Tumor Necrosis Factor-α mRNA Levels Research Articles

Anti-Tumoral Effect and Action Mechanism of Exosomes Derived From Toxoplasma gondii-Infected Dendritic Cells in Mice Colorectal Cancer.

Toxoplasma gondii is an obligate intracellular protozoan with anti-tumor activity against a variety of cancers. However, the therapeutic effect of T. gondii on colorectal cancer is unclear, and using direct Toxoplasma infection in immunotherapy involves safety concerns. This study investigated the anti-tumoral effect and mechanism of exosomes derived from dendritic cells (DCs) infected with T. gondii (Me49-DC-Exo). We used differential ultracentrifugation to isolate exosomes from uninfected DCs (DC-Exo) and T. gondii Me49-infected DCs (Me49-DC-Exo). The isolated exosomes were identified by transmission electron microscopy, nanoparticle tracking analysis, and western blotting. Me49-DC-Exo significantly inhibited the tumor growth and reduced the proportion of M2 macrophages in the blood of tumor-bearing mice. In vitro, Me49-DC-Exo suppressed macrophage (RAW264.7) polarization to M2 phenotype. miRNA sequencing revealed that multiple miRNAs in Me49-DC-Exo were differentially expressed compared with DC-Exo, among which miR-182-5p, miR-155-5p, miR-125b-2-3p, and miR-155-3p were up-regulated, while miR-9-5p was significantly down-regulated. Transfecting mimics or inhibitors of these differential miRNAs into RAW264.7 cells showed that miR-155-5p promoted M1 macrophage polarization while inhibiting M2 macrophage polarization. Bioinformatics prediction and dual-luciferase reporter assay confirmed the suppressor of cytokine signaling 1 (SOCS1) as a direct target of miR-155-5p. Silencing SOCS1 gene expression in RAW264.7 cells increased CD86 + CD206 − M1 macrophage proportion, and inducible nitric oxide synthase and tumor necrosis factor-α mRNA levels. However, arginase-1 and transglutaminase 2 expression levels decreased. These results suggest that the exosomes inhibit macrophage polarization to M2 phenotype and regulate SOCS1 expression by delivering functional miR-155-5p. These findings provide new ideas for colorectal cancer immunotherapy.

D-allulose protects against diabetic nephropathy progression in Otsuka Long-Evans Tokushima Fatty rats with type 2 diabetes.

d-allulose is a rare sugar that has been reported to possess anti-hyperglycemic effects. In the present study, we hypothesized that d-allulose is effective in attenuating the progression of diabetic nephropathy in the Otsuka Long-Evans Tokushima Fatty (OLETF) rat model of type 2 diabetes mellitus. Drinking water with or without 3% d-allulose was administered to OLETF rats for 13 weeks. Long-Evans Tokushima Otsuka rats that received drinking water without d-allulose were used as non-diabetic control rats. d-allulose significantly attenuated the increase in blood glucose levels and progressive mesangial expansion in the glomerulus, which is regarded as a characteristic of diabetic nephropathy, in OLETF rats. d-allulose also attenuated the significant increases in renal IL-6 and tumor necrosis factor-α mRNA levels in OLETF rats, which is a proinflammatory parameter. Additionally, we showed that d-allulose suppresses mesangial matrix expansion, but its correlation with suppressing renal inflammation in OLETF rats should be investigated further. Collectively, our results support the hypothesis that d-allulose can prevent diabetic nephropathy in rats.

Anti-Inflammatory Effect of Charantadiol A, Isolated from Wild Bitter Melon Leaf, on Heat-Inactivated Porphyromonas gingivalis-Stimulated THP-1 Monocytes and a Periodontitis Mouse Model.

Porphyromonas gingivalis has been identified as one of the major periodontal pathogens. Activity-directed fractionation and purification processes were employed to identify bioactive compounds from bitter melon leaf. Ethanolic extract of bitter melon leaf was separated into five subfractions by open column chromatography. Subfraction-5-3 significantly inhibited P. gingivalis-induced interleukin (IL)-8 and IL-6 productions in human monocytic THP-1 cells and then was subjected to separation and purification by using different chromatographic methods. Consequently, 5β,19-epoxycucurbita-6,23(E),25(26)-triene-3β,19(R)-diol (charantadiol A) was identified and isolated from the subfraction-5-3. Charantadiol A effectively reduced P. gingivalis-induced IL-6 and IL-8 productions and triggered receptors expressed on myeloid cells (TREM)-1 mRNA level of THP-1 cells. In a separate study, charantadiol A significantly suppressed P. gingivalis-stimulated IL-6 and tumor necrosis factor-α mRNA levels in gingival tissues of mice, confirming the inhibitory effect against P. gingivalis-induced periodontal inflammation. Thus, charantadiol A is a potential anti-inflammatory agent for modulating P. gingivalis-induced inflammation.

Animal model of intestinal anti-inflammatory effect of ginger-cinnamon complex.

This study evaluated the anti-inflammatory effect of ginger-cinnamon mixture using an animal model of dextran sulfate sodium (DSS)-induced intestinal inflammation. The mice were administered either distilled water or ginger extract (GE), cinnamon subcritical water extract (CSWE), low GE + CSWE (GCL), and high GE + CSWE (GCH) for 21days and drinking water containing 5% DSS for the final 7days to induce intestinal inflammation. We assessed the change of body weight, disease activity index (DAI), histopathological scores, myeloperoxidase (MPO) activity, and mRNA levels. Compared with the DSS group, the GCH group showed increased body weight, inhibited intestinal shortening, and decreased DAI and histopathological score of intestinal inflammation, which was similar to that for the control group. It inhibited MPO activity as well as interleukin (IL)-1β, IL-6, and tumor necrosis factor-α mRNA levels. Therefore, the ginger-cinnamon complex helps to improve intestine inflammation, which is beneficial for gut health.

Deoxynivalenol Induces Intestinal Damage and Inflammatory Response through the Nuclear Factor-κB Signaling Pathway in Piglets

Deoxynivalenol (DON) is highly toxic to animals and humans, but pigs are most sensitive to it. The porcine mucosal injury related mechanism of DON is not yet fully clarified. Here, we investigated DON-induced injury in the intestinal tissues of piglet. Thirty weanling piglets [(Duroc × Landrace) × Yorkshire] were randomly divided into three groups according to single factor experimental design (10 piglets each group). Piglets were fed a basal diet in the control group, while low and high dose groups were fed a DON diet (1300 and 2200 μg/kg, respectively) for 60 days. Scanning electron microscopy results indicated that the ultrastructure of intestinal epithelial cells in the DON-treated group was damaged. The distribution and optical density (OD) values of zonula occludens 1 (ZO-1) protein in the intestinal tissues of DON-treated groups were decreased. At higher DON dosage, interleukin (IL)-1β, IL-6, and tumor necrosis factor-α mRNA levels were elevated in the intestinal tissues. The mRNA and protein levels of NF-κB p65, IκB-α, IKKα/β, iNOS, and COX-2 in the small intestinal mucosa were abnormally altered with an increase in DON concentration. These results indicate that DON can persuade intestinal damage and inflammatory responses in piglets via the nuclear factor-κB signaling pathway.

Tumor necrosis factor-like weak inducer of apoptosis induces inflammation in Graves' orbital fibroblasts.

Tumor necrosis factor-like weak inducer of apoptosis (TWEAK), along with its receptor fibroblast growth factor-inducible (Fn)14, is associated with various biological activities including inflammation. However, its role in the pathogenesis of Graves’ orbitopathy (GO) is unknown. In this study, we investigated the mechanism by which TWEAK regulates inflammatory signaling in orbital fibroblasts from GO patients. We found that TWEAK and tumor necrosis factor-α (TNFA) mRNA levels were upregulated in GO as compared to non-GO tissue samples. TWEAK, TNF receptor (TNFR)1, TNFR2, and TNFR superfamily member 12A mRNA, and TWEAK and Fn14 protein levels were increased by interleukin (IL)-1β and TNF-α treatment. Treatment with exogenous recombinant TWEAK increased the transcript and protein expression of the pro-inflammatory cytokines IL-6, IL-8, and monocyte chemoattractant protein-1 to a greater extent in GO than in non-GO cells, while treatment with the anti-Fn14 antibody ITEM4 suppressed TWEAK-induced pro-inflammatory cytokine release and hyaluronan production. Additionally, the serum level of TWEAK was higher in Graves’ disease patients with (341.86 ± 86.3 pg/ml) as compared to those without (294.09 ± 41.44 pg/ml) GO and healthy subjects (255.33 ± 39.38 pg/ml), and was positively correlated with clinical activity score (r = 0.629, P < 0.001) and thyroid binding immunoglobulin level (r = 0.659, P < 0.001). These results demonstrate that TWEAK/Fn14 signaling contributes to GO pathogenesis. Moreover, serum TWEAK level is a potential diagnostic biomarker for inflammatory GO, and modulating TWEAK activity may be an effective therapeutic strategy for suppressing inflammation and tissue remodeling in GO.

Preventive effects of guanosine on intestinal inflammation in 2, 4-dinitrobenzene sulfonic acid (DNBS)-induced colitis in rats.

Guanosine, a guanine-based purine, is an extracellular signaling molecule exerting anti-inflammatory and antioxidative effects in several in vivo and in vitro injury models. We aimed to investigate its protective effects on 2, 4-dinitrobenzene sulfonic acid (DNBS)-induced colitis in rat. Rats were divided into five groups and colitis was induced by intracolonic instillation of DNBS (15mg/rat). Guanosine (4 or 8mg/kg) was administered for 6days i.p. starting the day of the colitis induction. Body weight loss, stool consistency, colon weight/length, histological analysis, myeloperoxidase activity (MPO) and pro-inflammatory cytokine levels were assessed. Immunoblotting of nuclear factor-κB (NF-κB) p65 protein levels and detection of oxidative and nitrosative stress markers were also performed. Guanosine, in a dose-dependent manner, significantly ameliorated the severity of DNBS-induced colitis, reducing body weight loss and diarrhea incidence, preventing the DNBS-induced macroscopic and microscopic damage to the colonic mucosa, and the MPO increase. Guanosine treatment also lowered interleukin-1β, interleukin-6, and tumor necrosis factor-α mRNA levels. Importantly, guanosine in DNBS rats down-regulated the expression of NF-κB p65 and the levels of reactive oxygen species and nitrite. In conclusion, guanosine exerts beneficial effects in DNBS-induced colitis in rats, through modulation of colonic inflammation, downregulating of NFκB-mediated signaling.

C-X-C Motif Chemokine 12 Enhances Lipopolysaccharide-Induced Osteoclastogenesis and Bone Resorption In Vivo.

C-X-C motif chemokine 12 (CXCL12) belongs to the family of CXC chemokines. Lipopolysaccharide (LPS) induces inflammation-induced osteoclastogenesis and bone resorption, and in recent years, stimulatory effects of CXCL12 on bone resorption have also been reported. In the present study, we investigated the effects of CXCL12 on LPS-induced osteoclastogenesis and bone resorption. LPS was administered with or without CXCL12 onto mouse calvariae by daily subcutaneous injection. Numbers of osteoclasts and bone resorption were significantly elevated in mice co-administered LPS and CXCL12 compared with mice administered LPS alone. Moreover, receptor activator of NF-kB ligand (RANKL) and tumor necrosis factor-α (TNF-α) mRNA levels were higher in mice co-administered LPS and CXCL12 compared with mice administered LPS alone. These in vitro results confirmed a direct stimulatory effect of CXCL12 on RANKL- and TNF-α-induced osteoclastogenesis. Furthermore, TNF-α and RANKL mRNA levels were elevated in macrophages and osteoblasts, respectively, co-treated in vitro with CXCL12 and LPS, in comparison with cells treated with LPS alone. Our results suggest that CXCL12 enhances LPS-induced osteoclastogenesis and bone resorption in vivo through a combination of increasing LPS-induced TNF-α production by macrophages, increasing RANKL production by osteoblasts, and direct enhancement of osteoclastogenesis.

Impact of diabetes on gingival wound healing via oxidative stress.

The aim of this study is to investigate the mechanisms linking high glucose to gingival wound healing. Bilateral wounds were created in the palatal gingiva adjacent to maxillary molars of control rats and rats with streptozotocin-induced diabetes. After evaluating postsurgical wound closure by digital imaging, the maxillae including wounds were resected for histological examinations. mRNA expressions of angiogenesis, inflammation, and oxidative stress markers in the surgical sites were quantified by real-time polymerase chain reaction. Primary fibroblast culture from the gingiva of both rats was performed in high glucose and normal medium. In vitro wound healing and cell proliferation assays were performed. Oxidative stress marker mRNA expressions and reactive oxygen species production were measured. Insulin resistance was evaluated via PI3K/Akt and MAPK/Erk signaling following insulin stimulation using Western blotting. To clarify oxidative stress involvement in high glucose culture and cells of diabetic rats, cells underwent N-acetyl-L-cysteine treatment; subsequent Akt activity was measured. Wound healing in diabetic rats was significantly delayed compared with that in control rats. Nox1, Nox2, Nox4, p-47, and tumor necrosis factor-α mRNA levels were significantly higher at baseline in diabetic rats than in control rats. In vitro study showed that cell proliferation and migration significantly decreased in diabetic and high glucose culture groups compared with control groups. Nox1, Nox2, Nox4, and p47 expressions and reactive oxygen species production were significantly higher in diabetic and high glucose culture groups than in control groups. Akt phosphorylation decreased in the high glucose groups compared with the control groups. Erk1/2 phosphorylation increased in the high glucose groups, with or without insulin treatment, compared with the control groups. Impaired Akt phosphorylation partially normalized after antioxidant N-acetyl-L-cysteine treatment. Thus, delayed gingival wound healing in diabetic rats occurred because of impaired fibroblast proliferation and migration. Fibroblast dysfunction may occur owing to high glucose-induced insulin resistance via oxidative stress.

Angiopoietin-like protein 2 regulates Porphyromonas gingivalis lipopolysaccharide-induced inflammatory response in human gingival epithelial cells.

Angiopoietin-like protein 2 (ANGPTL2) maintains tissue homeostasis by inducing inflammation and angiogenesis. It is produced in infiltrating immune cells or resident cells, such as adipocytes, vascular endothelial cells, and tumor cells. We hypothesized that ANGPTL2 might play an important role as a unique mediator in both systemic and periodontal disease. We demonstrated an increased ANGPTL2 concentration in gingival crevicular fluid from chronic periodontitis patients. Porphyromonas gingivalis lipopolysaccharide (LPS) treatment strongly induced ANGPTL2 mRNA and protein levels in Ca9-22 human gingival epithelial cells. Recombinant human ANGPTL2 increased interleukin 1β (IL-1β), IL-8, and tumor necrosis factor-α (TNF-α) mRNA and protein levels in Ca9-22 cells. Small-interfering (si)RNA-mediated ANGPTL2 knockdown in Ca9-22 cells reduced IL-1β, IL-8 and TNF-α mRNA and protein levels compared with control siRNA (p<0.01) in P. gingivalis LPS-stimulated Ca9-22 cells. Antibodies against integrin α5β1, an ANGPTL receptor, blocked induction of these inflammatory cytokines in P. gingivalis LPS-treated Ca9-22 cells, suggesting that secreted ANGPTL induces inflammatory cytokines in gingival epithelial cells via an autocrine loop. The classic sequential cascade of P. gingivalis LPS → inflammatory cytokine induction is well established. However, in the current study, we reveal a novel cascade comprising sequential P. gingivalis LPS → ANGPTL2 → integrin α5β1 → inflammatory cytokine induction, which might be responsible for inducing potent periodontal disorganization activity in gingival epithelial cells. Via this pathway, ANGPTL2 functions in the pathogenesis of periodontitis and contributes to prolonging chronic inflammation in patients with systemic disease.

Heme Oxygenase-1 Induction and Anti-inflammatory Actions of Atractylodes macrocephala and Taraxacum herba Extracts Prevented Colitis and Was More Effective than Sulfasalazine in Preventing Relapse.

Background/AimsIn inflammatory bowel disease (IBD), repeated bouts of remission and relapse occur in patients and can impose a risk of colitis-associated cancer. We hypothesized that plant extracts of Atractylodes macrocephala (AM) or Taraxacum herba (TH) may be better than sulfasalazine for treating this disease because these extracts can promote additional regeneration.MethodsMurine intestinal epithelial IEC-6 cells were pretreated with AM or TH before a lipopolysaccharide (LPS)-induced challenge. Acute colitis was induced with 7 days of dextran sulfate sodium (DSS) in male C57BL/6 mice, and extracts of AM and TH were administered for 2 weeks before DSS administration.ResultsIn vitro studies demonstrated that AM or TH treatment reduced LPS-induced COX-2 and tumor necrosis factor-α mRNA levels but increased heme oxygenase-1 (HO-1). Oral preadministration of AM and TH rescued mice from DSS-induced colitis by inhibiting inflammatory mediators via inactivated extracellular signal regulated kinase and repressed nuclear factor κB and signal transducer and activator of transcription 3, but the effect was weaker for sulfasalazine than that for the extracts. Anti-inflammatory activities occurred via the inhibition of macrophage and T lymphocyte infiltrations. Unlike sulfasalazine, which did not induce HO-1, TH extracts afforded significant HO-1 induction.ConclusionsBecause the AM or TH extracts were far superior in preventing DSS-induced colitis than sulfasalazine, AM or TH extracts can be considered natural agents that can prevent IBD relapse.

Effects of Exposure to Ozone on the Ocular Surface in an Experimental Model of Allergic Conjunctivitis.

Based on previous findings that ozone can induce an inflammatory response in the ocular surface of an animal model and in cultured human conjunctival epithelial cells, we investigated whether exposure to ozone exacerbates symptoms of allergic conjunctivitis. We evaluated the effects of exposure to ozone on conjunctival chemosis, conjunctival injection, corneal and conjunctival fluorescein staining scores, production of inflammatory cytokines in tears, and aqueous tear production in a mouse model of allergic conjunctivitis. To validate our in vivo results, we used interleukin (IL)-1α-pretreated conjunctival epithelial cells as an in vitro substitute for the mouse model. We evaluated whether exposure to ozone increased the inflammatory response and altered oxidative status and mitochondrial function in IL-1α-pretreated conjunctival epithelial cells. In the in vivo study, ozone induced increases in conjunctival chemosis, conjunctival injection, corneal and conjunctival fluorescein staining scores, and production of inflammatory cytokines, accompanied by a decrease in tear volume. In the in vitro study, exposure to ozone led to additional increases in IL-6 and tumor necrosis factor-α mRNA levels, which were already induced by treatment with IL-1α. Ozone did not induce any changes in cell viability. Pretreatment with IL-1α increased the expression of manganese superoxide dismutase, and exposure to ozone led to additional increments in the expression of this antioxidant enzyme. Ozone did not induce any changes in mitochondrial activity or expression of mitochondrial enzymes and proteins related to mitochondrial function, with the exception of phosphor-mammalian target of rapamycin. Treatment with butylated hydroxyanisole, a free radical scavenger, attenuated the ozone-induced increases in IL-6 expression in IL-1α-pretreated conjunctival epithelial cells. Therefore, we conclude that exposure to ozone exacerbates the detrimental effects on the integrity of the ocular surface caused by conjunctival allergic reactions, and further increases the inflammatory response in IL-1α-pretreated conjunctival epithelial cells.

High glucose induced-macrophage activation through TGF-β-activated kinase 1 signaling pathway.

Transforming growth factor-β-activated kinase 1 (TAK1) plays a pivotal role in innate immune responses and kidney disease, and is critically involved in macrophage activation. However, there is a paucity of data to explore the role of high glucose (HG) in the regulation of TAK1 signaling and its functional role in macrophage activation. We assume that TAK1 signaling in hyperglycemic condition could be a key factor leading to macrophage activation and inflammation response. Mice macrophages were seeded on a 96-well cell culture plate; cell viability was tested after treatment with different concentration of TAK1 inhibitors. Cells were divided into groups (OZ300; MC; NC; HG; HG+OZ30, 100, 300nM) and treated for given time course. Monocyte chemotactic protein1(MCP-1) and tumor necrosis factor-α (TNF-α) mRNA levels were evaluated by qRT-PCR. Flow cytometry and confocal microscopy are used to analyse the activated macrophage induced by HG. Expression levels of p-TAK1, TAB 1, p-JNK, p-p38MAPK, NF-κBpp65 were detected by western blot. Nuclear translocation of NF-κBp65 was assessed by confocal microscopy. Our data revealed that high glucose not only significantly increased macrophage activation and subsequently abnormal high-expression of MCP-1 and TNF-α, but likewise remarkably enhanced TAK1 activation, MAPK phosphorylation, NF-κB expression in macrophages. Furthermore, pharmacological inhibition of TAK1 attenuated high glucose-triggered signal pathways, macrophage activation and inflammatory cytokines in a simulated diabetic environment. Our findings suggested that high glucose activated macrophages mainly in TAK1/MAPKs and TAK1/NF-κB-dependent manners, which lead to the polarization of macrophages towards a pro-inflammatory phenotype, and finally lead to diabetic nephropathy. In sum, the study raises novel data about the molecular mechanisms involved in the high glucose-mediated inflammatory response in macrophages.

Ginkgolide B functions as a determinant constituent of Ginkgolides in alleviating lipopolysaccharide-induced lung injury.

Ginkgolides are the major bioactive components of Ginkgo biloba extracts, however, the exact constituents of Ginkgolides contributing to their pharmacological effects remain unknown. Herein, we have determined the anti-inflammatory effects of Ginkgolide B (GB) and Ginkgolides mixture (GM) at equivalent dosages against lipopolysaccharide (LPS)-induced inflammation. RAW 264.7 cell culture model and mouse model of LPS-induced lung injury were used to evaluate in vitro and in vivo effects of GB and GM, respectively. In RAW 264.7 cells, GB and GM at equivalent dosages exhibit an identical capacity to attenuate LPS-induced inducible nitric oxide synthase mRNA and protein expression and subsequent NO production. Likewise, GB and GM possess almost the same potency in attenuating LPS-induced expression and activation of nuclear factor kappa B (p65) and subsequent increases in tumor necrosis factor-α mRNA levels. In LPS-induced pulmonary injury, GB and GM at the equivalent dosages have equal efficiency in attenuating the accumulation of inflammatory cells, including neutrophils, lymphocytes, and macrophages, and in improving the histological damage of lungs. Moreover, GB and GM at equivalent dosages decrease the exudation of plasma protein to the same degree, whereas GM is superior to GB in alleviating myeloperoxidase activities. Finally, though GB and GM at equivalent dosages appear to reduce LPS-induced IL-1β mRNA and protein levels and IL-10 protein levels to the same degree, GM is more potent than GB to attenuate the IL-10 mRNA levels. Taken together, this study demonstrates that GB functions as the determinant constituent of Ginkgolides in alleviating LPS-induced lung injury.

Pioglitazone blocks ethanol induction of microglial activation and immune responses in the hippocampus, cerebellum, and cerebral cortex in a mouse model of fetal alcohol spectrum disorders.

Fetal alcohol spectrum disorders (FASD) result from fetal exposure to alcohol and are the leading cause of mental retardation in the United States. There is currently no effective treatment that targets the causes of these disorders. Thus, novel therapies are critically needed to limit the neurodevelopmental and neurodegenerative pathologies associated with FASD. A neonatal mouse FASD model was used to examine the role of the neuroimmune system in ethanol (EtOH)-induced neuropathology. Neonatal C57BL/6 mice were treated with EtOH, with or without pioglitazone, on postnatal days 4 through 9, and tissue was harvested 1 day post treatment. Pioglitazone is a peroxisome proliferator-activated receptor (PPAR)-γ agonist that exhibits anti-inflammatory activity and is neuroprotective. We compared the effects of EtOH with or without pioglitazone on cytokine and chemokine expression and microglial morphology in the hippocampus, cerebellum, and cerebral cortex. In EtOH-treated animals compared with controls, cytokines interleukin-1β and tumor necrosis factor-α mRNA levels were increased significantly in the hippocampus, cerebellum, and cerebral cortex. Chemokine CCL2 mRNA was increased significantly in the hippocampus and cerebellum. Pioglitazone effectively blocked the EtOH-induced increase in the cytokines and chemokine in all tissues to the level expressed in handled-only and vehicle-treated control animals. EtOH also produced a change in microglial morphology in all brain regions that was indicative of microglial activation, and pioglitazone blocked this EtOH-induced morphological change. These studies indicate that EtOH activates microglia to a pro-inflammatory stage and also increases the expression of neuroinflammatory cytokines and chemokines in diverse regions of the developing brain. Further, the anti-inflammatory and neuroprotective PPAR-γ agonist pioglitazone blocked these effects. It is proposed that microglial activation and inflammatory molecules expressed as a result of EtOH treatment during brain development contribute to the sequelae associated with FASD. Thus, pioglitazone and anti-inflammatory pharmaceuticals more broadly have potential as novel therapeutics for FASD.

Chromatin immunoprecipitation analysis of bortezomib-mediated inhibition of NFκB recruitment to IL-1β and TNFα gene promoters in human macrophages.

Interleukin-1β (IL-1) and tumor necrosis factor-α (TNF) are important pro-inflammatory cytokines involved in the mediation of the immune response, inflammation, tissue repair, and tumor progression. Regulation of IL-1 and TNF expression is mediated at the level of transcription by the transcription factor NFκB. Inhibition of NFκB activity by the proteasome inhibitor bortezomib (BZ) has been used as a frontline therapy in multiple myeloma and other hematological malignancies. In this chapter, we describe a protocol that uses chromatin immunoprecipitation (ChIP) to analyze the NFκB recruitment to endogenous IL-1 and TNF promoters in BZ-treated human macrophages. Corresponding to the BZ-suppressed mRNA levels of IL-1 and TNF, we show that BZ inhibits p65 NFκB recruitment to IL-1 and TNF promoters. This study specifically uses U937 macrophages, but the protocol could be easily modified to analyze the regulation of NFκB recruitment in other cell types.

Green tea (Camellia sinensis) administration induces expression of immune relevant genes and biochemical parameters in rainbow trout (Oncorhynchus mykiss)

Present study elucidates the efficacy of green tea (Camellia sinensis) on growth performance, immune and antioxidant systems and cytokine gene expression in rainbow trout tissues. Green tea was supplemented at 20, 100, and 500 mg kg−1 diet and fed to fish (average weight: 23.5 g) for 35 days. No remarkable changes in growth performance were observed among all test groups. Lower lipid peroxidation product and higher superoxide dismutase activity were noted in fish received the medium dose of green tea. Significant increase in serum bactericidal activity and total protein were recorded in all treatment groups. All doses of green tea up-regulated Interleukin-1β transcription in the spleen, while Interleukin-1β mRNA level decreased significantly in the kidney of low dose of green tea. Interleukin-6 mRNA level was up-regulated in the spleen of high dose of green tea and liver of middle and high doses of green tea. High dose and medium dose of green tea up-regulated the interleukin-8 transcription in the kidney and liver, respectively. Meanwhile, green tea inhibited the production of interleukin-10 in all treatment groups compared with control group. Medium dose of green tea up-regulated tumor necrosis factor-α transcription in all fish tissues, while high dose and low dose of green tea enhanced tumor necrosis factor-α mRNA levels in the kidney and spleen, respectively. Present study suggests that green tea especially at 100 mg kg−1 feed may effectively enhance the antioxidant system and immune system in rainbow trout.

KK/Ta Mice Administered Lactobacillus plantarum Strain No. 14 Have Lower Adiposity and Higher Insulin Sensitivity.

Excess accumulation of white adipose tissue can lead to obesity-related metabolic abnormalities such as insulin resistance. We previously reported that intragastric administration of Lactobacillus plantarum No. 14 reduced adipocyte size in diet-induced obese C57BL/6 mice. The present study tested whether L. plantarum No. 14 affects adiposity and insulin sensitivity in an animal model of type-2 diabetes mellitus. Male KK/Ta mice were fed a normal-fat diet and intragastrically given L. plantarum No. 14 (108 CFU/mouse) or vehicle daily for 10 weeks. Interscapular brown adipose tissue and inguinal, mesenteric, and retroperitoneal white adipose tissue weights, serum leptin and insulin concentrations, and insulin resistance index (HOMA-IR) were significantly lower in L. plantarum No. 14-fed mice than in vehicle-fed mice. The sum of the inguinal, epididymal, mesenteric and retroperitoneal white adipose tissue weights correlated with serum leptin and non-esterified fatty acid concentrations and HOMA-IR. The mesenteric adipose tissue mRNA levels of monocyte chemoattractant protein-1 and tumor necrosis factor-α were significantly lower in L. plantarum No. 14-fed mice than in vehicle-fed mice. Mesenteric adipose tissue weight correlated with interleukin-6, monocyte chemoattractant protein-1, and tumor necrosis factor-α mRNA levels. HOMA-IR correlated with monocyte chemoattractant protein-1 and tumor necrosis factor-α mRNA levels. These data suggest that L. plantarum No. 14 prevents the development of insulin resistance, which is at least partly attributable to the prevention of obesity, in KK/Ta mice.

Herpes Simplex Virus Vector Mediated Gene Therapy of Tumor Necrosis Factor-α Blockade for Bladder Overactivity and Nociception in Rats

We examined the effects of tumor necrosis factor-α blockade on bladder overactivity and nociception using replication defective HSV vectors expressing tumor necrosis factor-α soluble receptor. HSV vectors expressing tumor necrosis factor-α soluble receptor or β-galactosidase/green fluorescent protein as the control were injected into the bladder wall of female Sprague-Dawley® rats. Green fluorescent protein was observed with fluorescent microscopy in the bladder and L6 dorsal root ganglia. mRNA and protein expression of tumor necrosis factor-α, and interleukin-1β and 6 as well as myeloperoxidase activity in the bladder were determined by quantitative reverse transcriptase-polymerase chain reaction and enzyme-linked immunosorbent assay 4 hours after intravesical resiniferatoxin administration. c-Fos positive neurons were counted in the L6 spinal dorsal horn. Cystometry and behavioral analyses were also performed. Green fluorescent protein expression was confirmed in the bladder and L6 dorsal root ganglia. Resiniferatoxin administration significantly increased tumor necrosis factor-α mRNA and protein levels in the bladder in controls. Tumor necrosis factor-α mRNA was also increased in the tumor necrosis factor-α soluble receptor group, although tumor necrosis factor-α protein up-regulation was suppressed. The up-regulation of interleukin-1β and 6 mRNA and protein levels, and the myeloperoxidase activity seen in controls were suppressed in the tumor necrosis factor-α soluble receptor group. c-Fos positive cells in the L6 spinal dorsal horn were less prominent in the tumor necrosis factor-α soluble receptor group than in controls. On cystometry the significant decrease in intercontraction intervals after resiniferatoxin infusion detected in controls was not seen in the tumor necrosis factor-α soluble receptor group. On behavioral analyses freezing behavior was significantly decreased in the tumor necrosis factor-α soluble receptor group without affecting licking behavior. HSV vector mediated tumor necrosis factor-α blockade gene therapy in the bladder and bladder afferent pathways decreases the bladder pain and overactivity induced by nociceptive bladder stimuli.

TNF-Overexpression in Borna Disease Virus-Infected Mouse Brains Triggers Inflammatory Reaction and Epileptic Seizures

Proinflammatory state of the brain increases the risk for seizure development. Neonatal Borna disease virus (BDV)-infection of mice with neuronal overexpression of tumor necrosis factor-α (TNF) was used to investigate the complex relationship between enhanced cytokine levels, neurotropic virus infection and reaction pattern of brain cells focusing on its role for seizure induction. Viral antigen and glial markers were visualized by immunohistochemistry. Different levels of TNF in the CNS were provided by the use of heterozygous and homozygous TNF overexpressing mice. Transgenic TNF, total TNF (native and transgenic), TNF-receptor (TNFR1, TNFR2), IL-1 and N-methyl-D-aspartate (NMDA)-receptor subunit 2B (NR2B) mRNA values were measured by real time RT-PCR. BDV-infection of TNF-transgenic mice resulted in non-purulent meningoencephalitis accompanied by epileptic seizures with a higher frequency in homozygous animals. This correlated with lower weight gain, stronger degree and progression of encephalitis and early, strong microglia activation in the TNF-transgenic mice, most obviously in homozygous animals. Activation of astroglia could be more intense and associated with an unusual hypertrophy in the transgenic mice. BDV-antigen distribution and infectivity in the CNS was comparable in TNF-transgenic and wild-type animals. Transgenic TNF mRNA-expression was restricted to forebrain regions as the transgene construct comprised the promoter of NMDA-receptor subunit2B and induced up-regulation of native TNF mRNA. Total TNF mRNA levels did not increase significantly after BDV-infection in the brain of transgenic mice but TNFR1, TNFR2 and IL-1 mRNA values, mainly in the TNF overexpressing brain areas. NR2B mRNA levels were not influenced by transgene expression or BDV-infection. Neuronal TNF-overexpression combined with BDV-infection leads to cytokine up-regulation, CNS inflammation and glial cell activation and confirmed the presensitizing effect of elevated cytokine levels for the development of spontaneous epileptic seizures when exposed to additional infectious noxi.