Myeloperoxidase Assay Research Articles

Kaiso differentially regulates components of the Notch signaling pathway in intestinal cells

BackgroundIn mammalian intestines, Notch signaling plays a critical role in mediating cell fate decisions; it promotes the absorptive (or enterocyte) cell fate, while concomitantly inhibiting the secretory cell fate (i.e. goblet, Paneth and enteroendocrine cells). We recently reported that intestinal-specific Kaiso overexpressing mice (KaisoTg) exhibited chronic intestinal inflammation and had increased numbers of all three secretory cell types, hinting that Kaiso might regulate Notch signaling in the gut. However, Kaiso’s precise role in Notch signaling and whether the KaisoTg secretory cell fate phenotype was linked to Kaiso-induced inflammation had yet to be elucidated.MethodsIntestines from 3-month old Non-transgenic and KaisoTg mice were “Swiss” rolled and analysed for the expression of Notch1, Dll-1, Jagged-1, and secretory cell markers by immunohistochemistry and immunofluorescence. To evaluate inflammation, morphological analyses and myeloperoxidase assays were performed on intestines from 3-month old KaisoTg and control mice. Notch1, Dll-1 and Jagged-1 expression were also assessed in stable Kaiso-depleted colon cancer cells and isolated intestinal epithelial cells using real time PCR and western blotting. To assess Kaiso binding to the DLL1, JAG1 and NOTCH1 promoter regions, chromatin immunoprecipitation was performed on three colon cancer cell lines.ResultsHere we demonstrate that Kaiso promotes secretory cell hyperplasia independently of Kaiso-induced inflammation. Moreover, Kaiso regulates several components of the Notch signaling pathway in intestinal cells, namely, Dll-1, Jagged-1 and Notch1. Notably, we found that in KaisoTg mice intestines, Notch1 and Dll-1 expression are significantly reduced while Jagged-1 expression is increased. Chromatin immunoprecipitation experiments revealed that Kaiso associates with the DLL1 and JAG1 promoter regions in a methylation-dependent manner in colon carcinoma cell lines, suggesting that these Notch ligands are putative Kaiso target genes.ConclusionHere, we provide evidence that Kaiso’s effects on intestinal secretory cell fates precede the development of intestinal inflammation in KaisoTg mice. We also demonstrate that Kaiso inhibits the expression of Dll-1, which likely contributes to the secretory cell phenotype observed in our transgenic mice. In contrast, Kaiso promotes Jagged-1 expression, which may have implications in Notch-mediated colon cancer progression.

Anti-inflammatory Effects of Quercetin and Vitexin on Activated Human Peripheral Blood Neutrophils: - The effects of quercetin and vitexin on human neutrophils.

ObjectivesPolymorphonuclear neutrophils (PMNs) constitute the first line of defense against invading microbial pathogens. Early events in inflammation involve the recruitment of neutrophils to the site of injury or damage where changes in intracellular calcium can cause the activation of pro-inflammatory mediators from neutrophils including superoxide generation, degranulation and release of myeloperoxidase (MPO), productions of interleukin (IL)-8 and tumor necrosis factor α (TNF-α), and adhesion to the vascular endothelium. To address the anti-inflammatory role of flavonoids, in the present study, we investigated the effects of the flavonoids quercetin and vitexin on the stimulus-induced nitric oxide (NO), TNF-α, and MPO productions in human neutrophils.MethodsHuman peripheral blood neutrophils were isolated, and their viabilities were determined by using the Trypan Blue exclusion test. The polymorphonuclear leukocyte (PMNL) preparations contained more than 98% neutrophils as determined by morphological examination with Giemsa staining. The viabilities of cultured neutrophils with various concentrations of quercetin and vitexin (1 – 100 μM) were studied using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assays. Neutrophils were cultured in complete Roswell Park Memorial Institute (RPMI) medium, pre-incubated with or without quercetin and vitexin (25 μM) for 45 min, and stimulated with phorbol 12-myristate 13-acetate (PMA) (10−7 M). NO production was carried out through nitrite determination by using the Griess method. Also, the TNF-α and the MPO productions were measured using enzyme-linked immunosorbent assay (ELISA) kits and MPO assay kits.ResultsNeutrophil viability was not affected up to a concentration of 100 μM of quercetin or vitexin. Both quercetin and vitexin significantly inhibited TNF-α, NO, and MPO productions in human neutrophils (P < 0.001).ConclusionThe present study showed that both quercetin and vitexin had significant anti-inflammatory effects. Thus, treatment with either quercetin or vitexin may be considered as a therapeutic strategy for treating patients with neutrophil-mediated inflammatory diseases.

Cholecystokinin protects mouse liver against ischemia and reperfusion injury

BackgroundCholecystokinin (CCK), as a gastrointestinal hormone, has an important protective role against sepsis or LPS-induced endotoxic shock. We aim to address the role of CCK in hepatic ischemia followed by reperfusion (I/R) injury. Materials and methodsA murine model of 60min partial hepatic ischemia followed by 6h of reperfusion was used in this study. CCK and CCKAR Levels in blood and liver were detected at 3h, 6h, 12h and 24h after reperfusion. Then the mice were treated with CCK or proglumide, a nonspecific CCK-receptor (CCK-R) antagonist. Mice were randomly divided into four groups as follows: (1) sham group, in which mice underwent sham operation and received saline; (2) I/R group, in which mice were subjected to hepatic I/R and received saline; (3) CCK group, in which mice were subjected to hepatic I/R and treated with CCK (400μg/kg); (4) proglumide group (Pro), in which mice underwent hepatic I/R and treated with proglumide (3mg/kg); CCK and proglumide were administrated via tail vein at the moment of reperfusion. Serum AST (sAST) and serum ALT (sALT) were determined with a biochemical assay and histological analysis were performed with hematoxylin-eosin (H&E). Cytokines (IL-1β, IL-6, IL-10, TNF-α) expressions in blood were determined with enzyme-linked immunosorbent assay (ELISA). The MPO (myeloperoxidase) assay were used to measure neutrophils' infiltration into the liver. The apoptotic index (TUNEL-positive cell number/total liver cell number×100%) was calculated to assess hepatocelluar apoptosis. Finally, activation of NF-κB and phosphor-p38 expression in liver homogenates were analyzed with Western Blot (WB). ResultsOur findings showed that 1) CCK and CCK-AR were upregulated in our experimental model over time; 2) Treatment with CCK decreased sAST/sALT levels, inflammatory hepatic injury, neutrophil influx and hepatocelluar apoptosis, while proglumide aggravated hepatic injury. ConclusionThese findings support our hypothesis and suggest that CCK played a positive role in the ongoing inflammatory process leading to liver I/R injury.

Treatment with Rutin - A Therapeutic Strategy for Neutrophil-Mediated Inflammatory and Autoimmune Diseases: - Anti-inflammatory Effects of Rutin on Neutrophils.

Objectives:Neutrophils represent the front line of human defense against infections. Immediately after stimulation, neutrophilic enzymes are activated and produce toxic mediators such as pro-inflammatory cytokines, nitric oxide (NO) and myeloperoxidase (MPO). These mediators can be toxic not only to infectious agents but also to host tissues. Because flavonoids exhibit antioxidant and anti-inflammatory effects, they are subjects of interest for pharmacological modulation of inflammation. In the present study, the effects of rutin on stimulus-induced NO and tumor necrosis factor (TNF)-α productions and MPO activity in human neutrophils were investigated.Methods:Human peripheral blood neutrophils were isolated using Ficoll-Hypaque density gradient centrifugation coupled with dextran T500 sedimentation. The cell preparations containing > 98% granulocytes were determined by morphological examination through Giemsa staining. Neutrophils were cultured in complete Roswell Park Memorial Institute (RPMI) medium, pre-incubated with or without rutin (25 μM) for 45 minutes, and stimulated with phorbol 12-myristate 13-acetate (PMA). Then, the TNF-α, NO and MPO productions were analyzed using enzyme-linked immunosorbent assay (ELISA), Griess Reagent, and MPO assay kits, respectively. Also, the viability of human neutrophils was assessed using tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), and neutrophils were treated with various concentrations of rutin (1 - 100 μM), after which MTT was appended and incubated at 37ºC for 4 hour.Results:Rutin at concentrations up to 100 μM did not affect neutrophil viability during the 4-hour incubation period. Rutin significantly decreased the NO and TNF-α productions in human peripheral blood neutrophils compared to PMA-control cells (P < 0.001). Also, MPO activity was significantly reduced by rutin (P < 0.001).Conclusion:In this in vitro study, rutin had an anti-inflammatory effect due to its inhibiting NO and TNF-α productions, as well as MPO activity, in activated human neutrophils. Treatment with rutin may be considered as a therapeutic strategy for neutrophil-mediated inflammatory/ autoimmune diseases.

Targeting of 12/15-Lipoxygenase in retinal endothelial cells, but not in monocytes/macrophages, attenuates high glucose-induced retinal leukostasis

AimsOur previous studies have established a role for 12/15-lipoxygenase (LO) in mediating the inflammatory response in diabetic retinopathy (DR). However, the extent at which the local or systemic induction of 12/15-LO activity involved is unclear. Thus, the current study aimed to characterize the relative contribution of retinal endothelial versus monocytic/macrophagic 12/15-LO to inflammatory responses in DR. Materials & methodsWe first generated a clustered heat map for circulating bioactive lipid metabolites in the plasma of streptozotocin (STZ)-induced diabetic mice using liquid chromatography coupled with mass-spectrometry (LC–MS) to evaluate changes in circulating 12/15-LO activity. This was followed by comparing the in vitro mouse endothelium-leukocytes interaction between leukocytes isolated from 12/15-LO knockout (KO) versus those isolated from wild type (WT) mice using the myeloperoxidase (MPO) assay. Finally, we examined the effects of knocking down or inhibiting endothelial 12/15-LO on diabetes-induced endothelial cell activation and ICAM-1 expression. ResultsAnalysis of plasma bioactive lipids' heat map revealed that the activity of circulating 12/15-LO was not altered by diabetes as evident by no significant changes in the plasma levels of major metabolites derived from 12/15-lipoxygenation of different PUFAs, including linoleic acid (13-HODE), arachidonic acid (12- and 15- HETEs), eicosapentaenoic acid (12- and 15- HEPEs), or docosahexaenoic acid (17-HDoHE). Moreover, leukocytes from 12/15-LO KO mice displayed a similar increase in adhesion to high glucose (HG)-activated endothelial cells as do leukocytes from WT mice. Furthermore, abundant proteins of 12-LO and 15-LO were detected in human retinal endothelial cells (HRECs), while it was undetected (15-LO) or hardly detectable (12-LO) in human monocyte-like U937 cells. Inhibition or knock down of endothelial 12/15-LO in HRECs blocked HG-induced expression of ICAM-1, a well-known identified important molecule for leukocyte adhesion in DR. ConclusionOur data support that endothelial, rather than monocytic/macrophagic, 12/15-LO has a critical role in hyperglycemia-induced ICAM-1 expression, leukocyte adhesion, and subsequent local retinal barrier dysfunction. This may facilitate the development of more precisely targeted treatment strategies for DR.

Electrochemical Lateral Flow Devices: Towards Rapid Immunomagnetic Assays

AbstractPaper has been used for a long time in the production of low‐cost point‐of‐care (POC) detection devices, most of which rely on the detection of colored bands by the user. However, test strips rarely provide the quantitative detection of biomarkers in clinical samples. In contrast, electrochemical detection enables quantitative detection in paper‐based immunoassays. Here, we demonstrate the production of a simple and economical lateral‐flow electrode device using only screen printing. Contrary to previous work, this device is used to simplify the washing and detection of a short and sensitive immunomagnetic assay for myeloperoxidase (MPO) detection. As we show, after incubation of the sample with magnetic beads (MBs) and detection antibody (Ab) for only 5 min, the whole reaction mixture can be directly pipetted onto the strip. Washing and detection can then be performed, as the MBs are retained with the aid of a magnet, and minimal user intervention is needed. The obtained results reveal that MPO can be detected in 1 : 100 diluted serum with limits of detection and quantification of 0.18 and 0.62 ng mL−1, respectively, in less than 15 min.

Beneficial effects of anthocyanins from red cabbage (Brassica oleracea L. var. capitata L.) administration to prevent irinotecan-induced mucositis

Intestinal mucositis is a common side effect of Irinotecan (CPT-11). Anthocyanins (ACs) from red cabbage against CPT-11-induced mucositis were studied. Mice were given CPT-11 (75mg/kg, i.p. for 4days) and treated with ACs (50, 100mg/kg, i.g. for 7days). On day 8, diarrhea and leukocyte count in blood were assessed. Samples of intestine were obtained for morphometric analysis, myeloperoxidase (MPO), TBARS and GSH assay. The effects of ACs on cytoprotection and intestinal permeability were studied in Caco-2 cells. ACs effectively reversed the signs of intestinal injury, including MPO activity, length of ileum and colon, leukopenia, intestinal architecture, and quantity of ileum mucus. In vitro, pretreatment with ACs enhanced transepithelial electrical resistance (TER) and the expression of ZO-1. Furthermore, the protective effects of ACs were associated to modulate autophagy through mTOR pathway. In conclusion, ACs from red cabbage are candidate for supplementary therapy of CPT-11-induced mucositis.

ROLE OF ANTIOXIDANT AND MYELOPEROXIDASE LEVELS IN 7, 12-DIMETHYLBENZ [A] ANTHRACENE INDUCED EXPERIMENTAL RAT MODEL: EVIDENCE FOR OXIDATIVE DAMAGE IN ACTIVE ULCERATIVE COLITIS.

Objective: Ulcerative colitis known as inflammatory bowel disease (IBD) of unknown etiology. We examined the antioxidant and myeloperoxidase status in a murine model of 7,12-dimethylbenz[a]anthracene induced colitis to elucidate the exact mechanism behind the inflammation.Methods: Male Wistar rats were exposed to ulcerative colitis using various concentration of DMBA (7,12-Dimethylbenz[A]anthracene) were periodically analysed on 4th, 8th, 12th, 24th and 32nd week from the date of induction. To determine the disease activity index changes in body weight, food consumption, the presence of gross blood in stool and consistency of feces and diarrhea were observed. Macroscopic characters were elucidated based on clinical features of the colon and rectum using scoring pattern. Tissue inflammation status was noted through myeloperoxidase (MPO) assay. The antioxidant status in tissue samples was analysed by superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and total reduced glutathione (GSH).Results: Gavage intubation of DMBA induced colitis showed significant changes from 4th week and severity on 32nd week. The body weight was gradually reduced. Macroscopic scoring showed severe scoring pattern the inflammation was significantly heavier by week 4; and by the end of 32 w, inflammation in rats was double that of the controls, tissue myeloperoxidase (MPO) activity showed the steady increase of neutrophil infiltration and inflammation rate every week. A significant change was noted in tissue antioxidant status and it showed the oxidation level. Statistically, significant change was recorded from 4th week till 32nd week.Conclusion: The conventional biochemical changes in colitis induced animal model revealed the association between the oxidative stress and ulcerative colitis.

18F-fluoro-2-deoxyglucose PET informs neutrophil accumulation and activation in lipopolysaccharide-induced acute lung injury

IntroductionMolecular imaging of the earliest events related to the development of acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) could facilitate therapeutic development and patient management. We previously reported that 18F-fluoro-2-deoxyglucose (18F-FDG) PET identifies ALI/ARDS prior to radiographic abnormalities. The purpose of this study was to establish the time courses of 18F-FDG uptake, edema and neutrophil recruitment in an endotoxin-induced acute lung injury model and to examine molecular events required for 14C-2DG uptake in activated neutrophils. MethodsLung uptake of 18F-FDG was measured by PET in control male Sprague Dawley rats and at 2, 6 and 24h following the intraperitoneal injection of 10mg/kg LPS. Lung edema (attenuation) was measured by microCT. Neutrophil influx into the lungs was measured by myeloperoxidase assay. Control and activated human donor neutrophils were compared for uptake of 14C-2DG, transcription and content of hexokinase and GLUT isoforms and for hexokinase (HK) activity. ResultsSignificant uptake of 18F-FDG occurred by 2h following LPS, and progressively increased to 24h. Lung uptake of 18F-FDG preceded increased CT attenuation (lung edema). Myeloperoxidase activity in the lungs, supporting neutrophil influx, paralleled 18F-FDG uptake. Activation of isolated human neutrophils resulted in increased uptake of 14C-2DG, expression of GLUT 3 and GLUT 4 and expression and increased HK1 activity. ConclusionSystemic endotoxin-induced ALI results in very early and progressive uptake of 18F-FDG, parallels neutrophil accumulation and occurs earlier than lung injury edema. Activated neutrophils show increased uptake of 14C-2DG, expression of specific GLUT3, GLUT4 and HK1 protein and HK activity. Advances in knowledge and implications for patient care18F-FDG pulmonary uptake is an early biomarker of neutrophil recruitment in ALI and is associated with specific molecular events that mediate 14C-2DG uptake in activated neutrophils. 18F-FDG PET may provide a potential mechanism for early diagnosis and therapeutic assessment of ALI/ARDS.

Effects of Supernatants from Escherichia coli Nissle 1917 and Faecalibacterium prausnitzii on Intestinal Epithelial Cells and a Rat Model of 5-Fluorouracil-Induced Mucositis

ABSTRACTFaecalibacterium prausnitzii (Fp) and Escherichia coli Nissle 1917 (EcN) are probiotics, which have been reported to ameliorate certain gastrointestinal disorders. We evaluated the effects of supernatants (SN) derived from Fp and EcN on 5-fluorouracil (5-FU)-treated intestinal cells and in a rat model of mucositis. In vitro: IEC-6, Caco-2, and T-84 cells were analyzed for viability and monolayer permeability. In vivo: Female dark agouti rats were gavaged with Fp or EcN SN and injected intraperitoneally with saline (control) or 5-FU to induce mucositis. Rats were euthanized and intestinal tissues collected for myeloperoxidase assay and histological analyses. In vitro: Caco-2 cell viability was further reduced when treated with Fp SN + 5-FU compared to 5-FU controls. In both Caco-2 and T-84 cells, Fp SN partially prevented the decrease in transepithelial electrical resistance (TER) caused by 5-FU administration. In vivo: 5-FU-injected rats administered Fp SN or EcN SN partly prevented body weight loss and normalized water intake compared to 5-FU controls. These results suggest a growth inhibitory mechanism of Fp SN action on transformed epithelial cells that could be mediated by effects on tight junctions. Factors derived from Fp SN and EcN SN could have a role in reducing the severity of intestinal mucositis.

Methods for In Vivo/Ex Vivo Analysis of Antimicrobial Peptides in Bacterial Keratitis: siRNA Knockdown, Colony Counts, Myeloperoxidase, Immunostaining, and RT-PCR Assays.

Antimicrobial peptides (AMPs) are essential components of the innate immune response. They have direct killing ability as well as immunomodulatory functions. Here, we describe techniques to identify specific AMPs involved in the protection against microbial keratitis, a vision threatening infection of the cornea of the eye which is the most serious complication of contact lens wear. Specifically we detail the use of siRNA technology to temporarily knockdown AMP expression at the murine ocular surface in vivo and then describe ex vivo assays to determine the level of bacteria, relative number of neutrophils, and levels of cytokines, chemokines, and AMPs in infected corneas.

Anti-inflammatory effect of a fatty acid mixture with high ω-9:ω-6 ratio and low ω-6:ω-3 ratio on rats submitted to dental extraction

ObjectiveTo evaluate the anti-inflammatory effect of pretreatment for three days with a fatty acid mixture with high ω-9:ω-6 ratio and low ω-6:ω-3 ratio on rats submitted to dental extraction. Material and methodsThirty-two male Wistar rats (270–310g) were randomly distributed in four groups (n=8/group): the sham control group and the negative control group received saline; the high omega-6/low omega-9 group received isolipid fatty acid with high ω-6:ω-3 ratio and low ω-9:ω-6 ratio; the high omega-3/low omega-6 group received fatty acid with low ω-6:ω-3 ratio and high ω-9:ω-6 ratio. Saline and oils were administered by gavage for 4days before exodontia and 3days after surgery, followed by euthanasia. Masseter edema was evaluated clinically and tissue samples were submitted to osteoclast count (H&E), myeloperoxidase assay, and western blotting (tumor necrosis factor-alpha and interleukin-1beta). ResultsIn the high omega-3/low omega-6 group, a significant decrease was observed in masseter edema (p<0.0001), myeloperoxidase (p<0.0001), osteoclasts (p=0.0001) and TNF-α expression (p<0.0001), but not in IL-1β expression. ConclusionThe ingestion of fatty acid with high ω-9:ω-6 ratio and low ω-6:ω-3 ratio significantly reduced inflammatory response in rats submitted to dental extraction.

Glycyrrhizin Reduces HMGB1 and Bacterial Load in Pseudomonas aeruginosa Keratitis.

PurposeHigh mobility group box 1 (HMGB1) contributes to poor disease outcome in Pseudomonas aeruginosa keratitis. This study tests the prophylactic effect of treatment with HMGB1 inhibitors, glycyrrhizin (GLY) and its derivative, carbenoxolone (CBX), for Pseudomonas keratitis.MethodsWe treated C57BL/6 (B6) mice subconjunctivally with GLY or CBX, infected with a noncytotoxic clinical isolate (KEI 1025) or a cytotoxic strain (ATCC 19660) of P. aeruginosa, and injected intraperitoneally with either agent. Clinical score, photography with a slit lamp, real-time RT-PCR, ELISA, myeloperoxidase (MPO) assay, bacterial plate count, histopathology, and absorbance assays were used to assess treatment efficacy and bacteriostatic activity.ResultsAfter KEI 1025 infection, GLY treatment reduced HMGB1 (mRNA and protein levels) and improved disease outcome with significant reduction in mRNA levels of IL-1β, TLR4, CXCL2, and IL-12; protein expression (IL-1β, CXCL2); neutrophil infiltrate; and bacterial load. Treatment with GLY enhanced antimicrobial proteins, including CRAMP and mBD2, but not mBD3. Glycyrrhizin also reduced clinical scores and improved disease outcome in corneas infected with strain 19660. However, neither HMGB1 mRNA or protein levels were reduced, but rather, CXCL2 expression (mRNA and protein), neutrophil infiltrate, and bacterial load were reduced statistically. Treatment with GLY initiated 6 hours after infection reduced plate count; GLY also was bacteriostatic for KEI 1025 and ATCC 19660.ConclusionsGlycyrrhizin reduces HMGB1 and is protective against P. aeruginosa–induced keratitis with a clinical isolate that is noncytotoxic. It was similar, but less effective when used after infection with a cytotoxic strain, which did not reduce HMGB1.

Anti-Inflammatory Potential of Hecogenin in Experimental Animals: Possible Involvement of Inflammatory Cytokines and Myeloperoxidase.

Hecogenin is a steroidal sapogenin plays important role in treatment of variety of inflammatory diseases. We have investigated the anti-inflammatory effects of Hecogenin (50 µg/animal) (HG), Fluticasone (50 µg/animal) (FC) and Hecogenin+Fluticasone (HG+FC) combination (25 µg/animal, each) on various inflammatory models. The anti-inflammatory effect of HG, FC and HG+FC combination was studied on % inhibition of dry weight of granuloma tissue, Δ ear weight, myeloperoxidase assay, serum pro-inflammatory cytokines, colon weight to length ratio, macroscopic lesions, adhesion score, diarrhoea score and histopathological analysis of ear and colon tissue on Cotton pellets induced granuloma in rats, Croton oil induced ear edema in mice and TNBS induced granuloma in rats. Topical administration of HG and its combination with FC showed significant decrease (p<0.001) in the % inhibition of dry weight of granuloma tissue, Δ ear weight, myeloperoxidase level, serum pro-inflammatory cytokines levels, colon weigh to length ratio as compared with Cotton pellets treated with acetone groups and Croton oil treated animals. Further histopathological analysis of ear tissue showed significant decrease in dermal thickness and epidermal hyperplasia and colon tissue showed reduction of edema, infiltration of inflammatory cells and normalization of crypt structure compared to DC animals. Thus, the findings of present study suggest the possible role of HG in the treatment of inflammation by reducing the dose of FC in combination with HG.

Differential Effectiveness of Clinically-Relevant Analgesics in a Rat Model of Chemotherapy-Induced Mucositis.

Chemotherapy-induced intestinal mucositis is characterized by pain and a pro-inflammatory tissue response. Rat models are frequently used in mucositis disease investigations yet little is known about the presence of pain in these animals, the ability of analgesics to ameliorate the condition, or the effect that analgesic administration may have on study outcomes. This study investigated different classes of analgesics with the aim of determining their analgesic effects and impact on research outcomes of interest in a rat model of mucositis. Female DA rats were allocated to 8 groups to include saline and chemotherapy controls (n = 8). Analgesics included opioid derivatives (buprenorphine; 0.05mg/kg and tramadol 12.5mg/kg) and NSAID (carprofen; 15mg/kg) in combination with either saline or 5-Fluorouracil (5-FU; 150mg/kg). Research outcome measures included daily clinical parameters, pain score and gut histology. Myeloperoxidase assay was performed to determine gut inflammation. At the dosages employed, all agents had an analgesic effect based on behavioural pain scores. Jejunal myeloperoxidase activity was significantly reduced by buprenorphine and tramadol in comparison to 5-FU control animals (53%, p = 0.0004 and 58%, p = 0.0001). Carprofen had no ameliorating effect on myeloperoxidase levels. None of the agents reduced the histological damage caused by 5-FU administration although tramadol tended to increase villus length even when administered to healthy animals. These data provide evidence that carprofen offers potential as an analgesic in this animal model due to its pain-relieving efficacy and minimal effect on measured parameters. This study also supports further investigation into the mechanism and utility of opioid agents in the treatment of chemotherapy-induced mucositis.

Asiatic Acid Inhibits Lipopolysaccharide-Induced Acute Lung Injury in Mice.

Asiatic acid (AA), a major triterpene isolated from Centella asiatica (L.) Urban, is known to exert various pharmacological activities, including anti-inflammatory and antioxidant effects. The aim of this study was to evaluate the anti-inflammatory effects of AA on lipopolysaccharide (LPS)-induced acute lung injury (ALI) and clarify the underlying mechanisms. Lung pathological changes were assessed by H&E staining. The myeloperoxidase (MPO) activity was detected by MPO assay. The levels of inflammatory cytokines were measured by ELISA. TLR4 and NF-kB expression was detected by Western blot analysis. AA obviously inhibited LPS-induced lung histopathological changes, MPO activity, and inflammatory cell numbers in bronchoalveolar lavage fluid (BALF). Treatment of AA also inhibited LPS-induced TNF-α, IL-6, and IL-1β production. Furthermore, Western blot analysis showed that AA inhibited LPS-induced TLR4 expression and NF-kB activation. In conclusion, AA inhibited LPS-induced ALI in mice by inhibiting inflammatory cytokine production, which is mediated via blocking of the TLR4/NF-kB signaling pathway.

Polyphenols from Silybum marianum inhibit invitro the oxidant response of equine neutrophils and myeloperoxidase activity.

A recent study showed that silymarin, a standardized extract of S. marianum might be used in the prevention of equine laminitis. We investigated the effects of quercetin and some compounds found in silymarin (silybin, taxifolin and dehydrosilybin) on reactive oxygen species (ROS) production and myeloperoxidase (MPO) release by stimulated equine neutrophils (PMNs) and on MPO activity. All compounds (tested between 100nm and 100μm) inhibited superoxide anion production by stimulated PMNs in a dose-dependent manner. Dehydrosilybin and quercetin inhibited superoxide production and MPO release from 10μm. Classical MPO assay showed quercetin as the most potent inhibitor, followed by taxifolin, dehydrosilybin and silybin. SIEFED MPO assay highlighting the binding of tested compounds to MPO showed that only quercetin and taxifolin maintained an efficient inhibition above 90% at 10μm. Altogether, our results showed a strong inhibition of PMN activation by planar compounds such as quercetin and dehydrosilybin and a strong inhibition of MPO activity by the smallest molecules, quercetin and taxifolin. In conclusion, the compounds from silymarin may be useful for modulating the oxidative response of PMNs, involved in the pathogenesis of laminitis, but further invivo studies are needed.

Pentraxin 3, ischemia-modified albumin, and myeloperoxidase in predicting a cardiac damage in acute carbon monoxide poisoning

BackgroundCarbon monoxide (CO) poisoning is associated with cardiac injuries or manifestations, frequently attributing to direct hypoxic damage at cellular level. For this, the aims were to evaluate the role of serum pentraxin 3 (PTX 3), ischemia-modified albumin (IMA), and myeloperoxidase (MPO) as an early biomarker for cardiac damage when compared to cardiac troponin I (cTnI) and creatine kinase-MB fraction (CK-MB) in adult patients with acute CO poisoning. MethodsForty patients with acute CO poisoning admitted to the emergency department. The patients were divided into 2 main groups as follows: cardiac injury (group I, n=19) and nonsuspected cardiac injury (group II, n=21). Pentraxin 3, IMA, MPO, cTnI, CK-MB, and the other assays in the circulation were measured on admission. ResultsUpon measuring the serum PTX 3, IMA, MPO, cTnI, and CK-MB levels as well as large electrocardiography and echocardiography abnormalities of patients with cardiac injury on admission, no statistical difference for PTX 3, IMA, and MPO was found between the groups (P>.05). However, cTnI, CK-MB, and leukocyte count (white blood cell) were higher determined in patients of group I compared to group II (P<.05). Receiver operating characteristic curve was also performed to evaluate the diagnostic performance of these tests in patients with cardiac injury. ConclusionsOur results suggest that PTX, IMA, and MPO assays are not superior to cTnI and CK-MB in predicting a cardiac damage in patients with acute CO intoxication.

Aqueous garlic extract protects against sepsis induced toxicity in pulmonary and ileal tissues

Objective: Based on the potent antioxidant effects of aqueous garlic extract (AGE), the present study was designed to characterize the potential of AGE to modify blood coagulation parameters as well as and pulmonary and ileal injury in septic rats. Sepsis was induced using the caecal ligation and perforation (CLP) method. Methods: Twenty-four hours after sepsisinduction, rats were decapitated and trunk blood was collected for the measurement of platelet counts, fibrinogen, prothrombin time, activated partial thromboplastin time and D-dimer levels. Then, pulmonary and ileal tissue samples were immediately obtained and stored at – 70oC for malondialdehyde (MDA), glutathione, myeloperoxidase (MPO) and superoxide dismutase (SOD) activity assays. Results: Sepsis was associated with a decrease in platelet count and fibrinogen and an increase in APTT and INR. It also caused a significant decrease in GSH levels and SOD activity in both pulmonary and ileal tissue samples. On the other hand, AGE treatment in rats with CLP caused significantly augmented the level of these antioxidants. As a result of CLP induction increased MPO activity and MDA levels and decreased thromboplastic activity were reversed with AGE treatment. Conclusion: AGE treatment, through its antioxidant effects, protects against oxidative pulmonary and ileal injury and normalizes the impaired coagulation in sepsis.

Impact of reactive oxygen species (ROS) on the control of parasite loads and inflammation in Leishmania amazonensis infection.

BackgroundReactive oxygen species (ROS) protect the host against a large number of pathogenic microorganisms. ROS have different effects on parasites of the genus Leishmania: some parasites are susceptible to their action, while others seem to be resistant. The role of ROS in L. amazonensis infection in vivo has not been addressed to date.MethodsIn this study, C57BL/6 wild-type mice (WT) and mice genetically deficient in ROS production by phagocytes (gp91phox−/−) were infected with metacyclic promastigotes of L. amazonensis to address the effect of ROS in parasite control. Inflammatory cytokines, parasite loads and myeloperoxidase (MPO) activity were evaluated. In parallel, in vitro infection of peritoneal macrophages was assessed to determine parasite killing, cytokine, NO and ROS production.ResultsIn vitro results show induction of ROS production by infected peritoneal macrophages, but no effect in parasite killing. Also, ROS do not seem to be important to parasite killing in vivo, but they control lesion sizes at early stages of infection. IFN-γ, TNF-α and IL-10 production did not differ among mouse strains. Myeloperoxidase assay showed augmented neutrophils influx 6 h and 72 h post - infection in gp91phox−/− mice, indicating a larger inflammatory response in gp91phox−/− even at early time points. At later time points, neutrophil numbers in lesions correlated with lesion size: larger lesions in gp91phox−/− at earlier times of infection corresponded to larger neutrophil infiltrates, while larger lesions in WT mice at the later points of infection also displayed larger numbers of neutrophils.ConclusionROS do not seem to be important in L. amazonensis killing, but they regulate the inflammatory response probably by controlling neutrophils numbers in lesions.