The mechanism underlying CD4CD25Foxp3regulatory T cells (Tregs) promoting the development of colorectal cancer (CRC) was elucidated in the present study. Forty-eight cases of colorectal carcinomas, 22 cases of colon polyps and 21 cases of normal colorectal tissues were collected. The correlation among Foxp3, IL-10 and Stat3, and the clinical relevance of these three indexes were analyzed. The results showed that the levels of Foxp3 expressed in infiltrating CD4CD25Foxp3Tregs, and IL-10 and Stat3 in CRC tissues were all significantly higher than those in polypus tissues and normal colon tissues (P< 0.01). Pearson correlation analysis indicated that the expression level of Foxp3 was positively correlated with Stat3 at mRNA level (r=0.526, P=0.036), and was positively correlated with IL-10 at protein level (r=0.314, P=0.030). The Foxp3 expressed in CD4CD25Foxp3Tregs was correlated with the histological grade, lymph node metastasis and TNM stage of CRC (P<0.05 for all). The IL-10 expression was correlated with the histological grade and TNM stage (both P<0.05). The Stat3 expression was correlated with the lymph node metastasis and TNM stage (both P<0.05). It was concluded that CD4CD25Foxp3Tregs can inhibit tumor immunity in combination with some other related inhibitory cytokines and that Foxp3 expression in CD4CD25Foxp3Tregs correlates with CRC progression.
Adult ; Aged ; CD4-Positive T-Lymphocytes ; immunology ; Colorectal Neoplasms ; genetics ; immunology ; pathology ; Female ; Forkhead Transcription Factors ; biosynthesis ; genetics ; immunology ; Gene Expression Regulation, Neoplastic ; immunology ; Humans ; Immunity ; genetics ; Interleukin-10 ; biosynthesis ; immunology ; Interleukin-2 Receptor alpha Subunit ; immunology ; Lymphatic Metastasis ; Male ; Middle Aged ; STAT3 Transcription Factor ; biosynthesis ; immunology ; T-Lymphocytes, Regulatory ; immunology

OBJECTIVETo study the effect of oral administration of dimethyl dimethoxy biphenyl dicarboxylate (DDB) on adjusting angiogeneic/inflammatory mediators and ameliorating the pathology of bones in rats with collagen-induced arthritis (CIA).

METHODSWistar rat model of CIA was set up using bovine collagen type II. Fifty rats were divided into five groups randomly: normal, CIA model, DDB treatment, methotrexate (MTX) treatment, and combined DDB+MTX treatment. Ankle joints of rats were imaged with digital X-ray machine to show the destruction of joints. Fore and hind paw and knee joints were removed above the ankle joint then processed for haematoxylin and eosin staining. Plasma levels of vascular endothelial growth factor (VEGF), platelet derived growth factor, interleukin-8 (IL-8), IL-4, tumor necrosis factor α (TNF-α), and cyclooxygenase-2 (COX-2) were quantified by enzyme-linked immunosorbent assay. Nitric oxide levels were detected by Griess reagent.

RESULTSCompared with the CIA model group, a remarkable reduction in various angiogenic (VEGF and IL-8) and inflammatory mediators (TNF-α, IL-4 and COX-2) after treatment with DDB either alone or combined with MTX P<0.05 or P<0.01). Histopathological and X-ray findings were confirmatory to the observed DDB anti-arthritic effect. The DDB-treated group showed amelioration in signs of arthritis which appeared essentially similar to normal.

CONCLUSIONOur data shed light on the therapeutic efficacy of DDB in experimental rheumatoid arthritis (RA) compared with a choice drug (MTX) and it may be offered as a second-line drug in the treatment of RA.

Animals ; Arthritis, Experimental ; chemically induced ; diagnostic imaging ; drug therapy ; pathology ; Arthritis, Rheumatoid ; diagnostic imaging ; drug therapy ; pathology ; Collagen ; Cyclooxygenase 2 ; blood ; Dioxoles ; therapeutic use ; Enzyme-Linked Immunosorbent Assay ; Female ; Interleukin-4 ; blood ; Interleukin-8 ; blood ; Methotrexate ; therapeutic use ; Nitric Oxide ; biosynthesis ; Platelet-Derived Growth Factor ; analysis ; Radiography ; Rats ; Rats, Wistar ; Tumor Necrosis Factor-alpha ; blood ; Vascular Endothelial Growth Factor A ; blood

The relationship between T cell immunoglobulin domain and mucin domain protein 3 (Tim-3)/Galectin (Gal)-9 pathway and recurrent spontaneous abortion (RSA) was studied. Thirty-one pregnant women with RSA and 27 normal early gravidas were investigated to detect the levels of Tim-3 and Gal-9 in villi and deciduas by Western blotting. Meanwhile, the concentration of interleukin (IL)-4 and IL-12 in peripheral blood plasma was determined by ELISA in 25 healthy fertile non-pregnant controls, the normal early gravidas and pregnant women with RSA mentioned above, respectively. It was found that the relative expression levels of Tim-3 and Gal-9 in villi and deciduas were significantly increased in pregnant women with RSA as compared with those in the normal early gravidas. The concentration of IL-4 in peripheral blood plasma of pregnant women with RSA was lower than that of the normal early gravidas (P<0.05) and healthy fertile non-pregnant controls (P<0.05), but that of IL-2 in pregnant women with RSA was significantly higher than that of the normal early gravidas (P<0.05) and healthy fertile non-pregnant controls (P<0.05). It was suggested that the overexpression of Tim-3/Gal-9 pathway may be related to the pathogenesis of RSA.
Abortion, Spontaneous ; metabolism ; pathology ; Adolescent ; Adult ; Chorionic Villi ; metabolism ; pathology ; Female ; Galectins ; biosynthesis ; Hepatitis A Virus Cellular Receptor 2 ; Humans ; Interleukin-12 ; blood ; Interleukin-4 ; blood ; Membrane Proteins ; biosynthesis ; Pregnancy ; Pregnancy Proteins ; biosynthesis ; Up-Regulation

OBJECTIVEBuckwheat has been considered as a potential source of nutraceutical components on the world market of probiotic foodstuffs. The purpose of this study was to evaluate the effects of tartary buckwheat (Fagopyrum tataricum) sprouts on oxidation and pro-inflammatory mediators.

METHODSThe anti-oxidant effects of buckwheat extract (BWE) and rutin were evaluated by using 1,1-diphenyl-2-picrylhydrazyl (DPPH)- and nitric oxide (NO)-scavenging activities, serum peroxidation and chelating assays. Lipopolysaccharide (LPS)-stimulated RAW264.7 cells were used to evaluate anti-inflammatory activities of buckwheat and rutin. NO production in LPS-stimulated RAW264.7 cells was determined by using Griess reagent. The expressions of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), nuclear factor-kappa B (NF-κB) p65 subunit in cytosolic and nuclear portions were determined by Western blot analysis. Also, the production of inflammatory cytokines interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) was determined by enzyme-linked immunosorbent assay.

RESULTSInhibitory concentration 50 values for DPPH- and NO-scavenging activities of BWE were 24.97 and 72.54 μg/mL respectively. BWE inhibited serum oxidation and possessed chelating activity. Furthermore, BWE inhibited IL-6 and TNF-α production in LPS-stimulated RAW264.7 cells. Also, BWE inhibited iNOS and COX-2 expression and NF-κB p65 translocation.

CONCLUSIONBuckwheat sprouts possessed strong antioxidant activity and inhibited production of pro-inflammatory mediators in the applied model systems. Thus, buckwheat can be suggested to be beneficial in inflammatory diseases by inhibiting the free radicals and inflammatory mediators.

Animals ; Cells, Cultured ; Cyclooxygenase 2 ; analysis ; Fagopyrum ; Free Radical Scavengers ; pharmacology ; Inflammation Mediators ; antagonists & inhibitors ; Interleukin-6 ; biosynthesis ; Lipopolysaccharides ; pharmacology ; Macrophages ; drug effects ; metabolism ; Mice ; NF-kappa B ; metabolism ; Nitric Oxide ; biosynthesis ; Nitric Oxide Synthase Type II ; analysis ; Plant Extracts ; pharmacology ; Tumor Necrosis Factor-alpha ; biosynthesis

The paracaspase MALT1 is essential for the activation of NF-κB in response to T cell receptor (TCR) stimulation. It recruits downstream TRAF6 and activates the E3 ligase activity of TRAF6 to polyubiquitinate several targets, which ultimately leads to NF-κB activation. Here we identified ubiquitin-specific protease 2a (USP2a) as a MALT1-associated protein by biochemical affinity purification. Endogenous USP2a constitutively interacted with TRAF6, but dynamically interacted with MALT1 and CARMA1 in a stimulation-dependent manner. RNA interference (RNAi)-mediated silencing of USP2a attenuated TCR-induced NF-κB activation and production of interleukin-2 (IL-2). In addition, the ubiquitination of MALT1 and TRAF6 were both suppressed by USP2a knockdown. By knockdown and reconstitution assays, we found that USP2a mediated the interaction between MALT1 and TRAF6 in a catalytic activity-dependent manner. Furthermore, USP2a deSUMOylated TRAF6. Our findings implicate that USP2a plays an important role in TCR signaling by deSUMOylating TRAF6 and mediating TRAF6-MALT1 interaction.
Caspases ; metabolism ; Endopeptidases ; deficiency ; genetics ; metabolism ; Gene Knockdown Techniques ; HEK293 Cells ; Humans ; Interleukin-2 ; biosynthesis ; Jurkat Cells ; Mucosa-Associated Lymphoid Tissue Lymphoma Translocation 1 Protein ; NF-kappa B ; metabolism ; Neoplasm Proteins ; metabolism ; Receptors, Antigen, T-Cell ; metabolism ; Signal Transduction ; Sumoylation ; TNF Receptor-Associated Factor 6 ; metabolism

The aim of this study was to separate and amplify CD4(+)CD25(+)Treg cells from splenocytes of sensitized mice. The percentage of CD4(+)CD25(+)Treg cells was detected by flow cytometry in sensitized and normal control mice. CD4(+)T, CD4(+)CD25(+)Treg and CD4(+)CD25(-) T cells were isolated from mouse splenocytes by MACS. CD4(+)CD25(+)Treg cells were expanded in vitro cultures in addition of CD3/CD28 MACSiBead and IL-2. The activity of cells was detected with 0.4 trypan blue staining. The purity of cells after sorting, the main surface marker and the level of Foxp3 were detected by flow cytometry. The results showed that CD4(+)CD25(+)Treg cell proportion was higher in sensitized mice than normal control mice (P < 0.05). The average purity of CD4(+)CD25(+)Treg cells was 87. The activity of these cells was more than 97, and the expression of Foxp3 in these cells was high. The amplification multiples achieved 42 times after 2 weeks in vitro. The percentage of CD4(+)CD25(+) regulatory T cells was 85.32, and the expression of Foxp3 decreased from (76.92 ± 1.72) to (75.33 ± 2.11) (P > 0.05). It is concluded that the sorting of CD4(+)CD25(+)Treg cells is isolated successfully by MACS without affecting the vitality of target cells. The amplification of CD4(+)CD25(+)Treg cells is successful in vitro. Expression of surface markers and Foxp3 gene does not obviously change after amplification, so that to establish a practical method to recover and enlarge the amount of CD4(+)CD25(+)Treg cells in good condition.
Animals ; CD4 Antigens ; biosynthesis ; Flow Cytometry ; Forkhead Transcription Factors ; metabolism ; Immunomagnetic Separation ; methods ; Interleukin-2 Receptor alpha Subunit ; metabolism ; Lymphocyte Count ; Mice ; Mice, Inbred C57BL ; T-Lymphocytes, Regulatory ; cytology

In order to get a better understanding of the role of protease-activated receptor 2 (PAR2) in type 2 helper T (Th2) cell responses against Trichinella spiralis infection, we analyzed Th2 responses in T. spiralis-infected PAR2 knockout (KO) mice. The levels of the Th2 cell-secreted cytokines, IL-4, IL-5, and IL-13 were markedly reduced in the PAR2 KO mice as compared to the wild type mice following infection with T. spiralis. The serum levels of parasite-specific IgE increased significantly in the wild type mice as the result of T. spiralis infection, but this level was not significantly increased in PAR2 KO mice. The expression level of thymic stromal lymphopoietin, IL-25, and eotaxin gene (the genes were recently known as Th2 response initiators) of mouse intestinal epithelial cells were increased as the result of treatment with T. spiralis excretory-secretory proteins. However, the expression of these chemokine genes was inhibited by protease inhibitor treatments. In conclusion, PAR2 might involve in Th2 responses against T. spiralis infection.
Animals ; Antibodies, Helminth/blood ; Chemokine CCL11/biosynthesis ; Cytokines/biosynthesis ; Female ; Gene Expression Profiling ; Immunoglobulin E/blood ; Interleukin-13/secretion ; Interleukin-4/secretion ; Interleukin-5/secretion ; Interleukins/biosynthesis ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; Receptor, PAR-2/*metabolism ; Th2 Cells/*immunology ; Trichinella spiralis/*immunology ; Trichinellosis/*immunology

Recent clinical evidence indicates that the non-eosinophilic subtype of severe asthma is characterized by fixed airway obstruction, which may be related to emphysema. Transgenic studies have demonstrated that high levels of IFN-gamma in the airways induce emphysema. Fibroblast growth factor 2 (FGF2), which is the downstream mediator of TGF-beta, is important in wound healing. We investigated the role of FGF2 in IFN-gamma-induced emphysema and the therapeutic effects of recombinant FGF2 in the prevention of emphysema in a severe non-eosinophilic asthma model. To evaluate the role of FGF2 in IFN-gamma-induced emphysema, lung targeted IFN-gamma transgenic mice were cross-bred with FGF2-deficient mice. A severe non-eosinophilic asthma model was generated by airway application of LPS-containing allergens twice a week for 4 weeks. To evaluate protective effects of FGF2, recombinant FGF2 (10 microg) was injected subcutaneously during allergen challenge in the severe asthma model. We found that non-eosinophilic inflammation and emphysema induced by transgenic overexpression of IFN-gamma in the airways were aggravated by the absence of FGF2. Airway challenge with LPS-containing allergens induced more inflammation in mice sensitized with LPS-containing allergens compared to challenge with allergens alone. In addition, LPS-induced lung inflammation and emphysema depended on IFN-gamma but not on IL-13. Interestingly, emphysema in the severe asthma model was significantly inhibited by treatment with recombinant FGF2 during allergen challenge, whereas lung inflammation was unaffected. Therefore, our present data suggest that FGF2 may help protect against IFN-gamma-induced emphysema, and that recombinant FGF2 may help lessen the severity of emphysema.
Animals ; Asthma/drug therapy/*prevention & control ; Bronchoalveolar Lavage Fluid ; Disease Models, Animal ; Emphysema/drug therapy/*prevention & control ; Enzyme-Linked Immunosorbent Assay ; Fibroblast Growth Factor 2/deficiency/*metabolism/*therapeutic use ; Flow Cytometry ; Inflammation/immunology ; Interferon-gamma/*biosynthesis/genetics ; Interleukin-13 ; Lipopolysaccharides/administration & dosage/pharmacology ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; Pulmonary Eosinophilia ; Recombinant Proteins/administration & dosage/therapeutic use

OBJECTIVETo study the inhibitory effect of Fuzheng Yiliu Granule (FYG) on hepatocellular cancer (HCC) and investigate the mechanism mediating its bioactivity.

METHODSH22 tumor-bearing ICR mice were treated with FYG [3.6 g/(kg·d)] for 5 days. Tumor volume and tumor weight, percentages of CD3(+), CD4(+), CD8(+), and natural killer (NK) cells in peripheral blood, tumor apoptosis and serum levels of interleukin-2 (IL-2), and tumor necrosis factor-α (TNF-α) were evaluated. FYG-containing serum was prepared from SD rats treated for 7 days [high dose 3.6 g/(kg·d); middle dose 1.8 g/(kg·d); low dose 0.9 g/(kg·d)]. Cell cycle, cell viability, and apoptosis were evaluated after HepG2 cell line was cultured in FYG-containing serum for 48 h. The levels of IL-2 and TNF-α in FYG-containing serum were also determined.

RESULTSFYG produced a potent antitumor effect (P<0.01) and induced marked apoptosis of the tumor tissue (P<0.05). Mice treated with FYG had higher percentages of CD3(+) and CD4(+) (P<0.05), and more NK cells (P<0.01) in the peripheral blood than those in the animals treated with normal saline. Mice receiving FYG had the highest serum levels of IL-2 and TNF-α (P<0.01). High-dose FYG-containing serum significantly decreased HepG2 cell viability, inhibited cell proliferation (P<0.05), and induced apoptosis (P<0.01). In addition, the levels of IL-2 and TNF-α of high-dose-containing serum were higher than the blank serum (P<0.01).

CONCLUSIONFYG could inhibit HCC growth by regulating immune function and inducing apoptosis of tumor cells in vivo and in vitro.

Animals ; Apoptosis ; drug effects ; Carcinoma, Hepatocellular ; blood ; immunology ; pathology ; Cell Proliferation ; drug effects ; Cell Survival ; drug effects ; Drugs, Chinese Herbal ; pharmacology ; Hep G2 Cells ; Humans ; Interleukin-2 ; biosynthesis ; Liver Neoplasms ; blood ; immunology ; pathology ; Lymphocyte Count ; Male ; Mice ; Mice, Inbred ICR ; Rats ; Serum ; Tumor Burden ; drug effects ; Tumor Necrosis Factor-alpha ; biosynthesis ; Xenograft Model Antitumor Assays

Metastasis is the main cause of death in cancer patients. To improve the outcomes of patients undergoing a surgery, new adjuvant therapies that can effectively inhibit metastases have to be developed. Studies have shown that flavonoid naringenin, a natural product that is mainly present in grapes and citrus, may contribute to cancer prevention. It has many advantages compared to traditional chemotherapeutic drugs, such as low toxicity. To determine whether naringenin can also inhibit metastases, a breast cancer resection model that mimics clinical situations was established. We found that orally administered naringenin significantly decreased the number of metastatic tumor cells in the lung and extended the life span of tumor resected mice. Flow cytometry analysis revealed that T cells displayed enhanced antitumor activity in naringenin treated mice, with an increased proportion of IFN-γ and IL-2 expressing T cells. In vitro studies further demonstrated that relief of immunosuppression caused by regulatory T cells might be the fundamental mechanism of metastasis inhibition by naringenin. These results indicate that orally administered naringenin can inhibit the outgrowth of metastases after surgery via regulating host immunity. Thus, naringenin can be an ideal surgical adjuvant therapy for breast cancer patients.
Animals ; Anticarcinogenic Agents ; administration & dosage ; therapeutic use ; Antigens, CD ; analysis ; Breast Neoplasms ; drug therapy ; immunology ; pathology ; surgery ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Chemotherapy, Adjuvant ; Female ; Flavanones ; administration & dosage ; therapeutic use ; Humans ; Immunosuppressive Agents ; administration & dosage ; therapeutic use ; Interferon-gamma ; biosynthesis ; immunology ; Interleukin-2 ; biosynthesis ; immunology ; Lung Neoplasms ; drug therapy ; immunology ; prevention & control ; secondary ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; T-Lymphocytes, Regulatory ; drug effects ; immunology ; metabolism