Air Pouch Model Research Articles

Effect of microemulsion formulation on biodistribution of 99mTc-Aprotinin in acute pancreatitis models induced rats

Background: Aprotinin is a monomeric globular polypeptide, which derived from bovine lung tissue and theoretically attractive molecule in ameliorating the effects of acute pancreatitis. Acute pancreatitis is an inflammatory condition of the pancreas that is painful and at times deadly. Over the following two decades Aprotinin therapeutic potential on pancreatitis is proven experimentally, its clinical therapeutic success is limited due to low targeting to pancreas.Objective: The aim of this study was to evaluate the biodistribution of Technetium-99m (99mTc)-Aprotinin solution (99mTc-Aprotinin-S) and 99mTc-Aprotinin loaded microemulsion, which was prepared for the aim of treatment for acute pancreatitis.Method: Aprotinin was radiolabeled with 99mTc. Radiochemical purity was determined with radioactive thin layer chromatography studies. 99mTc-Aprotinin-S and 99mTc-Aprotinin loaded microemulsion (99mTc-Aprotinin-M) was administered to acute edematous, severe necrotizing pancreatitis and air pouch model induced rats. Tissue distribution of Aprotinin was investigated with gamma scintigraphy and biodistribution studies.Results: Aprotinin was radiolabeled by 99mTc with high radiochemical purity (95.430 ± 0.946%). The complex was found to be stable at room temperature up to 6 h. Animal studies have shown that similar to that of other small proteins Aprotinin is accumulated primarily in the kidney. The scintigraphy and biodistribution studies showed that, while i.v. administration of 99mTc-Aprotinin-S distributed mostly in kidneys and bladder, 99mTc-Aprotinin-M, with droplet size of 64.550 ± 3.217 nm, has high uptake in liver, spleen and pancreas.Conclusion: This might be concluding that microemulsions may be suggested as promising formulations for selectively targeting Aprotinin to pancreas inflammation.

Antipyretic, analgesic and anti-inflammatory effects of Kickxia ramosissima

Ethnopharmacological relevanceBranched cancerwort, Kickxia ramosissima (Wall.) Janchen (Scrophulariaceae) is traditionally used for the treatment of inflammatory disorders such as rheumatism, diabetes, jaundice and for activation of immune system. Local communities also used this plant for the treatment of spleen enlargement, as febrifuge and in dysmenorrhea. In this investigation antipyretic, analgesic and anti-inflammatory effects of K. ramosissima have been evaluated. MethodsDried powder of the whole plant of K. ramosissima was extracted with methanol (KRM) and partitioned with solvents to obtain the n-hexane (KRH), chloroform (KRC), ethyl acetate (KRE), n-butanol (KRB) and the residual aqueous (KRA) fraction. KRM and the derived fractions were analyzed for the phytochemical constituents, yeast induced pyrexia, analgesic and anti-inflammatory activities by using carrageenan and Freunds' complete adjuvant-induced paw edema model in rat. On account of appreciable effects of KRM in the aforesaid models, KRM was subjected to the carrageenan induced air pouch model in rat. The exudate of air pouch was analyzed for the count of neutrophils, monocytes, lymphocytes and WBCs and for the estimation of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), nitric oxide (NO) and prostaglandin (PGE2). ResultsPhytochemical investigation of KRM indicated the existence of tannins, flavonoids, alkaloids, coumarins, cardiac glycosides, saponins, terpenoids and phlobatannins. Maximum concentration of total phenolic was determined in KRB followed by KRM while reverse was true for total flavonoids contents. KRM (200mg/kg) distinctly decreased the rectal temperature in yeast induced pyrexia comparable to standard, paracetamol. Pain sensation was effectively inhibited at 200mg/kg p.o. of KRM and KRB as manifested by a decrease (P<0.001) in count of writhing induced with acetic acid and increase of latency time in hot plate. Anti-inflammatory effects of KRM were evident and edema formation induced with carrageenan and Freunds' complete adjuvant-induced paw edema in rat was significantly (P<0.001) inhibited. Count of neutrophils, monocytes, lymphocytes, and WBCs along with inflammatory mediators; IL-6, NO, TNF-α and PGE2 was significantly (P<0.001) decreased in subcutaneous air pouch exudate with KRM. HPLC analysis of KRM indicated the presence of gallic acid, caffeic acid, rutin, catechin and myricetin. ConclusionThe results suggested the anti-inflammatory effects of KRM in inflammation related disorders, might be attributed by the presence of active phytoconstituents; flavonoids, saponins and terpenoids.

Progranulin suppresses titanium particle induced inflammatory osteolysis by targeting TNFα signaling.

Aseptic loosening is a major complication of prosthetic joint surgery, characterized by chronic inflammation, pain, and osteolysis surrounding the bone-implant interface. Progranulin (PGRN) is known to have anti-inflammatory action by binding to Tumor Necrosis Factor (TNF) receptors and antagonizing TNFα. Here we report that titanium particles significantly induced PGRN expression in RAW264.7 cells and also in a mouse air-pouch model of inflammation. PGRN-deficiency enhanced, whereas administration of recombinant PGRN effectively inhibited, titanium particle-induced inflammation in an air pouch model. In addition, PGRN also significantly inhibited titanium particle-induced osteoclastogenesis and calvarial osteolysis in vitro, ex vivo and in vivo. Mechanistic studies demonstrated that the inhibition of PGRN on titanium particle induced-inflammation is primarily via neutralizing the titanium particle-activated TNFα/NF-κB signaling pathway and this is evidenced by the suppression of particle-induced IκB phosphorylation, NF-κB p65 nuclear translocation, and activity of the NF-κB-specific reporter gene. Collectively, these findings not only demonstrate that PGRN plays an important role in inhibiting titanium particle-induced inflammation, but also provide a potential therapeutic agent for the prevention of wear debris-induced inflammation and osteolysis.

Vascular changes and acute inflammation induced by agar in the air pouch model

Objective: was to examine agar as a phlogistic agent in the air pouch model. Method and Materials: Rats were divided into five groups and the effects of 1%, 2%, 3% and 4% agar in the air pouch model were compared with those of 1% carrageenan. The vascular inflammation induced by agar and the microvasculature of the air pouch membrane were analysed. In addition, the role of nitric oxide (NO)-dependent pathways and cellular migration in the responses to 2% agar were evaluated. To assess the mechanism of action underlying the inflammatory effects of agar, rats were treated with a standard anti-inflammatory drug, either celecoxib or acetylsalicylic acid. In addition, a differential leucocyte cell count, total cell count and NO and PGE2 concentrations were determined. Results: The 2% agar was chosen as the optimal concentration. Celecoxib or ASA both inhibited the inflammatory effects of agar, reducing the area of the microvasculature in the tissue lining the air pouch, as well as NO and PGE2 concentrations and cell migration in the exudate. Conclusion: The present study shows that a simple and alternative method (agar) produces consistent results in the air pouch model and can be used as an alternative experimental model of inflammation.

Extracellular Histones Induce Chemokine Production in Whole Blood Ex Vivo and Leukocyte Recruitment In Vivo.

The innate immune system relies to a great deal on the interaction of pattern recognition receptors with pathogen- or damage-associated molecular pattern molecules. Extracellular histones belong to the latter group and their release has been described to contribute to the induction of systemic inflammatory reactions. However, little is known about their functions in the early immune response to an invading pathogen. Here we show that extracellular histones specifically target monocytes in human blood and this evokes the mobilization of the chemotactic chemokines CXCL9 and CXCL10 from these cells. The chemokine induction involves the toll-like receptor 4/myeloid differentiation factor 2 complex on monocytes, and is under the control of interferon-γ. Consequently, subcutaneous challenge with extracellular histones results in elevated levels of CXCL10 in a murine air pouch model and an influx of leukocytes to the site of injection in a TLR4 dependent manner. When analyzing tissue biopsies from patients with necrotizing fasciitis caused by Streptococcus pyogenes, extracellular histone H4 and CXCL10 are immunostained in necrotic, but not healthy tissue. Collectively, these results show for the first time that extracellular histones have an important function as chemoattractants as their local release triggers the recruitment of immune cells to the site of infection.

Ferulic acid ethyl ester diminished Complete Freund's Adjuvant-induced incapacitation through antioxidant and anti-inflammatory activity.

Ferulic acid ethyl ester (FAEE) is a derivate from ferulic acid which reportedly has antioxidant effect; however, its role on inflammation was unknown. In this study, we investigated the orally administered FAEE anti-inflammatory activity on experimental inflammation models and Complete Freund's Adjuvant (CFA)-induced arthritis in rats. CFA-induced arthritis has been evaluated by incapacitation model and radiographic knee joint records at different observation time. FAEE (po) reduced carrageenan-induced paw edema (p < 0.001) within the 1st to 5th hours at 50 and 100 mg/kg doses. FAEE 50 and 100 mg/kg, po inhibited leukocyte migration into air pouch model (p < 0.001), and myeloperoxidase, superoxide dismutase, and catalase activities (p < 0.001) increased total thiol concentration and decreased the TNF-α and IL-1β concentrations, NO, and thiobarbituric acid reactive species. In the CFA-induced arthritis, FAEE 50 and 100 mg/kg significantly reduced the edema and the elevation paw time, a joint disability parameter, since second hour after arthritis induction (p < 0.001). FAEE presented rat joint protective activity in radiographic records (p < 0.001). The data suggest that the FAEE exerts anti-inflammatory activity by inhibiting leukocyte migration, oxidative stress reduction, and pro-inflammatory cytokines.

Prolonged cultures of unstimulated human neutrophils lead to the apparition and persistence of rest-in-plate structures (RIPs) recognized by professional phagocytes in vitro and in vivo

Polymorphonuclear neutrophil cells (PMNs) are known to spontaneously undergo apoptosis and then eliminated by professional phagocytes to prevent inflammation, a process called efferocytosis. However, when efferocytosis is impaired, PMNs will fall into secondary necrosis. Whether this state can persist for a certain period of time is unclear, since most of the studies investigating secondary necrosis are performed within 24h following induction by a proapoptotic agent. In this study, freshly isolated human PMNs were incubated without addition of exogenous agents in order to force them to undergo apoptosis and then secondary necrosis, an ideal experimental condition to study the behavior of secondary necrotic PMNs in absence of efferocytosis. By monitoring PMN cell morphology over time, we observed that an increasing proportion of cells harbored a ghost-like phenotype. Because these cellular remnants persist in plates for several days, we introduce here the terminology RIPs for ‘rest-in-plate’ structure. Heating of freshly isolated PMNs for 5min did not lead to the apparition of RIPs over time. In vivo administration of 7-days old RIPs in the murine air pouch model induced a slight inflammation resorbed within 24h. PKH26-stained RIPs were found to be ingested by professional phagocytes in vitro and in vivo in the murine air pouch and peritonitis models. Therefore, aged-PMNs have the potential to become RIPs in absence of efficient efferocytosis. Fortunately RIPs are recognized by professional phagocytes and, therefore, the concept of resolution of inflammation based on elimination of apoptotic and secondary necrotic PMNs could also be applied to RIPs.

Apremilast, a novel phosphodiesterase 4 (PDE4) inhibitor, regulates inflammation through multiple cAMP downstream effectors.

IntroductionThis work was undertaken to delineate intracellular signaling pathways for the PDE4 inhibitor apremilast and to examine interactions between apremilast, methotrexate and adenosine A2A receptors (A2AR).MethodsAfter apremilast and LPS incubation, intracellular cAMP, TNF-α, IL-10, IL-6 and IL-1α were measured in the Raw264.7 monocytic murine cell line. PKA, Epac1/2 (signaling intermediates for cAMP) and A2AR knockdowns were performed by shRNA transfection and interactions with A2AR and A2BR, as well as with methotrexate were tested in vitro and in the murine air pouch model. Statistical differences were determined using one or two-way ANOVA or Student’s t test. The alpha nominal level was set at 0.05 in all cases. A P value of < 0.05 was considered significant.ResultsIn vitro, apremilast increased intracellular cAMP and inhibited TNF-α release (IC50=104nM) and the specific A2AR-agonist CGS21680 (1μM) increased apremilast potency (IC50=25nM). In this cell line, apremilast increased IL-10 production. PKA, Epac1 and Epac2 knockdowns prevented TNF-α inhibition and IL-10 stimulation by apremilast. In the murine air pouch model, both apremilast and MTX significantly inhibited leukocyte infiltration, while apremilast, but not MTX, significantly inhibited TNF-α release. The addition of MTX (1 mg/kg) to apremilast (5 mg/kg) yielded no more inhibition of leukocyte infiltration or TNF-α release than with apremilast alone.ConclusionsThe immunoregulatory effects of apremilast appear to be mediated by cAMP through the downstream effectors PKA, Epac1, and Epac2. A2AR agonism potentiated TNF-α inhibition by apremilast, consistent with the cAMP-elevating effects of that receptor. Because the A2AR is also involved in the anti-inflammatory effects of MTX, the mechanism of action of both drugs involves cAMP-dependent pathways and is therefore partially overlapping in nature.Electronic supplementary materialThe online version of this article (doi:10.1186/s13075-015-0771-6) contains supplementary material, which is available to authorized users.

Comparison between inflammatory profiles during infections with Staphylococcus aureus and Escherichia coli in a murine air pouch model

Staphylococcus aureus along with Escherichia coli is the most common and one of the oldest human symbiotic bacteria. The Gram- positive bacterium, Staphylococcus aureus , is an important human pathogen that causes a wide variety of diseases, ranging from minor skin infections to life-threatening infections, well as some strains the Escherichia coli , a gram-negative bacterium. In this way, the aim of this study was evaluate inflammatory profiles during infections by Staphylococcus aureus and Escherichia coli in a murine air pouch model. Twelve BALB/c mice had a solution of sterile air inoculated into their dorsal region and were divided into three groups. One group of animals was infected with S. aureus , another group with E. coli and a third group received saline. Twenty-four hours after infection, systemic responses were evaluated by leukocyte counts in the blood, air pouch lavage and bone marrow. Samples of skin were evaluated by morphometry. The IFN-γ present in the air pouch lavage, homogenized spleen and bone marrow, was evaluated by ELISA. The results indicate that the group S. aureus promoted higher cell recruitment to the site of inflammation, confirmed by histopathological analysis. The animals infected with E. coli showed difference in the total blood leucocyte cells evaluation. The dosage of IFN-γ for the study groups was larger than the reference given by the control group. In synthesis, S. aureus induced greater local recruitment of leukocytes.

Preparation, characterization, and safety evaluation of poly(lactide-co-glycolide) nanoparticles for protein delivery into macrophages.

Following infection, HIV establishes reservoirs within tissues that are inaccessible to optimal levels of antiviral drugs or within cells where HIV lies latent, thus escaping the action of anti-HIV drugs. Macrophages are a persistent reservoir for HIV and may contribute to the rebound viremia observed after antiretroviral treatment is stopped. In this study, we further investigate the potential of poly(lactic-co-glycolic) acid (PLGA)-based nanocarriers as a new strategy to enhance penetration of therapeutic molecules into macrophages. We have prepared stable PLGA nanoparticles (NPs) and evaluated their capacity to transport an active molecule into the human monocyte/macrophage cell line THP-1 using bovine serum albumin (BSA) as a proof-of-concept compound. Intracellular localization of fluorescent BSA molecules encapsulated into PLGA NPs was monitored in live cells using confocal microscopy, and cellular uptake was quantified by flow cytometry. In vitro and in vivo toxicological studies were performed to further determine the safety profile of PLGA NPs including inflammatory effects. The size of the PLGA NPs carrying BSA (PLGA-BSA) in culture medium containing 10% serum was ~126 nm in diameter, and they were negatively charged at their surface (zeta potential =−5.6 mV). Our confocal microscopy studies and flow cytometry data showed that these PLGA-BSA NPs are rapidly and efficiently taken up by THP-1 monocyte-derived macrophages (MDMs) at low doses. We found that PLGA-BSA NPs increased cellular uptake and internalization of the protein in vitro. PLGA NPs were not cytotoxic for THP-1 MDM cells, did not modulate neutrophil apoptosis in vitro, and did not show inflammatory effect in vivo in the murine air pouch model of acute inflammation. In contrast to BSA alone, BSA encapsulated into PLGA NPs increased leukocyte infiltration in vivo, suggesting the in vivo enhanced delivery and protection of the protein by the polymer nanocarrier. We demonstrated that PLGA-based nanopolymer carriers are good candidates to efficiently and safely enhance the transport of active molecules into human MDMs. In addition, we further investigated their inflammatory profile and showed that PLGA NPs have low inflammatory effects in vitro and in vivo. Thus, PLGA nanocarriers are promising as a drug delivery strategy in macrophages for prevention and eradication of intracellular pathogens such as HIV and Mycobacterium tuberculosis.

Microemulsion-Based Topical Hydrogels of Tenoxicam for Treatment of Arthritis.

Tenoxicam (TNX) is a non-steroidal anti-inflammatory drug (NSAID) used for the treatment of rheumatoid arthritis, osteoarthritis, ankylosing spondylitis, backache and pain. However, prolonged oral use of this drug is associated with gastrointestinal adverse events like peptic ulceration, thus necessitating its development as topical formulation that could obviate the adverse effects and improve patient compliance. The present study was aimed at development of microemulsion-based formulations of TNX for topical delivery at the affected site. The pseudoternary phase diagrams were developed and microemulsion formulations were prepared using Captex 300/oleic acid as oil, Tween 80 as surfactant and n-butanol/ethanol as co-surfactant. Optimized microemulsions were characterized for drug content, droplet size, viscosity, pH and zeta potential. The ex vivo permeation studies through Laca mice skin were performed using Franz diffusion cell assembly, and the permeation profile of the microemulsion formulation was compared with aqueous suspension of drug and drug incorporated in conventional cream. Microemulsion formulations of TNX showed significantly higher (p < 0.001) mean cumulative percent permeation values in comparison to conventional cream and suspension of drug. In vivo anti-arthritic and anti-inflammatory activity of the developed TNX formulations was evaluated using various inflammatory models such as air pouch model, xylene-induced ear edema, cotton pellet granuloma and carrageenan-induced inflammation. Microemulsion formulations were found to be superior in controlling inflammation as compared to conventional topical dosage forms and showed efficacy equivalent to oral formulation. Results suggest that the developed microemulsion formulations may be used for effective topical delivery of TNX to treat various inflammatory conditions.

In vivo anti-inflammatory effects of isorhamnetin glycosides isolated from Opuntia ficus-indica (L.) Mill cladodes

Opuntia ficus-indica (L.) Mill [OFI] has been traditionally used to reduce inflammation. This plant possesses a varied composition of phytochemicals such as flavonoids. The aim of this investigation was to study the anti-inflammatory effects of an OFI extract and two isolated isorhamnetin glycosides using an air-pouch inflammation model. Isorhamnetin glycosides were isolated by semi-preparative chromatography and identified with LC/MSD TOF. Male Wistar rats were divided into five groups (n=6) receiving isorhamnetin-3-O-glucosyl-rhamnosyl-rhamnoside (IGRR), isorhamnetin-3-O-glucosyl-rhamnoside (IGR), OFI extract, indomethacin or vehicle intraperitoneally. IGR showed the best potential to decrease cell infiltration, mainly neutrophils (51.8±10.9%). IGRR and OFI extract inhibited the nitric oxide production (IGRR: 81.4%; OFI extract: 77.2%) and cyclooxygenase-2 activity (IGRR: 76.3%; OFI extract: 77.7%). OFI extract showed the highest inhibition of tumor necrosis factor (TNF)-α (85.2%) and interleukin (IL)-6 (53.0%). Results showed that OFI extract and isorhamnetin glycosides possess anti-inflammatory potential. IGRR showed better effect than IGR suggesting that the glycosylation profile affects the bioactivity.

Paracrine in vivo inhibitory effects of adipose tissue–derived mesenchymal stromal cells in the early stages of the acute inflammatory response

Background aimsExcessive or unresolved inflammation leads to tissue lesions. Adipose tissue–derived mesenchymal stromal cells (AMSCs) have shown protective effects that may be dependent on the modulation of inflammation by secreted factors. MethodsWe used the zymosan-induced mouse air pouch model at two time points (4 h and 18 h) to evaluate the in vivo effects of AMSCs and their conditioned medium (CM) on key steps of the early inflammatory response. We assessed the effects of AMSCs and CM on leukocyte migration and myeloperoxidase activity. The levels of chemokines, cytokines and eicosanoids in exudates were measured by use of enzyme-linked immunoassay or radio-immunoassay. In addition, the expression of cyclooxygenase-2 and microsomal prostaglandin E synthase-1 (mPGES-1) was studied by use of Western blotting and the phosphorylation of p65 nuclear factor-κB (NF-κB) by immunofluorescence. ResultsAll inflammatory parameters were significantly reduced by CM and AMSCs to a similar extent at 4 h after zymosan injection with lower effects at 18 h. The observed inhibition of leukocyte migration was associated with reduced levels of chemokines and leukotriene B4. Interleukin-1β, interleukin-6, tumor necrosis factor-α and tumor necrosis factor–stimulated gene 6 levels were significantly decreased. The downregulation of mPGES-1 was associated with inhibition of prostaglandin E2 production. Our results suggest that these anti-inflammatory effects are related, in part, to the inhibition of NF-κB activation. ConclusionsAMSCs dampen the early process of inflammation in the zymosan-induced mouse air pouch model through paracrine mechanisms. These results support the potential utility of these cells as a source of novel treatment approaches for inflammatory pathologies.

Etanercept administration prevents the inflammatory response induced by carrageenan in the murine air pouch model.

Rheumatoid arthritis (RA) is one of several inflammatory and autoimmune diseases that affect approximately 1% of world's population. The development of TNF inhibitors in the last decade represents a great advance in the treatment of mild and severe forms of RA. Etanercept is one of these drugs that is useful for RA treatment, but the mechanism of inhibition of the signaling pathway of inflammation was not completely elucidated. This study was conducted to evaluate the anti-inflammatory effect of etanercept in comparison to reference drugs (dexamethasone and indomethacin). Inflammation was induced by subcutaneal administration of carrageenan in the Swiss albino mice using the murine air pouch model. Exudation; leukocytes; myeloperoxidase (MPO); adenosine deaminase (ADA); nitric oxide metabolites (NOx); tumor necrosis factor (TNF); interferon gamma (IFN-γ); interleukins (IL) IL-6, IL-17, IL-10, IL-4, and IL-2; nuclear transcription factor kappa B (NF-κB) activation and apoptosis were evaluated 24 h after the induction of inflammation. Treatment with etanercept significantly inhibited exudate concentrations; leukocyte count; MPO and ADA activities; NOx, TNF, IFN-γ, and IL-17 levels; and NF-kappa B activation (p < 0.05). Etanercept induced apoptosis, reducing the number of viable neutrophils without increasing necrosis (p < 0.05). Our results suggest that the anti-inflammatory mechanism of action of etanercept may be via decrease of NF-κB activation. This effect promoted the reduction of pro-inflammatory cytokines and NOx and the induction of neutrophil apoptosis. The effect of etanercept upon neutrophils apoptosis may indicate the use of this drug therapy in the early stage of rheumatoid arthritis disease.

Definition of a Novel Pathway Centered on Lysophosphatidic Acid To Recruit Monocytes during the Resolution Phase of Tissue Inflammation

Blood-derived monocytes remove apoptotic cells and terminate inflammation in settings as diverse as atherosclerosis and Alzheimer’s disease. They express high levels of the proresolving receptor ALX/FPR2, which is activated by the protein annexin A1 (ANXA1), found in high abundance in inflammatory exudates. Using primary human blood monocytes from healthy donors, we identified ANXA1 as a potent CD14+CD16− monocyte chemoattractant, acting via ALX/FPR2. Downstream signaling pathway analysis revealed the p38 MAPK-mediated activation of a calcium independent phospholipase A2 with resultant synthesis of lysophosphatidic acid (LPA) driving chemotaxis through LPA receptor 2 and actin cytoskeletal mobilization. In vivo experiments confirmed ANXA1 as an independent phospholipase A2–dependent monocyte recruiter; congruently, monocyte recruitment was significantly impaired during ongoing zymosan-induced inflammation in AnxA1−/− or alx/fpr2/3−/− mice. Using a dorsal air-pouch model, passive transfer of apoptotic neutrophils between AnxA1−/− and wild-type mice identified effete neutrophils as the primary source of soluble ANXA1 in inflammatory resolution. Together, these data elucidate a novel proresolving network centered on ANXA1 and LPA generation and identify previously unappreciated determinants of ANXA1 and ALX/FPR2 signaling in monocytes.

Jobelyn® exhibited anti-inflammatory, antioxidant, and membrane-stabilizing activities in experimental models

Jobelyn® (JB) is an African sorghum-based food supplement claimed to be efficacious for the treatment of rheumatoid arthritis (RA). Although in vitro studies confirmed its anti-inflammatory property, no study had shown the effect of JB using in vivo animal models of inflammation. Thus, its effects on acute and chronic inflammation in rats were evaluated in this study. Its effect on rat red blood cell (RBC) lysis was also assessed. Acute inflammation was induced with intraplanter injection of carrageenan and increase in rat paw volume was measured using plethysmometer. The volume of fluid exudates, number of leukocytes, concentrations of malondialdehyde (MDA), and glutathione (GSH) in the fluid were measured on day 5 after induction of chronic inflammation with carrageenan in the granuloma air pouch model. RBC lysis induced by hypotonic medium as determined by release of hemoglobin was measured spectrophotometerically. JB (50-200 mg/kg) given orally produced a significant inhibition of acute inflammation induced by carrageenan in rats. It reduced the volume and number of leukocytes in inflammatory fluid in the granuloma air pouch model of chronic inflammation. It further decreased the levels of MDA in the fluid suggesting antioxidant property. JB elevated the concentrations of GSH in inflammatory exudates indicating free radical scavenging activity. It also significantly inhibited RBC lysis caused by hypotonic medium, suggesting membrane-stabilizing property. JB has in vivo anti-inflammatory activity, which may be related to its antioxidant and membrane-stabilizing properties, supporting its use for the treatment of arthritic disorder.

A transgenic mouse model for the in vivo bioluminescence imaging of the expression of the lysophosphatidic acid receptor 3: relevance for inflammation and uterine physiology research.

Lysophosphatidic acid (LPA) is a lipid-derived signaling molecule that plays key roles in diverse biological processes including inflammation and uterine remodeling. Although the function of LPA and its receptors has been extensively studied using knock-out mice, the temporal-spatial expression of LPA receptors is less well-characterized. To gain further insight into the dynamic regulation of LPA receptor 3 (Lpar3) expression in vivo by bioluminescence imaging, we generated and characterized mice transgenic for a putative Lpar3 promoter fragment. A non-coding region of the Lpar3 gene immediately upstream of the start site was subcloned adjacent to the luciferase gene. Promoter activity was determined by in vitro luciferase assays, in vivo bioluminescent imaging or by semi-quantitative real-time PCR. The air-pouch model was used to investigate Lpar3 promoter activity in the context of inflammation. The putative Lpar3 promoter fragment behaved similarly to the endogenous promoter in vitro and in vivo. In male mice, elevated levels of Lpar3-induced luciferase activity were observed in the testis. In female mice, the basal level of luciferase activity in the uterus significantly increased during pseudopregnancy. Moreover, luciferase activity was upregulated by TNF-α in the air-pouch model. We report the identification of a functional Lpar3 promoter fragment and the generation of a transgenic mouse model to investigate the regulation of Lpar3 promoter activity non-invasively in vivo by bioluminescence imaging. This mouse model is a valuable tool for reproductive biology and inflammation research as well as other biological processes in which this receptor is involved.

Osteopontin regulates Cxcr2 expression and neutrophil recruitment in endodontic infection (INC6P.302)

Abstract The integrin binding protein osteopontin (OPN) protects against bone destruction in a mouse model of endodontic infection. Here we have tested the hypothesis that this effect of OPN is mediated by effects on migration of innate immune cells to the site of infection. We used the subcutaneous chamber and air pouch as models of endodontic infection in mice. In vitro analysis of CXCR2 expression was determined by FACS and migration was assessed using transwell assay. Using both the chamber and a modified air-pouch infection model, we showed that neutrophil accumulation at the site of infection is significantly reduced in OPN KO mice, but there are very few macrophages and monocytes, and there is little effect of OPN on their accumulation. OPN does not affect neutrophil survival. Reduced neutrophil accumulation is accompanied by lower levels of myeloperoxidase activity and an increase in bacterial load in the absence of osteopontin. In vitro, OPN increases the ability of these cells to migrate in transwell assays when added in either to the upper or the lower chambers. Furthermore, OPN increases expression of CXCR2 on bone marrow neutrophils, in an alphav-integrin dependent manner. Biotinylation assays show that OPN suppresses the internalization of CXCR2. Together, these results support a model whereby OPN binding to the alphav integrin regulates CXCR2 internalization on neutrophils, increasing the ability of these cells to migrate in response to CXCR2 ligands.

Crystalline silica-induced leukotriene B4-dependent inflammation promotes lung tumour growth.

Chronic exposure to crystalline silica (CS) causes silicosis, an irreversible lung inflammatory disease that may eventually lead to lung cancer. In this study, we demonstrate that in K-rasLA1 mice, CS exposure markedly enhances the lung tumor burden and genetic deletion of leukotriene B4 receptor1 (BLT1−/−) attenuates this increase. Pulmonary neutrophilic inflammation induced by CS is significantly reduced in BLT1−/−K-rasLA1 mice. CS exposure induces LTB4 production by mast cells and macrophages independent of inflammasome activation. In an air pouch model, CS-induced neutrophil recruitment is dependent on LTB4 production by mast cells and BLT1 expression on neutrophils. In an implantable lung tumor model, CS exposure results in rapid tumor growth and decrease survival that is attenuated in the absence of BLT1. These results suggest that LTB4/BLT1 axis sets the pace of CS-induced sterile inflammation that promotes lung cancer progression. This knowledge will facilitate development of immunotherapeutic strategies to fight silicosis and lung cancer.

CD43 functions as an E‐selectin ligand for Th17 cells in vitro and is required for Th17 cell recruitment in vivo

Th17 cells are recruited at sites of inflammation and participate in important cell‐mediated inflammatory reactions. Their interactions with the vascular endothelium are highly dependent on Endothelial selectin (E‐sel). We hypothesized that Th17 cells use preferentially different E‐sel ligands than Th1 cells. We used real‐time live video microscopy to study the interactions of Th17 cells from different E‐sel ligand deficient mice with E‐sel under flow conditions and blot rolling assays, and the air pouch and experimental autoimmune encephalomyelitis (EAE) models of inflammation. We report that Th17 cells express the E‐sel ligands CD44, PSGL‐1 and CD43. Interestingly, CD43 functions as an E‐sel ligand for Th17 cells in vitro in a unique way that does not require cooperation with PSGL‐1. In vivo, CD43‐/‐ Th17 cells were not recruited into the air pouch in response to TNF‐α or CCL20. Interestingly, CD43‐/‐ mice were partially protected from EAE, and had reduced recruitment of Th17 cells to the central nervous system. Our results demonstrate the relevance of CD43 as an E‐sel ligand for Th17 cells in vitro, and its role in Th17 cell recruitment to sites of inflammation in vivo. Our results suggest the possibility to target Th17 cell recruitment specifically during inflammation. Future studies will unveil alternative mechanisms by which CD43 regulates Th17 cell recruitment in vivo in the context of disease.