Effects of interferon-γ on cell differentiation and cytokine production of a human monoblast cell line,U937

The U937 cell, a human monoblast cell line, has been used as a model to study the function of human monocytes. We investigated the effects of interferon- (IFN-) on Superoxide anion (O ₂ ^– ) production, cell surface antigens, and cytokine production of U937 cells. IFN- treatment enhanced O ₂ ^– production of fMLP or PMA-stimulated U937 cells. IFN- increased the ratio of CD23 and CD 11b positive cells. The fluorescence intensity of CD14 and CD25 was enhanced by IFN- treatment. U937 cells produced IL-1, IL-1, IL-6, and TNF- by lipopolysaccharide (LPS) stimulation. IFN- treatment enhanced TNF- production, but decreased IL-6 production. These results suggest that IFN- differentiates U937 cells to monocytelike cells and it regulates the production systems of IL-6 and TNF- separately in U937 cells.     

Effects of clofazimine analogues and tumor necrosis factor-α individually and in combination on human polymorphonuclear leukocyte functions in vitro

In the present study the individual and interactive effects of clofazimine, or three analogues of this agent (selected on the basis of similar or superior pro-oxidative properties: B669, B746 and B4021) and human recombinant TNF-α on the generation of antimicrobial oxidants by human polymorphonuclear leukocytes (PMNL), as well as release of granule enzymes from these cells, were investigated in vitro. All four riminophenazines at the concentrations tested (0.5 and 1.0 μg/ml) significantly increased myeloperoxidase (MPO)-mediated iodination, superoxide (0₂·⁻) generation, oxygen (0₂) consumption and chemiluminescence (CL), as well as the release of both primary and secondary granule contents (measured as the release of MPO, lysozyme and vitamin B₁₂-binding protein) by stimulated PMNL. Similar, but less impressive effects were observed with TNF-α (0.4–50.0 ng/ml). When PMNL were preincubated with both TNF-α and clofazimine or its analogues, the observed stimulation of cellular oxidative metabolism and granule enzyme release was at least additive in many assays. These data demonstrate that the spectrum of effects of clofazimine and its analogues on PMNL closely resemble those of TNF-α. Furthermore, TNF-α potentiates the pro-oxidative effects of clofazimine and its analogues on PMNL. Among the riminophenazines tested, clofazimine and B669 appear to be the most potent pro-oxidative agents for PMNL.     

Protocatechuic acid inhibits human dendritic cell functional activation: Role of PPARγ up-modulation

Polyphenols have been shown to exhibit anti-inflammatory, anti-oxidant and immunomodulatory activities. However, the effects of anthocyanins, flavonoids of great nutritional interest, in particular of their metabolite protocatechuic acid (PCA) on the phenotypic and functional maturation of human dendritic cells (DCs) are still largely unknown. In this study, we report that PCA is efficiently taken up and accumulated in human monocyte-derived DCs (MD-DCs). PCA exposure of MD-DCs markedly impaired the production of proinflammatory cytokines and chemokines (i.e. IL-6, IL-8 and CCL2) in response to bacterial endotoxin and leptin, and down-regulated the lipopolysaccharide (LPS)-induced migratory response of MD-DCs to CCL19. Conversely, the phenotypic profile induced by LPS-mediated activation as well as IL-12 production was not affected. Interestingly, we found that PPARγ is a main factor in the PCA-induced effects as blocking its activity abolish PCA capacity to down-regulate IL-6 and IL-8, but not CCL2, secretion and to inhibit MD-DC migration. In keeping with this observation, cytosol to nucleus translocation and PPARγ activity were found to be directly stimulated by PCA exposure of MD-DCs.     

Evaluation of direct and indirect effects of the PPARγ agonist troglitazone on mouse endothelial cell proliferation

Peroxisome proliferator-activated receptor gamma (PPARγ) agonists and PPARγ/α dual agonists are used in the treatment of type 2 diabetes mellitus and hyperlipidemias. In carcinogenicity studies, some of these agonists induced hemangiomas/hemangiosarcomas in mice, but not in rats. We hypothesized that increased endothelial cell (EC) proliferation may be involved in the mechanism of PPAR agonist-induced vascular tumors in mice. We previously showed that the sarcomagenic PPARγ agonist troglitazone (TG) increased EC proliferation in brown and white adipose tissue and liver in mice at sarcomagenic doses (400 and 800 mg/kg) after four weeks of treatment. In vitro, TG had a mitogenic effect on mouse microvascular mouse ECs by increasing cell proliferation and survival. The current studies showed that treatment of mouse ECs in vitro induced alterations in proliferation pathway gene expression, especially the expression of insulin-like growth factor-1, but had no effect on mouse oxidative stress pathways. In vivo, treatment with vitamin E did not inhibit TG-induced EC proliferation in liver and adipose tissue. In addition, no hypoxic effect was detected in adipose tissue of TG-treated mice; however, TG had a minor effect on hepatocellular hypoxia. These results provide additional evidence supporting a direct mitogenic effect in the mode of action of TG-induced hemangiosarcomas in mice.     

Effect of plasmid-mediated stable interferon-γ expression on proliferation and cell death in the SKOV-3 human ovarian cancer cell line

Introduction: High interferon-γ (IFN-γ) expression in tumors has been reported to be a favorable prognostic marker. Continuous exposure of ovarian cancer cells to IFN-γ was previously shown to result in significant growth inhibition and apoptosis. Our goal in this study was to evaluate the effect of plasmid-mediated stable IFN-γ expression on the SKOV-3 human ovarian carcinoma cell line.

Methods: SKOV-3 cells were stably transfected with the pEGFP-IFN-γ plasmid. IFN-γ mRNA was detected by RT-PCR and IFN-γ protein expression was measured by ELISA. Proliferation and cell death in transfected SKOV-3 cells were measured by methyl-thiazolyl tetrazolium (MTT) assay and Hoechst 33258 staining, respectively and compared with untransfected and empty vector-transfected cells.

Results: pEGFP-IFN-γ SKOV-3 cells efficiently expressed and secreted IFN-γ. They exhibited significantly decreased cellular proliferation when compared with control untransfected or empty vector-transfected cells (P?<?0.05). The mode of cell death was primarily apoptosis.

Conclusions: Stable expression of IFN-γ significantly inhibits the proliferation of ovarian carcinoma cells and has the potential to be used in clinical applications to treat ovarian carcinoma in the future.     

In vivo and in vitro evidence that PPARγ ligands are antagonists of leptin signaling in breast cancer

Obesity is a major risk factor for the development and progression of breast cancer. Leptin, a cytokine mainly produced by adipocytes, plays a crucial role in mammary carcinogenesis and is elevated in hyperinsulinemia and insulin resistance. The antidiabetic thiazolidinediones inhibit leptin gene expression through ligand activation of the peroxisome proliferator-activated receptor-γ (PPARγ) and exert antiproliferative and apoptotic effects on breast carcinoma. In this study, we investigated the ability of PPARγ ligands to counteract leptin stimulatory effects on breast cancer growth in either in vivo or in vitro models. The results show that activation of PPARγ prevented the development of leptin-induced MCF-7 tumor xenografts and inhibited the increased cell-cell aggregation and proliferation observed on leptin exposure. PPARγ ligands abrogated the leptin-induced up-regulation of leptin gene expression and its receptors in breast cancer. PPARγ-mediated repression of leptin gene involved the recruitment of nuclear receptor corepressor protein and silencing mediator of retinoid and thyroid hormone receptors corepressors on the glucocorticoid responsive element site in the leptin gene expression regulatory region in the presence of glucocorticoid receptor and PPARγ. In addition, PPARγ ligands inhibited leptin signaling mediated by MAPK/STAT3/Akt phosphorylation and counteracted leptin stimulatory effect on estrogen signaling. These findings suggest that PPARγ ligands may have potential therapeutic benefits in the treatment of breast cancer.     

The role of cell surface markers and enamel matrix derivatives on human periodontal ligament mesenchymal progenitor responses in vitro

Periodontitis is a chronic-, infectious-disease of the human periodontium that is characterized by the loss of supporting tissues surrounding the tooth such as the periodontal ligament (PDL), cementum and alveolar bone. Regeneration of the periodontium is dependent on the participation of mesenchymal stem/stromal cells (MSC) resident in the PDL. Enamel matrix derivative (EMD), an extract from immature porcine enamel rich in amelogenin protein but that also contain bone morphogenetic protein (BMP), is used to treat periodontal defects. The effects of EMD on MSC cells of the PDL are not well characterized. In this in?vitro study, we identify PDL progenitor cells from multiple individuals and demonstrate that EMD stimulates them. We show that the effect of EMD on cell proliferation and migration is mediated through the amelogenin it contains, while the differentiation of these progenitor cells to cell types of mineralized tissue is mainly due to BMP signaling.     

Increased expression of peroxisome proliferator-activated receptor (PPAR)-α and PPAR-γ in human atherosclerosis

To clarify the mechanism of atherosclerosis development in humans, we studied the mRNA and protein expression of PPAR subtypes in various types of atherosclerotic lesions and their correlation with cell proliferation and macrophage invasion. Human aortas were obtained from 35 autopsied cases, and each sample was divided into halves. One half was used for the analysis of mRNA or protein expression with RT-PCR or Western blotting, respectively. The other was microscopically classified into atheromatous plaque (AP), fatty streak (FS), and diffuse intimal thickening (DIT), and was analyzed immunohistochemically. The mRNA levels of both PPARs increased significantly in atherosclerosis and tended to increase in proportion to the severity of the lesion, and the expression of PPAR-α correlated with that of PPAR-γ in FS and AP. The PPAR-γ protein increased in AP. Monocytes/macrophages, as well as endothelial and smooth muscle cells, expressed the PPAR-γ protein in plaques. This expression in the DIT was noted mainly in macrophages but was not correlated with the density of macrophages, suggesting that only certain macrophages express the PPARs in DIT. Cell proliferation did not correlate with PPARs expression in any lesion type. These findings suggest that PPARs may be associated with atheromatous plaque formation, and that PPAR-γ may be involved in the early stages of human atherosclerosis.     

Effects of PPARα/PGC-1α on the energy metabolism remodeling and apoptosis in the doxorubicin induced mice cardiomyocytes in vitro

Dilated cardiomyopathy is the most frequent form of myocardial disease. Many factors contribute to dilated cardiomyopathy, for instance, long-term use of doxorubicin, one of the anthracyclines clinically used for cancer chemotherapy, result in dilated cardiomyopathy and congestive heart failure. However, the mechanism underlining doxorubicin-induced cardiomyocyte is still not fully understood. In this study, we evaluate the effects and their mechanisms of PPARα and PGC-1α pathways in doxorubicin induced mice cardiomyocytes. In vitro, cardiomyocytes isolated from hearts of adult FVB/NJ mice were treated with doxorubicin, GW 6471 (PPARα inhibitors) and WY14643 (PPARα agonists). The expression of PPARα and PGC-1α were detected via western blotting and Quantitative Real-Time PCR methods. Changes in energy and substrate metabolism were analyzed. MTT and flow cytometry were used for cell proliferation and apoptosis analysis. We detected expression of PPARα and PGC-1α was significantly higher in control group than doxorubicin group. Mitochondrial dysfunction was found in doxorubicin group including lower content of high-energy phosphates, significantly decreased mitochondrial ANT transport activity and markedly reduced mitochondrial membrane potential compared with control group. Metabolic remodeling existed in doxorubicin group because of higher concentration of free fatty acid and glucose consumption than of control group. More accumulations of reactive oxygen species were detected in doxorubicin group. The decreased cell viability and increased cell apoptosis observed in doxorubicin group. Severe apoptosis in doxorubicin group was verified by a set of markers including Bax, Bcl-2, cytosolic cytochrome c and caspase-3 up-regulation expression. These findings indicate that the PPARα and PGC-1α are closely involved in energy metabolism remodeling and apoptosis in cardiomyocytes.     

Role of proteinase-activated receptor-2 in anti-bacterial and immunomodulatory effects of interferon-γ on human neutrophils and monocytes

Recent studies show that proteinase-activated receptor-2 (PAR(2)) contributes to the development of inflammatory responses. However, investigations into the precise role of PAR(2) activation in the anti-microbial defence of human leucocytes are just beginning. We therefore evaluated the contribution of PAR(2) to the anti-microbial response of isolated human innate immune cells. We found that PAR(2) agonist, acting alone, enhances phagocytosis of Staphylococcus aureus and killing of Escherichia coli by human leucocytes, and that the magnitude of the effect is similar to that of interferon-γ (IFN-γ). However, co-application of PAR(2) -cAP and IFN-γ did not enhance the phagocytic and bacteria-killing activity of leucocytes beyond that triggered by either agonist alone. On the other hand, IFN-γ enhances PAR(2) agonist-induced monocyte chemoattractant protein 1 (MCP-1) secretion by human neutrophils and monocytes. Furthermore, phosphoinositide-3 kinase and janus kinase molecules are involved in the synergistic effect of PAR(2) agonist and IFN-γ on MCP-1 secretion. Our findings suggest a potentially protective role of PAR(2) agonists in the anti-microbial defence established by human monocytes and neutrophils.     

Effects of PPAR-γ Agonist Treatment on LPS-Induced Mastitis in Rats

PPAR-γ, a member of the nuclear receptor superfamily, plays an important role in lipid metabolism and inflammation. The aim of this study was to investigate the preventive effects of synthetic PPAR-γ agonist rosiglitazone on lipopolysaccharide (LPS)-induced mastitis in rats. The mouse model of mastitis was induced by the injection of LPS through the duct of the mammary gland. Rosiglitazone was injected 1 h before the induction of LPS intraperitoneally. The results showed that rosiglitazone attenuated the infiltration of inflammatory cells, the activity of myeloperoxidase (MPO), and the production of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-1β (IL-1β) in a dose-dependent manner. Additionally, Western blotting showed that rosiglitazone inhibited the phosphorylation of IκB-α and NF-κB p65. These results indicated that rosiglitazone has a protective effect on mastitis, and the anti-inflammatory mechanism of rosiglitazone on LPS-induced mastitis in rats may be due to its ability to inhibit NF-κB signaling pathways. PPAR-γ may be a potential therapeutic target against mastitis.     

Effects of NF-κB and hypoxia on the biological behavior of Y79 retinoblastoma cells

We aimed to investigate the influence of nuclear factor-κB (NF-κB) on the biological behavior of Y79 retinoblastoma cells exposed to hypoxia and its possible mechanism. The cells were administrated with hypoxia, and/or 5 μM pyrrolidine dithiocarbamate (PDTC) (a selective NF-κB inhibitor) to inhibit the NF-κB activity, expressions of NF-κB was measured by western blot, and the translocation of NF-κB was detected. To examine the proliferation of Y79 cells, MTT assay was applied. Transwell assay was used to detect the invasion and migration ability of cells. The expressions of molecules involved in invasion was analyzed including HIF-1α, MMP-2, 9, and VEGF. We found that hypoxia significantly activated NF-κB activity. While once the NF-κB was inhibited, the proliferation, invasion and migration ability of Y79 cells were also blocked. Interestingly, the expressions of invasion-involved molecules elevated by hypoxia induction were also decreased when NF-κB was inhibited. Hypoxia could significantly change the adhesive and invasive ability of Y79 retinoblastoma cells, NF-κB signal might be one of the main mediators for these hypoxia induced cell changes of biological behavior via downregulation of HIF-1α and the invasion related molecules, and the mechanism still needs further investigation.     

Effects of fibrin pad hemostat on the wound healing process in vivo and in vitro

Fibrin Pad is a hemostatic pad designed to control surgical-related bleeding. It consists of a fully absorbable composite matrix scaffold coated with human-derived active biologics that immediately form a fibrin clot upon contact with targeted bleeding surfaces. Studies were conducted to investigate the effect of Fibrin Pad and its biologics-free composite matrix component (Matrix) on the wound healing process in in vitro and in vivo models. Fibrin Pad was evaluated in solid organ, soft tissue defects, and subcutaneous tissues. Immunocompromised rodents were used to avoid xeno-mediated responses. Extracts created from both materials were evaluated for biological activity using in vitro cell culture assays. Neither Fibrin Pad nor Matrix alone showed any inhibition of the wound healing of treated defect sites. An apparent accelerated healing was noted in the soft tissue and subcutaneous tissue defects with Fibrin Pad as compared to Matrix. Both materials showed desirable properties associated with tissue scaffolds. The in vitro study results show that Fibrin Pad extract can induce dose-dependent increases in fibroblast proliferation and migration. These studies confirm that the biologic components of Fibrin Pad can enhance wound healing processes in in vitro assays and fully support wound healing at the site of in vivo application.     

In Vitro Effects of Benzophenone-2 and Octyl-Methoxycinnamate on the Production of Interferon-γ and Interleukin-10 by Murine Splenocytes

Chemical ultraviolet light absorbers (UV-filters) are nowadays widely used in cosmetic and plastic industry. Recent in vitro and in vivo studies have reported that certain chemical UV-filters possess estrogenic activity raising the question of whether these compounds are safe to human health. Work on estrogenic effects of these compounds, however, has focused mostly on reproductive organs, and as the presence of estrogen receptors has been identified in several cells of the immune system, UV screens also may have a great impact on immunity. Thus, we have studied the in vitro effects of two widely used UV-filters—benzophenone-2 (BP-2) and octyl-methoxycinnamate (OMC)—on the production of interferon (IFN)-γ and interleukin (IL)-10, two cytokines representing Th1- and Th2-type response, respectively, by activated murine splenocytes. Cells were cultured on 48-well plastic plates and stimulated with 12-miristate 13-acetate (PMA) (5 ng/ml) and ionomycin (50 ng/ml) in the presence of different concentrations (10^?5–10^?8M) of the studied substances or 17β-estradiol (E2). After 48 hr incubation the supernatants were collected and the levels of IFN-γ and IL-10 were measured using immunoenzymatic assay. Our results show that BP-2 and OMC at high concentrations (10^?5M) shifted the Th1/Th2 balance toward a Th2 response (lower IFN-γ production and higher IL-10). These effects were comparable to those of E2. Our results clearly show that UV-screens at high doses also may possess immunomodulatory effects some of which resemble those of E2.     

Preventive effects of siRNA targeting PPARγ gene on steroid-induced osteonecrosis in rabbits

Purposes/Aim: Glucocorticoid steroids can induce expression of PPARγ gene and enhance adipogenesis by bone marrow mesenchymal stem cells (BMSCs), which may result in osteonecrosis of the femoral head. Currently, there are no medications available to prevent steroid-induced osteonecrosis. We hypothesized that siRNA targeting PPARγ gene may prevent steroid-induced adipogenesis and osteonecrosis in rabbit. The purpose of this study was to evaluate the preventive effects of siRNA targeting PPARγ gene on steroid-induced adipogenesis and osteonecrosis.

Methods: Forty-eight healthy New Zealand rabbits were randomized into four groups with Group M treated with dexamethasone only, Group S with dexamethasone and a recombinant adenovirus shuttle vector carrying siRNA targeting PPARγ gene, Group Con with dexamethasone and a vector carrying irrelative sequence, and Group N with no treatment serving as control. Expressions of the PPARγ, osteocalcin and Runx2 genes, as well as histopathologic changes were evaluated.

Results: The levels of PPARγ gene expression were decreased while the levels of osteocalcin and Runx2 gene expression were increased in rabbits treated with dexamethasone and recombinant adenovirus shuttle vector carrying siRNA targeting PPARγ gene (Group S), compared to rabbits treated either with dexamethasone alone (Group M) or with both dexamethasone and a vector carrying irrelative sequence (Group Con). The marrow necrosis, adipocyte hypertrophy and proliferation, diminished hematopoiesis, thinner and sparse trabeculae, and increased empty osteocyte lacunae in the femoral head were observed in Group M and Group Con rabbits. However, no such changes were seen in Group S rabbits that were treated with dexamethasone and a recombinant adenovirus shuttle vector carrying siRNA targeting PPARγ gene.

Conclusion: siRNA targeting PPARγ gene can inhibit adipogenic differentiation of BMSCs and prevent steroid-induced osteonecrosis in rabbit. The inhibition of bone-marrow adipogenesis and concomitant enhancement of osteogenesis with RNAi may provide a novel approach to the prevention of steroid-induced osteonecrosis.