This paper aims to explore the effects of chicken interferon-γ (ChIFN-γ) and interleukin-2 (ChIL-2) on type 1 helper (Th1) T lymphocyte differentiation. To be specific, ChIFN-γ and ChIL-2 were first expressed in Escherichia coli competent cells and then purified by Ni-NTA affinity chromatography. Different concentration of ChIFN-γ and ChIL-2 were employed to stimulate the lymphocytes in chicken peripheral blood which had been activated by concanavalin A (Con A), and the mRNA levels of cytokines related to Th1 cell differentiation were detected by real-time quantitative PCR (RT-qPCR). The results showed that both ChIFN-γ and ChIL-2 can significantly up-regulate mRNA levels of cytokines related to Th1 cell differentiation and the optimal concentration was 12.5 μg/mL and 25.0 μg/mL, respectively. In addition, specific-pathogen-free (SPF) chickens were immunized with ChIL-2 or ChIFN-γ together with H9N2 vaccine, or H9N2 vaccine alone by oral administration or intramuscular injection, respectively. The mRNA levels of cytokines related to Th1 cell differentiation were detected after immunization. The results showed that ChIFN-γ and ChIL-2 significantly up-regulated the mRNA levels of cytokines related to Th1 cell differentiation induced by H9N2 vaccine compared with H9N2 vaccine alone, and that the intramuscular injection was better than oral administration. In this study, we verified that ChIFN-γ and ChIL-2 can significantly enhance mRNA levels of cytokines related to Th1 cell differentiation induced by ConA or H9N2 vaccine in vitro and in vivo. The results of this study can lay a theoretical basis for using ChIFN-γ and ChIL-2 as vaccine adjuvants.
Animals ; Cell Differentiation ; Chickens ; Concanavalin A ; Cytokines/genetics* ; Influenza A Virus, H9N2 Subtype/genetics* ; Interferon-gamma/metabolism* ; Interleukin-2/genetics* ; RNA, Messenger

Viscum plants,the evergreen perennial parasitic shrubs or subshrubs,are mainly distributed in tropical and subtropical regions. There are about 70 Viscum species around the world,including 11 species and one variety in China. Mistletoe lectins are typeⅡ ribosome-inactivating proteins( RIPs) extracted from Viscum plants with anticancer and immunoregulatory activities. Many studies have focused on the mistletoe lectins isolated from V. album in Europe and V. album var. coloratum distributed in South Korea,respectively,and several preparations,such as Iscucin Ⓡ,were developed and clinically applied for cancer treatment. Although Viscum plants are widely distributed in China,only a few studies of mistletoe lectins have been reported. The recent progress of mistletoe lectins was reviewed from extraction,purification,quantitative/qualitative detection,molecular structure,pharmacological activities,toxicities,and clinical application,aiming at providing a reference for in-depth research and utilization of mistletoe lectins produced in China.
Humans ; Lectins ; Plant Extracts ; Plant Lectins ; Plant Proteins/genetics* ; Toxins, Biological ; Viscum

The study aimed to investigate the effect of processing on lectin protein in four toxic Chinese medicines tubers of Pinellia ternata,P. pedatisecta,Arisema heterophyllum and Typhonium giganteum. Western blot was used to semi-quantitatively analyze the content of lectin in the four kinds of toxic Chinese medicines and their different processed products. Raw products and lectin were treated by heating or soaking in ginger juice or alum solution. The effects of different excipients and the heating methods on lectin proteins were investigated. The results showed that the content of lectin in raw products of P. pedatisecta,P. ternata,A. heterophyllum,and T. giganteum were 7. 3%,4. 9%,2. 7%,2. 3%,respectively. And the content of lectin in Pinelliae Rhizoma praeparatum cum alumine was 0. 027%. Lectin was not detected in the Pinelliae Rhizoma Praeparatum cum Zingibere et Alumine,Arisaematis Rhizma Praeparatum and Typhonii Rhizoma Praeparatum,which indicated that processing could significantly reduce the content of active lectin in raw products. The results also showed that with the prolongation of soaking and heating time,the content of lectin in raw products decreased gradually,while the content was almost unchanged when soaked in ginger juice alone. The effects of different excipients and heating on lectin were the same as those on raw products. Therefore,the method with alum soaking and heating can reduce the content of active lectin,which is the key to reduce the toxicity of toxic Chinese medicines. In this paper,Western blot was used to study the content of toxic protein in Araceae toxic Chinese medicines as an evaluation method of the processing degree.
Araceae/chemistry* ; Chemistry, Pharmaceutical/methods* ; Drugs, Chinese Herbal/analysis* ; Lectins/analysis* ; Plant Tubers/chemistry* ; Rhizome/chemistry*

Indoor animal husbandry environments are inevitably contaminated with endotoxins. Endotoxin exposure is associated with various inflammatory illnesses in animals. This cross-sectional study evaluated the relationship between the degree of endotoxin exposure and the cellular and humoral immune profiles of fattening pigs. Blood samples were taken from the jugular vein of 47 pigs from ten pig farms in Korea. Whole blood cell counts and plasma immunoglobulin (Ig) classes were determined. Peripheral-blood mononuclear cells were stimulated in vitro with concanavalin A for 48 h, and cytokines released into culture supernatants were measured. The barns in which the pigs lived were assessed for endotoxin levels in the total and respirable dust by using the limulus amebocyte lysate kinetic QCL method. Low and high endotoxin exposures were defined as ≤ 30 and > 30 EU/m³, respectively. Compared to pigs with low endotoxin exposure (n = 19), highly exposed pigs (n = 28) had higher circulating neutrophil and lymphocyte (particularly B cells) counts, IgG and IgE levels, interferon-gamma (IFNγ) and interleukin (IL)-4 productions, and lower IgA levels and tumor necrosis factor-alpha (TNFα) production. The IL-4, IFNγ, and TNFα levels significantly correlated with endotoxin level and/or pig age. Constant exposure of pigs to high levels of airborne endotoxins can lead to aberrant immune profiles.
Agriculture ; Animal Husbandry ; Animals ; Blood Cell Count ; Concanavalin A ; Cross-Sectional Studies ; Cytokines ; Dust ; Endotoxins ; Horseshoe Crabs ; Housing ; Immunity, Cellular ; Immunoglobulin A ; Immunoglobulin E ; Immunoglobulin G ; Immunoglobulins ; In Vitro Techniques ; Interferon-gamma ; Interleukin-4 ; Interleukins ; Jugular Veins ; Korea ; Lymphocytes ; Methods ; Neutrophils ; Plasma ; Swine ; Tumor Necrosis Factor-alpha

Thapsigargin (TGN) is a potent and selective inhibitor of sarco-endoplasmic Ca²⁺-ATPase, leading to rapid elevation of cytoplasmic Ca2+ concentration. Previous reports have shown that TGN increases the production of various cytokines from macrophages and dendritic cells. Here, we examine the effects of TGN on murine T cells. Nanomolar concentrations of TGN are a significant inducer of IL-2 production with full activity at 50 nM. Micromolar concentrations of TGN, however, are inhibitory to IL-2 production and T cell proliferation. The IL-2 production-inducing activity of TGN is much more prominent when T cells are primed with concanavalin A or anti-CD3 mAb, and is due to the increase of cytoplasmic Ca²⁺ concentration. TGN at 50 nM does not affect interferon-gamma or IL-4 production from T cells. Thus, the present study shows that low nanomolar concentrations of TGN could be useful in potentiating IL-2 production from antigen-primed T cells.
Cell Proliferation ; Concanavalin A ; Cytokines ; Cytoplasm ; Dendritic Cells ; Interferon-gamma ; Interleukin-2* ; Interleukin-4 ; Macrophages ; T-Lymphocytes* ; Tetradecanoylphorbol Acetate ; Thapsigargin*

The expression of immunogenic markers after differentiation of umbilical cord blood (UCB)-derived mesenchymal stem cells (MSC) has been poorly investigated and requires extensive in vitro and in vivo testing for clinical application. The expression of human leukocyte antigen (HLA) classes on UCB-derived MSC was tested by Fluorescence-activated cell sorting analysis and immunocytochemical staining. The undifferentiated MSC were moderately positive for HLA-ABC, but almost completely negative for HLA-DR. The MSC differentiated to chondrocytes expressed neither HLA-ABC nor HLA-DR. The proliferation of MSC was not significantly affected by the allogeneic lymphocytes stimulated with concanavalin A. The responder lymphocytes showed no significant decrease in proliferation in the presence of the MSC, but the apoptosis rate of the lymphocytes was increased in the presence of MSC. Taken together, these findings indicate that UCB-derived MSC differentiated to chondrocytes expressed less HLA class I and no class II antigens. The MSC showed an immunomodulatory effect on the proliferation and apoptosis of allogeneic lymphocytes. These data suggest that the differentiated and undifferentiated allogeneic MSC derived from umbilical cord blood can be a useful candidate for allogeneic cell therapy and transplantation without a major risk of rejection.
Apoptosis ; Cell- and Tissue-Based Therapy ; Chondrocytes ; Concanavalin A ; Fetal Blood* ; Flow Cytometry ; Histocompatibility Antigens Class II ; HLA-DR Antigens ; Humans ; In Vitro Techniques ; Leukocytes ; Lymphocytes ; Mesenchymal Stromal Cells* ; Umbilical Cord*

OBJECTIVEThis study was to expand the cytotoxic T lymphocytes (CTL) through inducing the differentiation of umbilical blood monomuclear cells (UBMNC) by using various combination of cytokines, and to investigate the functions of expanded CTL.

METHODSThe MNC were isolated by ficoll density gradient centrifugation. Then, the PHA-P, IFN-γ combined with IL-2, IL-15 and other cytokines were used for induction and expansion of the cord blood-derived CTL. The biological function of CTL was examined by phenotype analysis, cytotoxic tests and real-time fluorescence quantitative PCR.

RESULTSAfter expansion for 15 days, the cell number increased by 1522% ± 137%. The content of CD3(-)CD8(-) cells in uncultured cord blood MNC was 95%, and the CD3(+)CD8(+) CTL cells reached 82.77% in cultured cord blood MNC after expansion for 15 days. The expanded CTL cell showed the cytotoxic activity against K562 and HeLa cell line. The killing rate of MNC was 61.88 ± 1.08%. After expansion, the killing rate could reach to 90% with the average value of 90.33 ± 2.02%. The expanded CTL cells highly expressed some key cytokines, such as granzyme A, granzyme B, GM-CSF, granulysin, IFN-γ, TGF-β, TNF-α and perforin. Compared with the control group, the expression of IFN-γ and TGF-β significantly increased (P < 0.05), and the other factors dramatically increased (P < 0.01).

CONCLUSIONThe cord blood-derived CTL can be expanded by different combinations of cytokines. These protocols may provide alternative choices for CTL cell expansion in tumor adoptive immunotherapy.

Cytokines ; Fetal Blood ; Granulocyte-Macrophage Colony-Stimulating Factor ; Granzymes ; Histocompatibility Antigens Class I ; Histocompatibility Antigens Class II ; Humans ; Immunotherapy, Adoptive ; Perforin ; Phytohemagglutinins ; T-Lymphocytes, Cytotoxic

OBJECTIVETo investigate anti-attachment effect of ethyl acetate extract of Huanglian Jiedu decoction (EAHD) on Candida glabrata.

METHODSerial 2-fold dilution assay was used to determine the minimum inhibitory concentrations MICs of EAHD to C. glabrata. XTT assay was used to evaluate the effect of EAHD against adhesion of C. glabrata. Inverted microscope, scanning electron microscope (SEM) and fluorescein diacetate (FDA) staining were applied to observe the morphological changes of C. glabrata in adhesion. PCR was adopted to inspect the expression of attachment-related genes such as EPA1, EPA6 and EPA7.

RESULTThe MIC of EAHD and fluconazole to C. glabrata were 320 mg · L(-1) and 1 mg · L(-1) respectively. The total cells including budding cells decreased in a dose-dependent manner following EAHD treatment. The expressions of EPA1, EPA6 and EPA7 were downregulated dramatically after EAHD treatment.

CONCLUSIONEAHD could effectively inhibit adherence of C. glabrata.

Acetates ; Candida glabrata ; drug effects ; physiology ; Drugs, Chinese Herbal ; pharmacology ; Fungal Proteins ; genetics ; Lectins ; genetics ; Plant Extracts ; pharmacology

GV1001 is a peptide derived from the human telomerase reverse transcriptase (hTERT) sequence that is reported to have anti-cancer and anti-inflammatory effects. Enolase1 (ENO1) is a glycolytic enzyme, and stimulation of this enzyme induces high levels of pro-inflammatory cytokines from concanavalin A (Con A)-activated peripheral blood mononuclear cells (PBMCs) and ENO1-expressing monocytes in healthy subjects, as well as from macrophages in rheumatoid arthritis (RA) patients. Therefore, this study investigated whether GV1001 downregulates ENO1-induced pro-inflammatory cytokines as an anti-inflammatory peptide. The results showed that GV1001 does not affect the expression of ENO1 in either Con A-activated PBMCs or RA PBMCs. However, ENO1 stimulation increased the production of pro-inflammatory cytokines such as tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, and IL-6, and these cytokines were downregulated by pretreatment with GV1001. Moreover, p38 mitogen-activated protein kinase (MAPK) and nuclear factor (NF)-kappaB were activated when ENO1, on the surface of Con A-activated PBMCs and RA PBMCs, was stimulated, and they were successfully suppressed by pre-treatment with GV1001. These results suggest that GV1001 may be an effective anti-inflammatory peptide that downregulates the production of pro-inflammatory cytokines through the suppression of p38 MAPK and NF-kappaB activation following ENO1 stimulation.
Arthritis, Rheumatoid ; Concanavalin A ; Cytokines ; Down-Regulation* ; Humans ; Inflammation ; Interleukin-6 ; Interleukins ; Macrophages ; Monocytes ; NF-kappa B ; p38 Mitogen-Activated Protein Kinases ; Protein Kinases ; Telomerase ; Tumor Necrosis Factor-alpha

OBJECTIVE: The imbalance between pro-inflammatory and anti-inflammatory cytokines may underlie different pain states. Although ascorbic acid is the most important physiological antioxidant that affects host defense mechanisms and immune homeostasis, there is limited information on the effects of ascorbic acid on the production of cytokines. METHODS: In this study, we investigated the in vitro effect of L-ascorbic acid (AA) on the production of pro-inflammatory and anti-inflammatory cytokines by stimulating C57BL/6 mouse splenocytes with the polyclonal activators lipopolysaccharide or concanavalin A. RESULTS: AA significantly downregulated the expression of IL-6, IL-12, and TNF-alpha at 48 h and 72 h in mouse splenocytes treated with a combination of polyclonal activators and AA. AA treatment also resulted in upregulation of IL-4 and IL-10 at 72 h. These findings demonstrated that AA significantly potentiated production of anti-inflammatory cytokines whereas there was an inverse association between AA and expression of pro-inflammatory cytokines in mouse splenocytes. CONCLUSION: AA may have potential applications in the reduction of inflammatory pain because of its function in modulating the production of cytokines. However, further in vivo investigations are necessary to elucidate the mechanisms involved.
Animals ; Ascorbic Acid* ; Concanavalin A ; Cytokines* ; Defense Mechanisms ; Homeostasis ; Interleukin-10 ; Interleukin-12 ; Interleukin-4 ; Interleukin-6 ; Interleukins ; Mice* ; Tumor Necrosis Factor-alpha ; Up-Regulation