A-stimulated Cells Research Articles

Anti-inflammatory effects of Athyrium yokoscense extract via inhibition of the Erk1/2 and NF-κB pathways in bisphenol A-stimulated A549 cells.

Bisphenol A is an environmental endocrine disruptor that has similar functions to estrogen in humans. However, few studies have investigated pulmonary inflammation induced by BPA, and the effect of Athyrium yokoscense extract on this inflammatory response is unknown. In this study, we investigated this effect in A549 human alveolar epithelial cells. BPA at concentrations higher than 100 µM were cytotoxic to A549 cells at 24 and 48h after treatment; however, AYE (100µg/mL) had a protective effect against BPA-induced cytotoxicity. AYE also inhibited the generation of intracellular reactive oxygen species, expressions of cyclooxygenase-2 and extracellular signal-regulated kinase1/2 proteins, activities of phospholipase A2, COX-2, nuclear factor kappa-light-chain-enhancer of activated B cells, and proinflammatory mediators including prostaglandin E2, tumor necrosis factor-α, and interleukin-6 induced by BPA in A549 cells. This study demonstrated that BPA, which induces chronic lung disease, causes oxidative stress and inflammatory response in lung epithelial cell line, and found that AYE reduces BPA-induced oxidative stress and inflammatory response by down-regulating the Erk1/2 and NF-κB pathways.

Identification of ANXA2 on epithelial cells as a new receptor for secretory IgA using immunoprecipitation and mass spectrometry.

Secretory immunoglobulin A plays an important role in the protection against exogenous pathogens and antigens, but it has also been reported to have pathogenic potential. We previously found that secretory immunoglobulin A accumulated in the peripheral lungs during idiopathic pulmonary fibrosis and that transferrin receptor/CD71 was partially involved in secretory immunoglobulin A-induced inflammatory cytokine production in A549 cells. This study aimed to identify the receptor responsible for the induction of cytokine production by secretory immunoglobulin A-stimulated airway epithelial cells. To this end, immunoprecipitation followed by time-of-flight mass spectrometry and peptide mass fingerprinting were performed and Annexin A2 was detected as a novel receptor for secretory immunoglobulin A. Enzyme-linked immunosorbent assay demonstrated binding of secretory immunoglobulin A to Annexin A2, and flow cytometry showed robust expression of Annexin A2 on the surface of BEAS-2B cells, A549 cells, and normal human bronchial/tracheal epithelial cells. Experiments in A549 cells using Annexin A2 small interfering RNA and neutralizing antibodies suggested that Annexin A2 was partially involved in the production of interleukin-8/CXCL8 and C-C motif chemokine ligand 2/monocyte chemoattractant protein-1 induced by secretory immunoglobulin A. Immunohistochemistry using lung sections revealed clear expression of Annexin A2 on airway epithelial cells, although the staining remained equivalent in idiopathic pulmonary fibrosis, asthma, and healthy control lungs. In conclusion, we identified that Annexin A2 expressed in airway epithelial cells is a novel receptor for secretory immunoglobulin A, which is involved in cytokine synthesis.

The mRNA and miRNA profiles of goat bronchial epithelial cells stimulated by Pasteurella multocida strains of serotype A and D.

Pasteurella multocida (P. multocida) is a zoonotic bacterium that predominantly colonizes the respiratory tract and lungs of a variety of farmed and wild animals, and causes severe respiratory disease. To investigate the characteristics of the host immune response induced by P. multocida strains of serotype A and D, high-throughput mRNA-Seq and miRNA-Seq were performed to analyze the changes in goat bronchial epithelial cells stimulated by these two serotypes of P. multocida for 4 h. Quantitative RT-PCR was used to validate the randomly selected genes and miRNAs. The results revealed 204 and 117 differentially expressed mRNAs (|log2(Fold-change)| ≥ 1, p-value < 0.05) in the P. multocida serotype A and D stimulated groups, respectively. Meanwhile, the number of differentially expressed miRNAs (|log2(Fold-change)| > 0.1, p-value < 0.05) were 269 and 290, respectively. GO and KEGG enrichment analyses revealed 13 GO terms (p-value < 0.05) and four KEGG pathways (p-value < 0.05) associated with immunity. In the serotype A-stimulated group, the immune-related pathways were the GABAergic synapse and Toll-like receptor signaling pathways, while in the serotype D-stimulated group, the immune-related pathways were the phagosome and B cell receptor signaling pathways. Based on the predicted results of TargetScan and miRanda, the differentially expressed mRNA–miRNA network of immune-related GO terms and KEGG pathways was constructed. According to the cell morphological changes and the significant immune-related KEGG pathways, it was speculated that the P. multocida serotype D strain-stimulated goat bronchial epithelial cells may induce a cellular immune response earlier than serotype A-stimulated cells. Our study provides valuable insight into the host immune response mechanism induced by P. multocida strains of serotype A and D.

Cytokine Expression in Peripheral Blood Mononuclear Cell Cultures Obtained from Cattle with Different Stages of Natural Mycobacterium bovis Infection

In bovine tuberculosis (bTB), cellular, humoral, or both types of immune responses have been observed. The purpose of this study was to examine the immune status of tuberculous cows based on the differential cytokine gene expression associated with Th1 (IFN-γ, IL-2), or Th2 (IL-4, IL-10) responses. Twenty-three (23) cows belonging to a dairy herd located in a rural region of the State of Hidalgo, México, were selected for the study. Single Intradermal Comparative Cervical Tuberculin (SICCT) Test, Interferon-Gamma (IFN-γ) Release Assay (BOVIGAM), and Enzyme-Linked Immunosorbent Assay (ELISA) were used for detection of cattle infected by M. bovis. Thirteen cows were positive to all the tests (Group 1); ten cows were positive only to ELISA (Group 2), and the remaining Group (Group 3, control) included cows negative to all the tests. Peripheral blood mononuclear cells (PBMC) from animals were in vitro stimulated by bovin purified protein derivative (PPD), avian PPD, and Concanavalin A (Con A) mitogen for 72h. Changes in the levels of expression of mRNA of the respective cytokines was measured by Reverse Transcription-Polymerase Chain Reaction (RT-PCR) using β-actin gene as internal control. In group 1, PPD bovis and Con A-stimulated cells exhibited high production of IFN-γ, IL-2 and IL-4, but not IL-10. In contrast, PPD avium-stimulated cells displayed a low production of cytokine transcripts. In group 2, cells showed a significant production of IL-10 in response to bovine PPD (P&lt; 0.001). In the control group, a high production of IFN-γ and IL-2 was observed only in Con A-stimulated cells. Post-mortem examinations in animals of group 1 showed slight and medium lesions in lymph nodes, whereas in group 2, the lesions were more extensive. Results indicate differences on gene expression levels of cytokines considered to determine balance in Th1/Th2 response among the evaluated groups. In addition, high levels of antibodies against M. bovis and high IL-10 expression in PBMC together are indicators of progressive bTB when both tuberculin test and IFN-γ assay are negative in tuberculous anergic cattle. Inclusion of serology and IL-10 cytokine expression in in the diagnosis checklist improves detection of infected cattle to help control bovine tuberculosis.

Determination of phenolic profiles of Herniaria polygama and Herniaria incana fractions and their in vitro antioxidant and anti-inflammatory effects

The study is based on phytochemical profiling and in vitro evaluation of biological effects of phenolic acid derivatives-rich Herniaria fractions, isolated from two rupturewort (Herniaria L.) species, i.e. Herniaria incana Lam. (syn. H. besseri Fisch. ex Hornem) and H. polygama J. Gay (syn. H. odorata). For the first time, the composition of phenolic compounds of these species was extensively evaluated by both LC-HR-QTOF-ESI-MS and Nuclear Magnetic Resonance spectroscopy (NMR). LC-MS analyses of H. polygama revealed 72 tentatively identified compounds, while H. incana – 63. Only 8% of the metabolites reported in this work have been previously described for Herniaria spp. Most of the identified specialized metabolites were cinnamic and benzoic acid derivatives. Phenolic fraction of H. incana herb contained flavonoids as well. A multi-step chromatographic separation of phenolic fractions from H. polygama yielded three known cinnamic and one benzoic acid derivates, and from H. incana – 4 known flavonoids and one previously undescribed, i.e. rhamnocitrin-3-O-[3-hydroxy-3-methylglutaryl-(1 → 6′′)]-[α-rhamnopyranosyl-(1 → 2′′)]-β-glucopyranoside.Antioxidant properties of the examined fractions (1–50 μg/ml) were assessed in human blood plasma under the conditions of peroxynitrite-induced oxidative stress. Measurements of well-known biomarkers such as 3-nitrotyrosine, protein thiol groups, thiobarbituric acid-reactive substances and the ferric reducing ability of blood plasma revealed the protective effect of Herniaria fractions against oxidative damage to blood plasma components. Furthermore, the examined fractions effectively ameliorated the inflammatory response of the concanavalin A-stimulated human peripheral blood mononuclear cells (PBMCs). Additionally, cellular safety of the fractions was confirmed in PBMCs.

Effect of Roasting and Alkalization on the Chemical Composition and In Vitro Anti-Inflammatory Effects of Cocoa

Abstract Objectives Cocoa beans undergo fermentation, roasting, and possibly alkalization prior to consumption. Cocoa and chocolate have been shown to exert anti-inflammatory effects. Previous studies have shown that roasting and alkalization can adversely affect the total phenolic content (TPC) of cocoa, but the effect of these steps on bioactivity has not been well-studied. Our objective was to prepare cocoa powders using different roasting and alkalization protocols and measure their chemical composition and in vitro anti-inflammatory activity. Methods Cocoa beans were roasted (110–150°C) and alkali-treated (0 – 120 min) using a 22 + center point study design. Cocoa nibs were defatted and extracted with 70% aqueous acetone. The extract was dried prior to analysis. TPC was determined using the Folin-Ciocalteu method. Select polyphenols were quantified by liquid chromatography. In vitro anti-inflammatory activity was assessed as inhibition of phospholipase A2 (PLA2) and inhibition of interleukin 8 (IL8) production by tumor necrosis factor a-stimulated HT-29 human colon cells. Results Roasting and alkalization led to decreased TPC, but alkalization had a greater effect. Cocoa beans roasted at 130°C and alkalized for 120 min had 44% lower TPC than those roasted that same way but without alkalization. In the absence of alkalization, beans roasted at 150°C had only a 13% lower TPC than beans roasted at 110°C. Roasting and alkalization also influenced the levels of individual polyphenols, but the effects varied based on the analyte of interest. Roasting tended to enhance the PLA2, inhibitory potency of the cocoa whereas alkalization reduced inhibitory potency. Cocoa that had been roasted at 150°C but not alkalized had the lowest IC50 (14 mg/mL) whereas cocoa that had been roasted at 150°C and alkalized for 120 min had the highest (&amp;gt;100 mg/mL). Similar results were observed for inhibition of IL8 production. Conclusions Roasting and alkalization are important for achieving desired sensory characteristics in cocoa, but these processes adversely affect the levels of polyphenols in cocoa and has been considered inconsistent with maintaining bioactivity. Our results suggest that it is possible to identify processing protocols that balance the sensory characteristics of cocoa with its anti-inflammatory activity. Funding Sources This work was funded by USDA AFRI.

Concentration-dependent effect of silymarin on concanavalin A-stimulated mouse spleen cells in vitro

AbstractAims: Silymarin (SIL), a mixture of phenolic compounds, has a pleiotropic mode of action on various cell types, including immune cells. In this study, we investigated the concentration-dependent effect of SIL on proliferation of concanavalin A (CoA)-stimulated mouse spleen T lymphocytes, their viability, and secretion of IFN-g and IL-4 cytokinesex vivoin relation to gene expressions of transcription factors nuclear factor kappa B and Foxp3. In addition, metabolic activity of T cells was determined as changes in the mitochondrial membrane potential and apoptosis.Material/Methods: Isolated splenocytes were stimulated with lectin CoA and treated with SIL atthe concentrations of 5, 10, 20, and 40 μg/ml for 70 h and unstimulated cells served as the control. Cultures of splenocytes were evaluated for proliferation index following BrdU incorporation and viability of cells after trypan blue staining. Gene expressions of transcription factors and cytokines were assessed using real-time PCR, whereas ELISA test was applied to measure cytokine secretion. Mitochondrial membrane potential and apoptosis were determined by flow cytometry.Results: We demonstrated that CoA-activated mouse spleen T lymphocytes show different susceptibilities to low (£10 μg/ml) and higher (20 and 40 μg/ml) SIL concentrations. Low concentrations resulted in increased proliferation, cytokine secretion, and mitochondrial membrane potential and reduced transition of cells to apoptosis. High concentration of SIL had the opposite effect without exerting significant cytotoxicity and upregulated genes for cytokines and transcription factors on mRNA level. It is possible that individual subpopulations of T cells induced by CoA were differentially affected by the various SIL concentrations and the dose of 40 μg/ml had the profound suppressive effect. This correlated with the highest expression of Foxp3 factor, indicating that this dose stimulated preferential differentiation to Tregs lymphocytes.Conclusions: Treatment with suitable doses of SIL can provide potential benefits in the modulation of host immune functions in various diseases.

GABA potentiate the immunoregulatory effects of Lactobacillus brevis BGZLS10-17 via ATG5-dependent autophagy in vitro

The characterization of mechanisms involved in the positive effects of probiotic bacteria in various pathophysiological conditions is a prerogative for their safe and efficient application in biomedicine. We have investigated the immunological effects of live bacteria-free supernatant collected from GABA-producing Lactobacillus brevis BGZLS10-17 on Concanavalin A-stimulated mesenteric lymph node cells (MLNC), an in vitro model of activated immune cells. We have shown that GABA containing and GABA-free supernatant of Lactobacillus brevis BGZLS10-17 have strong immunoregulatory effects on MLNC. Further, GABA produced by this strain exhibit additional inhibitory effects on proliferation, IFN-γ and IL-17 production by MLNC, and the expression of MHCII and CD80 on antigen presenting cells. At the other hand, GABA-containing supernatants displayed the strongest stimulatory effects on the expression of immunoregulatory molecules, such as Foxp3+, IL-10, TGF-β, CTLA4 and SIRP-α. By looking for the mechanisms of actions, we found that supernatants produced by BGZLS10-17 induce autophagy in different MLNC, such as CD4+ and CD8+ T lymphocytes, NK and NKT cells, as well as antigen presenting cells. Further, we showed that the stimulation of Foxp3+, IL-10 and TGF-β expression by BGZLS10-17 produced GABA is completely mediated by the induction of ATG5 dependent autophagy, and that other molecules in the supernatants display GABA-, ATG5-, Foxp3+-, IL-10- and TGF-β- independent, immunoregulatory effects.

Cytokine Profile and Expression of FYN, ZAP-70 and LAT During Concanavalin A Stimulation in Patients with Resistant Bronchial Asthma

Background: Bronchial asthma (BA) is one of the most spreading chronic lung pathology in the world. The disease is characterized by high heterogeneity of clinical phenotypes including resistant forms which provoke significant clinical problem. Immune shift from Th2 to alternative immunological response is considered to be a mechanism of drug-resistance in BA treatment but this issue is not considerably studied yet. Aims: Detection of distinctive patterns in cytokine secretion and genetic expression (ZAP-70, FYN and LAT) of naïve and concanavalin A stimulated lymphocytes in patients with resistant BA. Materials and methods: The study enrolled ten patients in each group: subjects with treatment resistant BA, severe BA, and controls (30 in total). During the experiment, all patients with BA received treatment according to the condition. For each participant lymphocytes isolation from venous blood was performed. Cells were cultured with concanavalin A and without stimulation. Concentrations of cytokines IL-2, IL-12, TNF-α, IL-4, IL-5, and IL-6 in supernatants were measured with ELISA. Reverse transcription polymerase chain reaction was used to detect the mRNA expression of LAT, ZAP-70, and FYN genes. Results: Significant disease contribution to the lymphocyte secretion profile was established without concanavalin A stimulation: increased levels of IL-2 and IL-4 was observed in lymphocytes of patients with resistant BA if compared to the results of gorup with severe BA. Patients with resistant BA were characterized by weak cytokine response to the stimulation: only TNF-α and IL-5 levels were significantly increased whereas in group with severe BA all cytokines concentrations increased except IL-12, in controls — except IL-12 and IL-2. Significant FYN upregulation was identified in resistant BA group if compared with other groups, and in severe BA patients if compared with controls. The concanavalin A-stimulated cells showed increased expression of ZAP-70 in cells of patients with resistant BA compared to control group. Conclusions: Lymphocytes from patients with resistant BA are characterized by lack of cytokine response to concanavalin A stimulation, alteration of cytokine secretion, and genetic expression profile similar to cells with low sensitivity to apoptosis. The FYN gene is a perspective target for finding approaches to overcome resistance to steroid drugs in bronchial asthma.

Effect of different loads of treadmill exercise on Th1/Th2 cytokine balance in rat splenocytes

The effect of moderate and overtraining exercise on Th1/Th2 balance was evaluated in rat splenocytes. Male Wistar rats were divided into sedentary control (C), moderately trained (MT; V=20m/min, 30min/day, 8 weeks), overtrained (OT; V=25m/min, 60min/day, 11 weeks) and recovered after overtraining (OR) (OT plus 2 weeks recovery) groups. At the end of study, cell viability, proliferation, interleukin 4 (IL-4) and interferon-γ (IFN-γ) secretion were evaluated in non-stimulated, phytohemagglutinin (PHA) and concavaline A (Con A)-stimulated splenocytes. Cell viability increased in MT and OR groups compared to control. Cell proliferation was higher in OR group than other groups. IL-4 concentration in PHA-stimulated cells from MT and OT groups, and IL-4 concentration in Con A-stimulated cells from OR and OT groups, were higher than the control group, but not for IFN-γ. In non-stimulated cells, IFN-γ/ IL-4 ratio was higher than MT and OT groups. In PHA and Con A-stimulated cells, IFN-γ/ IL-4 ratio was lower in exercise groups than control. We previously showed that moderate exercise increases Th1 cytokines in serum, but in splenocytes, Th2 or Th1 response may increase depending on the type of mitogen stimulation. Two-week recovery restored Th1/Th2 balance, only in non-stimulated splenocytes of overtrained animals.

Up-regulation of μ-, δ- and κ-opioid receptors in concanavalin A-stimulated rat spleen lymphocytes

Regulation of μ-, δ- and κ-opioid receptor protein level in spleen lymphocytes when stimulated by mitogen is not known. To answer the question whether these cells do express opioid receptor (OR) proteins, primary, fresh rat spleen lymphocytes were prepared and stimulated for 48 h with mitogenic dose of Con A. The unstimulated lymphocytes did not express μ- and δ-OR proteins in detectable amounts, however, stimulation with Con A resulted in appearance of clearly detectable immunoblot signals of both μ-OR and δ-OR. κ-OR were detected already in primary cells and increased 2.4-fold in Con A-stimulated cells. These results were supported by data obtained by flow cytometry analysis indicating a dramatic increase in number of μ-, δ- and κ-OR expressing cells after mitogen stimulation. The newly synthesized μ-, δ- and κ-OR in Con A-stimulated spleen lymphocytes were present in the cells interior and not functionally mature, at least in terms of their ability to enhance activity of trimeric G proteins determined by three different protocols of agonist-stimulated, high-affinity [35S]GTPγS binding assay. The up-regulation of μ-, δ- and κ-OR was associated with specific decrease of their cognate trimeric G proteins, Gi1α/Gi2α; the other Gα and Gβ subunits were unchanged. The level of β-arrestin-1/2 was also decreased in Con A-stimulated splenocytes. We conclude that up-regulation of OR expression level in spleen lymphocytes by Con A proceeds in conjunction with down-regulation of their intracellular signaling partners, Gi1α/Gi2α proteins and β-arrestin-1/2. These regulatory proteins are expressed in high amounts already in unstimulated cells and decreased by mitogen stimulation.

Pretreatment of Low-Dose and Super-Low-Dose LPS on the Production of In Vitro LPS-Induced Inflammatory Mediators

Pretreatment of low-dose lipopolysaccharide (LPS) induces a hyporesponsive state to subsequent secondary challenge with high-dose LPS in innate immune cells, whereas super-low-dose LPS results in augmented expression of pro-inflammatory cytokines. However, little is known about the difference between super-low-dose and low-dose LPS pretreatments on immune cell-mediated inflammatory and hepatic acute-phase responses to secondary LPS. In the present study, RAW 264.7 cells, EL4 cells, and Hepa-1c1c7 cells were pretreated with super-low-dose LPS (SL-LPS: 50 pg/mL) or low-dose LPS (L-LPS: 50 ng/mL) in fresh complete medium once a day for 2~3 days and then cultured in fresh complete medium for 24 hr or 48 hr in the presence or absence of LPS (1∼10 µg/mL) or concanavalin A (Con A). SL-LPS pretreatment strongly enhanced the LPS-induced production of tumor necrosis factor (TNF)-α, interleukin (IL)-6, TNF-α/IL-10, prostaglandin E2 (PGE2), and nitric oxide (NO) by RAW 264.7 cells compared to the control, whereas L-LPS increased IL-6 and NO production only. SL-LPS strongly augmented the Con A-induced ratios of interferon (IFN)-γ/IL-10 in EL4 cells but decreased the LPS-induced ratios of IFN-γ/IL-10 compared to the control, while L-LPS decreased the Con A- and LPS-induced ratios of IFN-γ/IL-10. SL-LPS enhanced the LPS-induced production of IL-6 by Hepa1c1c-7 cells compared to the control, while L-LPS increased IL-6 but decreased IL-1β and C reactive protein (CRP) levels. SL-LPS pretreatment strongly enhanced the LPS-induced production of TNF-α, IL-6, IL-10, PGE2, and NO in RAW 264.7 cells, and the IL-6, IL-1β, and CRP levels in Hepa1c1c-7 cells, as well as the ratios of IFN-γ/IL-10 in LPS- and Con A-stimulated EL4 cells compared to L-LPS. These findings suggest that pre-conditioning of SL-LPS may contribute to the mortality to secondary infection in sepsis rather than pre-conditioning of L-LPS.

Metabolite of ellagitannins, urolithin A induces autophagy and inhibits metastasis in human sw620 colorectal cancer cells

Autophagy is an evolutionarily conserved pathway in which cytoplasmic contents are degraded and recycled. This study found that submicromolar concentrations of urolithin A, a major polyphenol metabolite, induced autophagy in SW620 colorectal cancer (CRC) cells. Exposure to urolithin A also dose‐dependently decreased cell proliferation, delayed cell migration, and decreased matrix metalloproteinas‐9 (MMP‐9) activity. In addition, inhibition of autophagy by Atg5‐siRNA, caspases by Z‐VAD‐FMK suppressed urolithin A‐stimulated cell death and anti‐metastatic effects. Micromolar urolithin A concentrations induced both autophagy and apoptosis. Urolithin A suppressed cell cycle progression and inhibited DNA synthesis. These results suggest that dietary consumption of urolithin A could induce autophagy and inhibit human CRC cell metastasis. Urolithins may thus contribute to CRC treatment and offer an alternative or adjunct chemotherapeutic agent to combat this disease.

Emodin induces apoptosis of concanavalin A-stimulated murine splenocytes.

Emodin, one of the major compounds in the herb Reynoutria elliptica, is known to maintain immunosuppressive, anti-allergic, anti-cancer, and anti-inflammatory effects. In this study, we assessed the possibility of using emodin to induce apoptosis in stimulated immune cells in vitro. After treatment with emodin and concanavalin A (Con A), we observed DNA damage-induced apoptosis in splenocytes. Moreover, treatment with emodin and Con A increased the number of apoptotic splenocytes compared with untreated controls. Emodin also diminished the size of CD45R/B220+ cells, CD19+CD69+ cells, and cDC populations. These results indicate that emodin-induced apoptosis was involved in attenuating the immune activity promoted by DNA damage and in decreasing the number of CD45R/B220+ B cells and CD19+CD69+ activating B cells. This demonstration of emodin inducing apoptosis of Con A-stimulated immune cells indicates its potential utility as a therapy for diseases caused by abnormally activated immune cells.

Anti-inflammatory potential of ursolic acid in Mycobacterium tuberculosis-sensitized and concanavalin A-stimulated cells.

Ursolic acid (3-β-3-hydroxy-urs-12-ene-28-oic-acid; UA) is a triterpenoid carboxylic acid with various pharmaceutical properties. It is commonly found in apples, basil, berries, rosemary, peppermint, lavender, oregano, thyme, hawthorn and prunes. In the present study, the activities of UA against the Mycobacterium tuberculosis H37Rv‑induced release of a panel of inflammatory cytokines, including tumor necrosis factor-α (TNF-α), interleukin (IL)-1β and IL-6 from RAW 264.7 murine macrophages, A549 alveolar epithelial cells and in concanavalin A (Con A)-stimulated rat splenocytes were investigated. In addition, the present study examined the ability of UA to reduce the expression levels of the inflammatory mediators, cyclooxygenase‑2 (COX‑2) and inducible nitric oxide synthase (iNOS) in the stimulated cells. The reduction of nitric oxide (NO) release by UA was also examined in the stimulated cells. UA significantly inhibited the mRNA expression levels of TNF‑α, IL‑1β and IL‑6 in the stimulated cells. The expression levels of COX‑2 and iNOS were also suppressed by UA, as was the release of NO at a significant level. The data indicated the potency of UA on different cell types, which may assist in the development of anti‑inflammatory drugs. In the case of adjunct host‑directed immune therapy for tuberculosis, UA may be used, in addition to established antibiotic therapies, to improve treatment efficacy and outcome due to their anti‑inflammatory potential. Further detailed investigations are required to establish its use as an anti-inflammatory.

Immunomodulatory and cytotoxic effects of Nigella sativa and thymoquinone on rat splenocytes

Three different concentrations of Nigella sativa (N. sativa) ethanolic extract, thymoquinone (TQ), dexamethasone, and saline were examined to see whether they had any effects on cell viability, proliferation, and interleukin 4 (IL-4) and interferon-γ (IFN-γ) secretion in non-stimulated, phytohemagglutinin (PHA) and concavaline A (Con A)-stimulated splenocytes. In PHA and Con A-stimulated splenocytes, cell viability and proliferation were increased and Con A shifted cytokine profile towards Th2 balance. Dexamethasone treatment showed a suppression in viability, IFNγ and IL-4 secretion in non-stimulated and stimulated splenocytes. Extract and TQ reduced the viability and inhibited the proliferation of stimulated and non-stimulated splenocytes concentration-dependently. Higher concentrations of N. sativa (1000 mg/ml) and TQ (5 and 10 mg/ml) reduced the secretion of IL-4 in stimulated cells. Two higher concentrations of N. sativa had decreased IFNγ secretion in both stimulated and non-stimulated cells. In non-stimulated cells, only the highest and in Con A-stimulated cells, all TQ concentrations had inhibited IFNγ secretion. The highest concentration of N. sativa increased IFNγ/IL-4 ratio in both stimulated and non-stimulated cells while higher concentrations of TQ only had the same effect on stimulated cells. N. sativa and TQ showed cytotoxic inhibitory effect on rat splenocytes and on Th1/Th2 cytokines concentration-dependently. Higher concentrations of extract and TQ increased cytokines balance in Th1/Th2.

Modulation of immune function by milk fat globule membrane isolates

The nutritional value and characterization of minor milk components on mammalian immune function are not fully understood. The aim of this research was to test the ability of a milk fat globule membrane (MFGM) isolate to modulate murine immune function in vitro, by studying its effects on splenocyte proliferation, apoptosis, and cytokine production. Proliferation of spleen cells was not affected by the MFGM isolate; however, in the presence of polyclonal activators, the MFGM isolate suppressed cell proliferation. Results obtained by flow cytometry did not support programmed cell death as the cause of the MFGM immune-modulating capacity. A mode of suppression on the splenocyte activation process was suggested from a marked decrease in the production of IFN-γ and tumor necrosis factor-α cytokines, typical indicators of immune cell activation. The effect of MFGM on IL-4 secretion was significantly less than that for the other 2 cytokines. The activity exerted by the MFGM over concanavalin A-stimulated cells differed from that observed in cells treated with lipopolysaccharide, suggesting a different mode of action depending on the activator used. These results indicate the potential of MFGM extracts as functional ingredients with bioactive modulating capacity.

Mitogen-activated protein kinase/IκB kinase/NF-κB-dependent and AP-1-independent CX3CL1 expression in intestinal epithelial cells stimulated with Clostridium difficile toxin A

Clostridium difficile toxin A causes acute colitis associated with inflammatory cell infiltration and increased production of proinflammatory mediators. Although CX3CL1 (fractalkine) plays a role in chemoattracting monocytes/macrophages, NK cells, and T cells, little information is available on the regulated expression of CX3CL1 in response to toxin A stimulation. In this study, we investigated the role of C. difficile toxin A on CX3CL1 induction in intestinal epithelial cells. Stimulation of murine intestinal epithelial cells with toxin A resulted in the upregulation of CX3CL1. Expression of CX3CL1 was dependent on nuclear factor-kappaB (NF-κB) and IκB kinase (IKK) activation, while the suppression of activator protein-1 (AP-1) did not affect toxin A-induced CX3CL1 expression. Suppression of p38 mitogen-activated protein kinase (MAPK) significantly inhibited IKK-NF-κB signaling leading to CX3CL1 induction in C. difficile toxin A-stimulated cells. CX3CL1 was mainly secreted from the basolateral surfaces in toxin A-treated cells. Furthermore, inhibition of p38 activity attenuated the toxin A-induced upregulation of CX3CL1 in the mouse ileum in vivo. These results suggest that a pathway, including p38 MAPK, IKK, and NF-κB activation, is required for CX3CL1 induction in intestinal epithelial cells exposed to C. difficile toxin A and may regulate the development of intestinal inflammation induced by infection with toxigenic C. difficile. C. difficile toxin A causes colitis with inflammatory cell infiltration. CX3CL1 plays a role in chemoattracting immune cells. MAPK-NF-κB signaling is required for CX3CL1 induction in toxin A-exposed cells. CX3CL1 is mainly secreted from the basolateral surfaces. CX3CL1 may contribute to the regulation of toxigenic C. difficile infection.

FOXL2-induced follistatin attenuates activin A-stimulated cell proliferation in human granulosa cell tumors

Human granulosa cell tumors (GCTs) are rare, and their etiology remains largely unknown. Recently, the FOXL2 402C>G (C134W) mutation was found to be specifically expressed in human adult-type GCTs; however, its function in the development of human GCTs is not fully understood. Activins are members of the transforming growth factor-beta superfamily, which has been shown to stimulate normal granulosa cell proliferation; however, little is known regarding the function of activins in human GCTs. In this study, we examined the effect of activin A on cell proliferation in the human GCT-derived cell line KGN. We show that activin A treatment stimulates KGN cell proliferation. Treatment with the activin type I receptor inhibitor SB431542 blocks activin A-stimulated cell proliferation. In addition, our results show that cyclin D2 is induced by treatment with activin A and is involved in activin A-stimulated cell proliferation. Moreover, the activation of Smad signaling is required for activin A-induced cyclin D2 expression. Finally, we show that the overexpression of the wild-type FOXL2 but not the C134W mutant FOXL2 induced follistatin production. Treatment with exogenous follistatin blocks activin A-stimulated cell proliferation, and the overexpression of wild-type FOXL2 attenuates activin A-stimulated cell proliferation. These results suggest that FOXL2 may act as a tumor suppressor in human adult-type GCTs by inducing follistatin expression, which subsequently inhibits activin-stimulated cell proliferation.

Molecular Design of a Highly Selective and Strong Protein Inhibitor against Matrix Metalloproteinase-2 (MMP-2)

Synthetic inhibitors of matrix metalloproteinases (MMPs), designed previously, as well as tissue inhibitors of metalloproteinases (TIMPs) lack enzyme selectivity, which has been a major obstacle for developing inhibitors into safe and effective MMP-targeted drugs. Here we designed a fusion protein named APP-IP-TIMP-2, in which the ten amino acid residue sequence of APP-derived MMP-2 selective inhibitory peptide (APP-IP) is added to the N terminus of TIMP-2. The APP-IP and TIMP-2 regions of the fusion protein are designed to interact with the active site and the hemopexin-like domain of MMP-2, respectively. The reactive site of the TIMP-2 region, which has broad specificity against MMPs, is blocked by the APP-IP adduct. The recombinant APP-IP-TIMP-2 showed strong inhibitory activity toward MMP-2 (Ki(app) = 0.68 pm), whereas its inhibitory activity toward MMP-1, MMP-3, MMP-7, MMP-8, MMP-9, or MT1-MMP was six orders of magnitude or more weaker (IC50 > 1 μm). The fusion protein inhibited the activation of pro-MMP-2 in the concanavalin A-stimulated HT1080 cells, degradation of type IV collagen by the cells, and the migration of stimulated cells. Compared with the decapeptide APP-IP (t&frac12; = 30 min), APP-IP-TIMP-2 (t&frac12; ≫ 96 h) showed a much longer half-life in cultured tumor cells. Therefore, the fusion protein may be a useful tool to evaluate contributions of proteolytic activity of MMP-2 in various pathophysiological processes. It may also be developed as an effective anti-tumor drug with restricted side effects.