of the interalveolar septa, and reduction of α-antitrypsin in the lung tissue was more pronounced in series with inhalation with LPS ultradispersions in water-salt phase and lipid phase, and damage to the alveolar epithelium, in the series with the inhalation of the lipid phase.

Antigen presenting cells targeting and stimulation potential of lipoteichoic acid functionalized lipo-polymerosome: a chemo-immunotherapeutic approach against intracellular infectious disease.

Antigen presenting cells (APC) are well-recognized therapeutic targets for intracellular infectious diseases, including visceral leishmaniasis. These targets have raised concerns regarding their potential for drug delivery due to overexpression of a variety of receptors for pathogen associated molecular pathways after infection. Since, lipoteichoic acid (LTA), a surface glycolipid of Gram-positive bacteria responsible for recognition of bacteria by APC receptors that also regulate their activation for pro-inflammatory cytokine secretion, provides additive and significant protection against parasite. Here, we report the nanoarchitechture of APC focused LTA functionalized amphotericin B encapsulated lipo-polymerosome (LTA-AmB-L-Psome) delivery system mediated by self-assembly of synthesized glycol chitosan-stearic acid copolymer (GC-SA) and cholesterol lipid, which can activate and target the chemotherapeutic agents to Leishmania parasite resident APC. Greater J774A and RAW264.7 macrophage internalization of FITC tagged LTA-AmB-L-Psome compared to core AmB-L-Psome was observed by FACSCalibur cytometer assessment. This was further confirmed by higher accumulation in macrophage rich liver, lung and spleen during biodistribution study. The LTA-AmB-L-Psome overcame encapsulated drug toxicity and significantly increased parasite growth inhibition beyond commercial AmB treatment in both in vitro (macrophage-amastigote system; IC50, 0.082 ± 0.009 µg/mL) and in vivo (Leishmania donovani infected hamsters; 89.25 ± 6.44% parasite inhibition) models. Moreover, LTA-AmB-L-Psome stimulated the production of protective cytokines like interferon-γ (IFN-γ), interleukin-12 (IL-12), tumor necrosis factor-α (TNF-α), and inducible nitric oxide synthase and nitric oxide with down-regulation of disease susceptible cytokines, like transforming growth factor-ß (TGF-ß), IL-10, and IL-4. These data demonstrate the potential use of LTA-functionalized lipo-polymerosome as a biocompatible lucrative nanotherapeutic platform for overcoming toxicity and improving drug efficacy along with induction of robust APC immune responses for effective therapeutics of intracellular diseases.

Distress do paciente oncológico: prevalência e fatores associados na opinião de familiares / Distress of cancer patients: prevalence and associated factors in the opinion of family / Angustia de los pacientes con cáncer: prevalencia y factores asociados en la opinión de la familia

Este estudo verificou a prevalência e fatores associados ao distress de pacientes oncológicos, na opinião de familiares. Foram entrevistados 140 familiares responsáveis pelo cuidado de pacientes com câncer. O Termômetro de Distress foi adaptado para uso em familiares. Estes consideraram que 72,9% dos pacientes estavam com distress relacionado a preocupações (80,4%), nervosismo (78,4%), tristeza (74,5%), dor (67,6%), fadiga (67,6%) e problemas com alimentação (57,8%). Modelos de regressão logística hierárquica mostraram que, enquanto familiares do sexo masculino (OR=0,025) e idades mais avançadas (OR=0,006 a 0,059) tiveram menor risco de perceber o distress, indivíduos protestantes, comparados a católicos, tiveram chance 12,77 vezes maior de percebê-lo. Quanto aos fatores associados, nervosismo (OR=10,8) contribuiu significativamente mais com a percepção de distress pelos familiares quando comparado a fadiga (OR=3,38) ou ter plano de saúde privado (OR=2,55). Familiares podem ser grandes aliados na avaliação e acompanhamento do distress de pacientes com câncer.

The study aimed to verify the opinion of family members about distress on cancer patients and the factors associated with it. Interviews with 140 family members of cancer patients were conducted. The Distress Thermometer was adapted to be used with family members. Approximately 72.9% of patients were considered in distress, related to concern (80.4%), nervousness (78.4%), sadness (74.5%), pain (67.6%), fatigue (67.6%) and problems with eating (57.8%). The hierarchical logistic regression models showed that while male (OR=0.025) and older ages (OR=0.006 to 0.059) had lower risk of perceiving the distress, individuals Protestants, compared to Catholics, were 12.77 times more likely to perceive it. About the associated factors, nervousness (OR=10.8) contributed significantly more to the perception of distress for family members when compared to fatigue (OR=3.38) or have private health insurance (OR=2.55). Family can be great allies in the evaluation and monitoring of distress in patients with cancer.

Este estudio examinó la prevalencia y los factores asociados con el distress de los pacientes de cáncer, de acuerdo con los familiares. Fueran entrevistados 140 cuidadores familiares de pacientes con cáncer. El Termómetro de Distress fue adaptado para el uso en los familiares. Ellos encontraron que el 72,9% de los pacientes estaban con distress relacionado con preocupaciones (80,4%), nerviosismo (78,4%), tristeza (74,5%), dolor (67,6%), fatiga (67, 6%) y problemas con la alimentación (57,8%). Modelos de regresión logística jerárquica mostraran que, mientras los familiares de sexo masculino (OR=0,025) y de edades más avanzadas (OR=0,006 a 0,059) tuvieron un menor riesgo de percibir el distress, los individuos protestantes, comparados a los católicos, tuvieron oportunidad 12,77 veces mayor para percibirlo. En cuanto a los factores asociados, el nerviosismo (OR=10,8) contribuyó significativamente más a la percepción del distress de los familiares, en comparación con la fatiga (OR=3,38) o tener un seguro de salud privado (OR=2,55). Familiares pueden ser grandes aliados en la evaluación y el seguimiento de sufrimiento en los pacientes con cáncer.

Deoxynivalenol and E.coli lipopolysaccharide alter epithelial proliferation and spatial distribution of apical junction proteins along the small intestinal axis.

We investigated a proposed synergistic effect of deoxynivalenol (DON) and lipopolysaccharides (LPS) on small intestinal architecture and epithelial barrier integrity in pigs. Crypt depth and intestinal cell proliferation were analyzed, as well as expression of zonula occludens protein-1 (ZO-1) and ß-catenin of the apical junction complex along the small intestine. Barrows (26.2±4.1 kg) were fed restrictedly either a control diet (CON) or a diet naturally contaminated with 3.1 mg DON/kg feed (DON) for 37 d. At d 37, the control group was infused for 1 h either with 100 µg/kg BW of DON (CON-DON, n=6), 7.5 µg/kg BW of LPS (CON-LPS, n=6), a combination of both (CON-DON+LPS, n=7), or 0.9% NaCl (CON-CON, n=6) and the DON group with 7.5 µg/kg BW of LPS (DON-LPS, n=8) or 0.9% NaCl (DON-CON, n=6). Pigs were euthanized 3.25 h after start of infusion. Immunohistochemistry (5'-bromo-2'-deoxyuridine for proliferation) and immunofluorescence (ZO-1 and ß-catenin) from duodenum, proximal jejunum, mid-jejunum, proximal ileum, and terminal ileum were analyzed for crypt depth, cell proliferation, and apical junction proteins. Duodenal crypts were deeper compared with the other 4 intestinal regions, and proximal jejunal crypts were deeper than those of mid-jejunum and proximal ileum (P<0.001). Epithelial proliferation showed a bell-shaped distribution along the small intestinal axis. Duodenal proliferating cells had the least number compared with jejunal sections and proximal ileum (P<0.001). Neither DON nor LPS affected these variables. Zonula occludens-1 displayed a distinct spatial distribution in the epithelium with an apical and a cytosolic component. Apical expression of ZO-1 was severely damaged in the mid-jejunum (P<0.001) of CON-DON compared with animals treated with LPS. Also, in all animals receiving LPS systemically, the cytosolic ZO-1 fraction in the 3 upper gut sections disappeared completely. This effect was independent of DON presence. Control pigs had a greater basolateral ß-catenin accumulation (P<0.05) in the cells, whereas the protein distribution did not differ in CON-DON pigs. In conclusion, results of this experiment demonstrated that epithelial proliferation has a distinct pattern along the small intestine and is not necessarily positively linked to crypt depth in pigs. Furthermore, results indicate that LPS changed the spatial distribution of ZO-1. A synergistic effect of DON and LPS on intestinal architecture could not be verified in the present study.

Nadph oxidase regulates alveolar epithelial sodium channel activity and lung fluid balance in vivo via O⁻2 signaling.

To define roles for reactive oxygen species (ROS) and epithelial sodium channel (ENaC) in maintaining lung fluid balance in vivo, we used two novel whole animal imaging approaches. Live X-ray fluoroscopy enabled quantification of air space fluid content of C57BL/6J mouse lungs challenged by intratracheal (IT) instillation of saline; results were confirmed by using conventional lung wet-to-dry weight ratios and Evans blue as measures of pulmonary edema. Visualization and quantification of ROS produced in lungs was performed in mice that had been administered a redox-sensitive dye, hydro-Cy7, by IT instillation. We found that inhibition of NADPH oxidase with a Rac-1 inhibitor, NSC23766, resulted in alveolar flooding, which correlated with a decrease in lung ROS production in vivo. Consistent with a role for Nox2 in alveolar fluid balance, Nox2(-/-) mice showed increased retention of air space fluid compared with wild-type controls. Interestingly, fluoroscopic analysis of C57BL/6J lungs IT instilled with LPS showed an acute stimulation of lung fluid clearance and ROS production in vivo that was abrogated by the ROS scavenger tetramethylpiperidine-N-oxyl (TEMPO). Acute application of LPS increased the activity of 20 pS nonselective ENaC channels in rat type 1 cells; the average number of channel and single-channel open probability (NPo) increased from 0.14 ± 0.04 to 0.62 ± 0.23. Application of TEMPO to the same cell-attached recording caused an immediate significant decrease in ENaC NPo to 0.04 ± 0.03. These data demonstrate that, in vivo, ROS has the capacity to stimulate lung fluid clearance by increasing ENaC activity.

Oral administration of lipopolysaccharides for the prevention of various diseases: benefit and usefulness.

It is well known that intravenous administration of lipopolysaccharide (LPS) induces severe toxicity in mammals. The maximum tolerated dose of intravenous administration of LPS in humans is reported to be only 1 to 4 ng/kg body weight. However, oral administration of a high dose of LPS caused no toxicity or systemic inflammation in other mammals, birds, or fish. Two weeks of oral administration of a high dose of LPS (2 mg/kg) did not induce toxicity in a rat experiment. Moreover, several experiments have reported that oral administration of LPS had preventative and curative properties against various diseases, including allergic, and lifestyle-related diseases. These results demonstrate that mucosal administration of LPS acts via a different regulatory mechanism in biological responses from that of parenteral administration. Mucosal administration of LPS is thought to be quite promising for prevention of diseases, but LPS is rarely used. In order to expand the usage of oral administration of LPS for preventing lifestyle and allergic diseases, it will be necessary to clarify the mechanisms that arouse immune responses after oral administration of LPS. This short review presents a recent observation of the usefulness of orally administered LPS.

Differential ex vivo and in vivo endotoxin tolerance kinetics following human endotoxemia.

OBJECTIVES: Endotoxin (lipopolysaccharide) tolerance is characterized by a transient refractory state to a subsequent lipopolysaccharide challenge. Following human endotoxemia, ex vivo tolerance of circulating leukocytes to lipopolysaccharide resolves within 24 hrs. However, the duration of in vivo tolerance, assumed to be primarily mediated by tissue-resident macrophages, is unknown. DESIGN, SETTING, SUBJECTS, AND INTERVENTIONS: Clinical experimental study in 16 healthy male volunteers at an intensive care research unit. To compare ex vivo and in vivo tolerance kinetics, whole blood from healthy volunteers was stimulated with lipopolysaccharide before, 4 hrs after, and 1, 2, 3, and 4 wks following in vivo endotoxin (2 ng/kg; lipopolysaccharide derived from Escherichia coli O:113) administration. Furthermore, we compared the inflammatory response during two subsequent endotoxemia experiments in healthy volunteers with an interval of 2 wks. The cytokines tumor necrosis factor-α, interleukin-6, interleukin-10, interleukin-1 receptor antagonist, and transforming growth factor-ß were measured. MEASUREMENTS AND MAIN RESULTS: Four hours after in vivo lipopolysaccharide administration, production of tumor necrosis factor-α, interleukin-6, and interleukin-10, but not interleukin-1 receptor antagonist in ex vivo lipopolysaccharide-stimulated whole blood was diminished. Ex vivo lipopolysaccharide tolerance completely resolved within 1 week. In contrast, in vivo lipopolysaccharide tolerance was still apparent after 2 wks. Compared to the first lipopolysaccharide administration, plasma peak levels of tumor necrosis factor-α, interleukin-6, interleukin-10, interleukin-1 receptor antagonist, and transforming growth factor-ß were attenuated by 46%, 36%, 45%, 10%, and 14%, respectively (all p < .05). CONCLUSIONS: While ex vivo lipopolysaccharide tolerance quickly resolves, in vivo lipopolysaccharide tolerance persists for at least 2 wks. These findings strengthen the notion that the in vivo response to lipopolysaccharide is mediated by tissue-resident macrophages and that ex vivo stimulation does not accurately reflect the in vivo innate immune response. Intervention studies utilizing the human endotoxemia model should be performed using parallel groups rather than a crossover design.

Peritoneal resorption capacity for lipopolysaccharide and interleukin-6 in acute zymosan-induced chemical peritonitis.

AIM: To evaluate peritoneal resorption capacity for lipopolysaccharide (LPS) and interleukin-6 (IL-6) in a model of chemical peritonitis. METHODS: Zymosan peritonitis was induced in anesthetized rats. LPS was injected intraperitoneally to different groups at 4 h (n = 10), 8 h (n = 9), 12 h (n = 9), and 24 h (n = 9) after peritonitis and to a control group (n = 8). Similarly, IL-6 was injected intraperitoneally to different groups at 4 h (n = 9), 8 h (n = 10), 12 h (n = 10), and 24 h (n = 10) after peritonitis, and to a control group (n = 10). Plasma levels of LPS or IL-6 were measured immediately after intraperitoneal injections of LPS or IL-6, respectively, and at 5, 15, 30, 45, and 60 min later. RESULTS: There was no change over time in plasma LPS levels in the groups receiving LPS intraperitoneally (p = 0.4). There was highly significant change over time in the IL-6 level in the studied time periods in the groups receiving IL-6 intraperitoneally (p < 0.0001). There was an increase in the plasma IL-6 level when sampled at 4 h after peritonitis. CONCLUSION: There was a reduction of resorption capacity of inflamed peritoneum for inflammatory mediators in acute chemical peritonitis.

Toll-like receptors and their adaptors are regulated in macrophages after phagocytosis of lipopolysaccharide-coated titanium particles.

Macrophages phagocytose metallic wear particles and produce mediators, which can induce cellular host response and aseptic implant loosening. Lipopolysaccharide (LPS) on the wear debris can stimulate macrophages via Toll-like receptor 4 (TLR4) and enhance the response. However, the precise functional role and interaction of TLRs and their adaptor molecules is still unclear. Rat bone marrow macrophages were stimulated with titanium particle (Ti) coated by LPS (Ti/LPS+) and LPS-free Ti (Ti/LPS-). mRNA levels of cytokines, TLRs and their adaptor molecules were measured using real time PCR. mRNA levels of TNF-α, IL-1ß, and IL-6 increased in Ti/LPS+ than Ti/LPS-. In contrast, mRNA levels of TLR4, TLR5, and TLR9 decreased in Ti/LPS+ compared to Ti/LPS-. mRNA levels of MyD88, IRAK1, IRAK4 decreased gradually, and TRAF6 underwent an initial transient increase, followed by suppression in Ti/LPS+. However, mRNA levels of TLR2 and IRAK2 increased after phagocytosis of Ti/LPS+ than Ti/LPS-. The increased expressions of proinflammatory cytokines found in Ti/LPS+ indicated that their productions cytokines could be enhanced by phagocytosis of LPS-coated particles. Subsequent down-regulation of TLR4, TLR5, TLR9, MyD88, IRAK1, and IRAK4 suggests that self-protective mechanisms to regulate excessive host responses are activated in macrophages. Increase of TLR2 and IRAK2 and a transient increase of TRAF6 in Ti/LPS+ suggest that another possible pathway to modulate TLR-mediated cellular response to prolong inflammatory response in foreign body reaction of aseptic loosening. This down- and/or up-regulation of the potential TLR-mediated responses to LPS-coated particles reflects the proactive behavior of effector cells.

Lipopolysaccharide induces apoptotic insults to human alveolar epithelial A549 cells through reactive oxygen species-mediated activation of an intrinsic mitochondrion-dependent pathway.

Alveolar type II epithelial cells can regulate immune responses to sepsis-induced acute lung injury. Lipopolysaccharide (LPS), an outer membrane component of Gram-negative bacteria, can cause septic shock. This study was designed to evaluate the cytotoxic effects of LPS on human alveolar epithelial A549 cells and its possible molecular mechanisms. Exposure of A549 cells to LPS decreased cell viability in concentration- and time-dependent manners. In parallel, LPS concentration- and time-dependently induced apoptosis of A549 cells. Meanwhile, LPS only at a high concentration of 10 µg/ml caused mildly necrotic insults to A549 cells. In terms of the mechanism, exposure of A549 cells to LPS increased the levels of cellular nitric oxide and reactive oxygen species (ROS). Pretreatment with N-acetylcysteine (NAC), an antioxidant, significantly lowered LPS-caused enhancement of intracellular ROS in A549 cells and simultaneously attenuated the apoptotic insults. Sequentially, treatment of A549 cells with LPS caused significant decreases in the mitochondrial membrane potential and biosynthesis of adenosine triphosphate. In succession, LPS triggered the release of cytochrome c from the mitochondria to the cytoplasm. Activities of caspase-9 and caspase-6 were subsequently augmented following LPS administration. Consequently, exposure of A549 cells induced DNA fragmentation in a time-dependent manner. Pretreatment of A549 cells with NAC significantly ameliorated LPS-caused alterations in caspase-9 activation and DNA damage. Therefore, this study shows that LPS specifically induces apoptotic insults to human alveolar epithelial cells through ROS-mediated activation of the intrinsic mitochondrion-cytochrome c-caspase protease mechanism.

Emulsified lipids increase endotoxemia: possible role in early postprandial low-grade inflammation.

Low-grade inflammation is a risk factor for the onset of atherosclerosis. Little is known about the involvement of endotoxin absorption from the gut during the digestion of lipids. In the present study, we first investigated in humans the impact of a mixed meal containing dispersed lipids on postprandial endotoxemia and inflammation. We then investigated the effect of (i) oil emulsification in vivo in rats and (ii) fatty acid amounts in vitro using Caco-2 cells on postprandial endotoxemia. In humans, postprandial endotoxemia increased early after the meal. Moreover, we evidenced that the endotoxin receptor sCD14 increased during digestion and that chylomicrons could contribute to absorbed endotoxin transport. This could explain the significant peak of inflammatory cytokine IL-6 that we observed 2 h after the mixed meal. Interestingly, in rats, the emulsion led to both higher endotoxemia and hypertriglyceridemia than oil and compared to a control saline load. In vitro, incubation of Caco-2 cells with increasing fatty acid concentrations enhanced epithelial absorption of endotoxin. To our knowledge, this is the first study evidencing in healthy humans that, following a mixed meal containing lipids, increased endotoxemia is associated with raised sCD14 and a peak of IL-6. On a repeated basis, this may thus be a triggering cascade for the onset of atherosclerosis. In this respect, optimizing both dietary fat amount and structure could be a possible strategy to limit such low-grade endotoxemia and inflammation by the control of postprandial lipemia.

Combined chemo-immunotherapy as a prospective strategy to combat cancer: a nanoparticle based approach.

The prime objective of this study was to develop a combined chemo-immunotherapeutic formulation which could directly kill cancer cells as well as activate the immunosuppressed tumor microenvironment to mount a robust antitumor immune response. Paclitaxel (PTX) and SP-LPS (nontoxic derivative of lipopolysaccharide) were selected as anticancer drug and immunostimulant respectively. Poly(lactic-co-glycolic acid) (PLGA) based PTX and SP-LPS containing nanoparticles (TLNP) were prepared by the double-emulsion method (w/o/w) and characterized in terms of size, zeta potential and transmission electron microscopy (TEM). The release behavior of PTX and SP-LPS from the TLNP exhibited a biphasic pattern characterized by an initial burst followed by slow continuous release. In vitro anticancer activity of TLNP was found to be higher compared to PTX when studied in a tumor cell-splenocyte coculture system. TLNP activated murine monocytes induced the secretion of various proinflammatory cytokines. After iv administration of TLNP in tumor bearing C57BL/6 mice, the amount of PTX in the tumor mass was found to be higher in TLNP treated mice as compared to commercial Taxol group at all time points studied. In vitro studies suggest that nanoparticles containing PTX and SP-LPS have both direct cytotoxicity and immunostimulatory activity. Hence this might have potential as a chemo-immunotherapeutic formulation against cancer with advantage over present day chemotherapy with Taxol, in terms of tumor targeting, less toxicity and immunostimulation.

Human cholesteryl ester transfer protein expression enhances the mouse survival rate in an experimental systemic inflammation model: a novel role for CETP.

Mice expressing human cholesteryl ester transfer protein (huCETP) are more resistant to Escherichia coli bacterial wall LPS because death rates 5 days after intraperitoneal inoculation of LPS were higher in wild-type than in huCETP+/+ mice, whereas all huCETP+/+ mice remained alive. After LPS inoculation, plasma concentrations of TNF-alpha and IL-6 increased less in huCETP+/+ than in wild-type mice. LPS in vitro elicited lower TNF-alpha production by CETP expressing than by wild-type macrophages. In addition, TNF-alpha production by RAW 264.7 murine macrophages increased on incubation with LPS but decreased in a dose-dependent manner when human CETP was added to the medium. Human CETP in vitro enhanced the LPS binding to plasma high-density lipoprotein/low-density lipoprotein. The liver uptake of intravenous infused 14C-LPS from Salmonella typhimurium was greater in huCETP+/+ than in wild-type mice. Present data indicate for the first time that CETP is an endogenous component involved in the first line of defense against an exacerbated production of proinflammatory mediators.