Monovalent antibody-conjugated lipid-polymer nanohybrids for active targeting to desmoglein 3 of keratinocytes to attenuate psoriasiform inflammation.

To improve the treatment of psoriasiform inflammation, we developed actively targeted nanocarriers loaded with the phosphodiesterase 4 inhibitor AN2728. Methods: Phospholipid-poly(lactic-co-glycolic acid) nanohybrids were prepared and conjugated with monovalent anti-desmoglein 3 antibody to bind keratinocytes. Results: The actively targeted nanohybrids were 229 nm in mean size with a nearly neutral surface charge. Flow cytometry and confocal microscopy showed a 9-fold increase in keratinocyte uptake of targeted nanohybrids relative to non-targeted nanoparticles. The nanoparticles localized mainly in lysosomes after internalization. AN2728-loaded antibody-conjugated nanocarriers inhibited cytokine/chemokine overexpression in activated keratinocytes without affecting cell viability. The targeted nanohybrids also suppressed neutrophil migration by reducing CXCL1 and CXCL2 release from keratinocytes. Following subcutaneous administration in mice, the nanohybrids distributed to the epidermis and hair follicles. In a psoriasis-like skin mouse model, the actively targeted nanoparticles were superior to free drug and non-targeted nanoparticles in mitigating skin inflammation. Intervention with the targeted nanosystem reduced the epidermal thickness of the psoriasiform lesion from 191 to 42 µm, decreased the Psoriasis Area Severity Index by 74%, restored barrier function, and returned chemokine levels to baseline. Conclusions: Our developed nanosystem was safe and demonstrated efficient targeting properties for the treatment of cutaneous inflammation.

[Effect of resveratrol on the expression of MIP-2 in P.e-LPS-induced mouse osteoblasts].

PURPOSE: To investigate the effect of lipopolysaccharides(LPS) extracted from Porphyromonas endodontalis(P.e) on the expression of macrophage inflammatory protein-2 (MIP-2) mRNA and protein levels in MC3T3-E1 cells and the influence of resveratrol on the expression of MIP-2 protein in P.e-LPS induced cells. METHODS: MC3T3-E1 cells were treated with different concentrations of P.e-LPS(0-50 mg/L) and 20 mg/L P.e-LPS for different time (0-48 h). The expression of MIP-2 mRNA and protein was detected by real-time RT-PCR and enzyme linked immunosorbent assay (ELISA). MC3T3-E1 cells were pretreated with resveratrol for 1 h in the presence of 20 mg/L P.e-LPS for 24 h，which was detected by ELISA. Statistical analysis was performed using one-way ANOVA and Dunnett t test with SPSS 13.0 software package. RESULTS: Treatment of MC3T3-El cells with different concentrations of P.e-LPS(0-50 mg/L) caused a significantly increase in MIP-2 mRNA and protein expression in dose-dependent manners.The expression of MIP-2 protein increased from (41.86±2.49) ng/L to (3126.74±158.30) ng/L, and the difference was significant(P<0.05). In the observation time (0-48 h), the impact of 20 mg/L P.e-LPS on induction of MIP-2 in MC3T3-El cells exhibited a time-dependent manner. At 48 h, the maximal induction of MIP-2 protein expression was (2102.55±123.27) ng/L(P<0.01). Incubation of cells with 10 µmol/L resveratrol for 1h significantly decreased the expression of MIP-2 protein from (1805.33±67.54) ng/L to（813.82±47.21) ng/L, and the difference was significant(P<0.05). CONCLUSIONS: The results suggest that P.e-LPS may mediate MIP-2 expression in MC3T3-E1 cells, and resveratrol has a significant inhibitory effect on this process.

Local release of pioglitazone (a peroxisome proliferator-activated receptor γ agonist) accelerates proliferation and remodeling phases of wound healing.

Peroxisome proliferator-activated receptor γ (PPARγ) is a member of the nuclear receptor superfamily known for its anti-inflammatory and macrophage differentiation effects, as well as its ability to promote fat cell differentiation and reduce insulin resistance. Pioglitazone (Pio) is a PPARγ agonist used clinically as an anti-diabetic agent for improving insulin sensitivity in patients with diabetes. The objective of this study was to develop a drug delivery system (DDS) for the local release of Pio to promote wound healing. Pio of low aqueous solubility was water-solubilized by micelles formed from gelatin grafted with L-lactic acid oligomers, and incorporated into a biodegradable gelatin hydrogel. An 8-mm punch biopsy tool was used to prepare two skin wounds on either side of the midline of 8-week-old mice. Wounds were treated by the hydrogels with (Pio-hydrogel group) or without (control group) Pio, and the wound area were observed 1, 4, 7, and 14 days after treatment. In addition, a protein assay and immunohistological stain were performed to determine the effects of the Pio-hydrogel on inflammation and macrophage differentiation. The Pio-hydrogels promote wound healing. Moreover, Western blotting analysis demonstrated that treatment with Pio-hydrogels resulted in decreased levels of the cytokines MIP-2 and TGF-ß, and increased levels of glucose-regulating adiponectin. It is concluded that Pio-incorporated hydrogels promote the proliferation and remodeling phases of wound healing, and may prove to be effective as wound dressings.

Efficacy of purified lactonase and ciprofloxacin in preventing systemic spread of Pseudomonas aeruginosa in murine burn wound model.

Pseudomonas aeruginosa is an important etiological agent associated with systemic infection in burn patients. Quorum sensing (QS) mechanism of P. aeruginosa contributes to its virulence. Inhibition of QS signals can serve as an effective anti-virulence strategy. Aim of the present study was to evaluate the efficacy of lactonase alone and in combination with ciprofloxacin in treating P. aeruginosa murine burn wound infection. Topical application of lactonase alone and ciprofloxacin alone prevented systemic spread of P. aeruginosa through burned skin and also reduced the mortality. When lactonase (topical) and ciprofloxacin (I/P) were used in combination, zero mortality was observed. It not only significantly reduced systemic dissemination and severity of histopathologic lesions but also resulted in skin regeneration. Decreased production of pathologic index factors (malondialdehyde and reactive nitrogen intermediates) and interleukins (IL-10, IL-6 and MIP-2) was also observed in comparison to control. The results of present study suggest that combination of lactonase and ciprofloxacin can potentially attenuate the virulence of P. aeruginosa. This is the first report of topical administration of lactonase along with antibiotic for the efficient control of burn wound infection.

NFATc3 regulates trypsinogen activation, neutrophil recruitment, and tissue damage in acute pancreatitis in mice.

BACKGROUND & AIMS: The signaling mechanisms that regulate trypsinogen activation and inflammation in acute pancreatitis (AP) are unclear. We explored the involvement of the calcium- and calcineurin-dependent transcription factor nuclear factor of activated T cells (NFAT) in development of AP in mice. METHODS: We measured levels of myeloperoxidase and macrophage inflammatory protein 2 (CXCL2), trypsinogen activation, and tissue damage in the pancreas 24 hours after induction of AP by retrograde infusion of taurocholate into the pancreatic ducts of wild-type, NFAT luciferase reporter (NFAT-luc), and NFATc3-deficient mice. We isolated acinar cells and measured NFAT nuclear accumulation, trypsin activity, and expression of NFAT-regulated genes. RESULTS: Infusion of taurocholate increased the transcriptional activity of NFAT in the pancreas, aorta, lung, and spleen of NFAT-luc mice. Inhibition of NFAT with A-285222 blocked taurocholate-induced activation of NFAT in all organs. A-285222 also reduced taurocholate-induced increases in levels of amylase, myeloperoxidase, and CXCL2; activation of trypsinogen; necrosis of acinar cells; edema; leukocyte infiltration; and hemorrhage in the pancreas. NFATc3-deficient mice were protected from these effects of taurocholate. Similar results were obtained using an l-arginine-induced model of AP. Reverse-transcription polymerase chain reaction and confocal immunofluorescence analyses showed that NFATc3 is expressed by acinar cells. NFATc3 expression was activated by stimuli that increase intracellular calcium levels, and activation was prevented by the calcineurin blocker cyclosporin A or A-285222. Activation of trypsinogen by secretagogues in acinar cells was prevented by pharmacologic inhibition of NFAT signaling or lack of NFATc3. A-285222 also reduced expression of inflammatory cytokines such as CXCL2 in acinar cells. CONCLUSIONS: NFATc3 regulates trypsinogen activation, inflammation, and pancreatic tissue damage during development of AP in mice and might be a therapeutic target.

Mineral trioxide aggregate stimulates macrophages and mast cells to release neutrophil chemotactic factors: role of IL-1beta, MIP-2 and LTB(4).

OBJECTIVE: In the present study, the role of macrophages and mast cells in mineral trioxide aggregate (MTA)-induced release of neutrophil chemotactic factor was investigated. STUDY DESIGN: MTA suspension (50 mg/mL) was plated over inserts on macrophages or mast cells for 90 minutes. Untreated cells served as controls. Cells were washed and cultured for 90 minutes in RPMI without the stimuli. Macrophages and mast cell supernatants were injected intraperitoneally (0.5 mL/cavity), and neutrophil migration was assessed 6 hours later. In some experiments, cells were incubated for 30 minutes with dexamethasone (DEX, 10 muM/well), BWA4C (BW, 100 muM/well) or U75302 (U75, 10 muM/well). The concentration of Leukotriene B(4) (LTB(4)) in the cell-free supernatant from mast cells and macrophage culture was measured by ELISA. RESULTS: Supernatants from MTA-stimulated macrophages and mast cells caused neutrophil migration. The release of neutrophil chemotactic factor by macrophages and mast cells was significantly inhibited by DEX, BW, or U75. Macrophages and mast cells expressed mRNA for interleukin-1 (IL-1)beta and macrophage inflammatory protein-2 (MIP-2) and the pretreatment of macrophages and mast cells with DEX, BW, or U75 significantly altered IL-1beta and MIP-2 mRNA expression. LTB(4) was detected in the MTA-stimulated macrophage supernatant but not mast cells. CONCLUSIONS: MTA-induces the release of neutrophil chemotactic factor substances from macrophages and mast cells with participation of IL-1beta, MIP-2, and LTB(4).

The temporal expression, cellular localization, and inhibition of the chemokines MIP-2 and MCP-1 after traumatic brain injury in the rat.

The expression of the neutrophil chemokine macrophage inflammatory protein-2 (MIP-2/CXCL2) and the monocyte chemokine monocyte chemotactic protein-1 (MCP-1/CCL2) have been described in glial cells in vitro but their origin following TBI has not been established. Furthermore, little is known of the modulation of these chemokines. Chemokine expression was investigated in male Sprague-Dawley rats following moderate lateral fluid percussion injury (LFPI). At 0, 4, 8, 12, and 24 h after injury, brains were harvested and MIP-2/CXCL2 and MCP-1/CCL2 levels measured by ELISA. To investigate the inhibition of chemokine expression a second cohort of animals received dexamethasone (1-15mg/kg), FK506 (1mg/kg), or vehicle, systemically, immediately after injury. These animals were sacrificed at the time of peak chemokine expression. A third cohort of animals was also sacrificed at the time of peak chemokine expression and immunohistochemistry performed for MIP-2/CXCL2 and MCP-1/CCL2. Following LFPI, chemokines were increased in the ipsilateral hemisphere, MIP-2/CXCL2 peaking at 4 h and MCP-1/CCL2 peaking at 8-12 h post-injury. Dexamethasone significantly reduced cortical MCP-1/CCL2, but not MIP-2/CXCL2 concentrations. FK506 did not inhibit chemokine expression. In undamaged brain, chemokine expression was localized to cells with a neuronal morphology. For MIP-2/CXCL2 this was supported by double staining for the neuronal antigen NeuN. In contused tissue, increased MIP-2/CXCL2 and MCP-1/CCL2 staining was visible in cells with the morphology of degenerating neurons. MIP-2/CXCL2 and MCP-1/CCL2 are increased after injury, and neurons appear to be the source of this expression. Chemokine expression was selectively inhibited by dexamethasone. The implications of this are discussed.

Characterization of the pharmacokinetics of human recombinant erythropoietin in blood and brain when administered immediately after lateral fluid percussion brain injury and its pharmacodynamic effects on IL-1beta and MIP-2 in rats.

This study sought to determine the bio-availability of recombinant human erythropoietin (EPO) in the brain and blood and its effects on the cerebral concentrations of the inflammatory mediators interleukin-1beta (IL-1beta) and macrophage-inflammation protein-2 (MIP-2) following lateral fluid percussion brain injury (FPI) in the rat. After induction of moderate FPI (1.6-1.8 atm), EPO was injected intraperitoneally (IP) or intravenously (IV) at doses of 1000-5000 U/kg in a randomized and blinded manner. Animals were then sacrificed at time points (4, 8, 12, 24 h) post-trauma, and the brain concentrations of EPO, IL-1beta, and MIP-2 were determined. EPO administration leads to a dose-dependent increase in the brain concentration of the drug; however, this could only be detected at doses of 3000 and 5000 U/kg. The cerebral concentration peaked in the first 4 h following trauma. EPO concentrations were significantly higher and decreased more slowly in the traumatized cortex compared to the contralateral side (p<0.0125). IV EPO (5000 U/kg) produced slightly higher concentrations of EPO than same doses injected IP; however, this was not significant. At a dose of 5000 U/kg, EPO significantly reduced the increase in IL-1beta at 8 and 12 h in both cortical sides. It also reduced the increase in MIP-2 but only after 8 h, on the contralateral side and after 12 h on the ipsilateral side. Our results suggest that EPO crosses the blood-brain barrier (BBB) by 4 h after trauma and is localized primarily in the traumatized cortex. Further, it has biological efficacy at 8 h on several inflammatory proteins, yet must be employed at high doses to cross the BBB.

Caspofungin modulates inflammatory responses to Aspergillus fumigatus through stage-specific effects on fungal beta-glucan exposure.

Echinocandins target fungal beta-1,3 glucan synthesis and are used clinically to treat invasive aspergillosis. Although echinocandins do not completely inhibit in vitro growth of Aspergillus fumigatus, they do induce morphological changes in fungal hyphae. Because beta-1,3 glucans activate host antifungal pathways via the Dectin-1 receptor, we investigated the effect of echinocandins on inflammatory responses to A. fumigatus. Caspofungin- or micafungin-treated conidia and germlings induced less secretion of tumor necrosis factor (TNF) and CXCL2 by macrophages than did their untreated counterparts. Diminished secretion of TNF and CXCL2 correlated with diminished beta-glucan exposure on echinocandin-treated germ tubes. In contrast to treated conidia and germlings, echinocandin-treated hyphae stimulated increased release of TNF and CXCL2 by macrophages and demonstrated intense staining with a beta-glucan-specific antibody, particularly at hyphal tips. Our experiments demonstrate that echinocandin-induced morphological changes in A. fumigatus hyphae are accompanied by increased beta-glucan exposure, with consequent increases in Dectin-1-mediated inflammatory responses by macrophages.

Effect of oleic and linoleic acids on the inflammatory phase of wound healing in rats.

Inflammation is a crucial step for the wound healing process. The effect of linoleic and oleic acids on the inflammatory response of the skin during the healing process and on the release of pro-inflammatory cytokines by rat neutrophils in vitro was investigated. A wound in the dorsal surface of adult rats was performed and fatty acids were then topically administered. Both oleic and linoleic acids increased the wound healing tissue mass. The total protein and DNA contents of the wounds were increased by the treatment with linoleic acid. The treatments with oleic and linoleic acids did not affect vascular permeability. However, the number of neutrophils in the wounded area and air pouches was increased and the thickness of the necrotic cell layer edge around the wound was decreased. A dose-dependent increase in vascular endothelial growth factor-alpha (VEGF-alpha) and interleukin-1beta (IL-1beta) by neutrophils incubated in the presence of oleic and linoleic acid was observed. Oleic acid was able to stimulate also the production of cytokine-induced neutrophil chemoattractant in inflammation 2 alpha/beta (CINC-2alpha/beta). This pro-inflammatory effect of oleic and linoleic acids may speed up the wound healing process.