OBJECTIVETo establish a new method for screening active ingredients of Chinese herbs by determining different bio-thermodynamic effects of 3 genosides on splenic lymphocyte of mice.

METHODSUsing a thermal bioactivity monitoring system, the maximum heat output (mHO), average metabolic heat (MH) and constant of decrease rate (DR) of lymphocyte were determined based on the growth metabolic power-time curve, and the outcomes were verified by MIT.

RESULTSThe mHO and MH increased and the DR decreased after lymphocytes being exposed to the 3 genosides in different concentrations, arranged upon their potency as genoside Rg3 > genoside Rg2 > genoside Rg1 (merely insignificant effect). MTT showed the same results.

CONCLUSIONHeat activity monitoring system could precisely display the different bio-thermal dynamic effects of 3 genosides on splenic lymphocyte.

Animals ; Cell Survival ; drug effects ; Cells, Cultured ; Energy Metabolism ; drug effects ; Ginsenosides ; pharmacology ; Lymphocytes ; cytology ; drug effects ; metabolism ; Male ; Mice ; Mice, Inbred BALB C ; Spleen ; cytology ; Thermodynamics

This study was aimed to establish the quantitative analysis of hIL-2 in culture supernatant by multifunctional Luminex 100. The lymphocytes were separated from ACD-anticoagulated peripheral blood by density gradient method. The lymphocytes were stimulated with PHA for 48 hours, and frozen at -20 degrees C The relative fluorescence units of standard preparations and samples were detected by multifunctional Luminex 100, and the sample concentrations were calculated by standard curve. The results indicated that the regression equation of standard preparation is Lg (RFU) = 1.547 + 0.867 LgC. ANOVA F = 301.7427, p < 0.05 (nu = 6). The analysis of variance showed F = 301.7427, p < 0.05 (nu = 6). The test of regression coefficient showed t = 17.3707 (nu = 6), p < 0.05. It is concluded that method for induction and measurement of human IL-2 in vitro is established. The standard curve established by this way is statistically significant. There is linear relationship between the concentration of hIL-2 and fluorescence intensity.
Cell Separation ; methods ; Humans ; Interleukin-2 ; analysis ; Lymphocytes ; cytology ; drug effects ; metabolism ; Phytohemagglutinins ; pharmacology

OBJECTIVETo study how the way in which betaine promotes the proliferation of mouse spleen lymphocytes is related to calcium channels.

METHODBALB/c mice were used for this experiment. Mouse spleen lymphocytes were obtained through in vitro cultivation after they had been separated, and were divided into a negative control group, a Con A group, and 0.04, 0.4, 4, and 20 mmol x L(-1) betaine groups. MTT was used to observe the effect of betaine on the proliferation of mouse spleen lymphocytes; flow cytometry was used to measure the changes in the cell cycle of mouse spleen lymphocytes; and laser confocal scanning microscopy was used to observe the changes in the intracellular [Ca2+]i of mouse spleen lymphocytes after betaine or different calcium channel blockers were applied.

RESULTBetaine was found to promote the proliferation of mouse spleen lymphocytes 12 h after it had been applied in vitro in concentrations of 4 and 20 mmol x L(-1). It was also found to promote the proliferation of mouse spleen lymphocytes 24 h and 48 h after it had been applied in vitro in concentrations of 0.04, 0.4, 4, and 20 mmol x L(-1), with the effect being most marked for the 4 mmol x L(-1) group 24 h after its application. It was found to facilitate the entry of mouse spleen lymphocytes from the G0/G1 to the S phase 4, 6, 18, and 24 h after it had been applied to mouse spleen lymphocytes in a concentration of 4 mmol x L(-1), with the effect being most marked at 18 h after its application. Intracellular [Ca2+]i in mouse spleen lymphocytes increased significantly (P < 0.01) 6, 12, 18 h after 4 mmol x L(-1) betaine had acted on the lymphocytes, with the effect being most marked at 6 h. The calcium channel blockers nifidipine, diltiazem, mibefradil, and genistein had no effect on the increase of the intracellular [Ca2+]i in mouse spleen lymphocytes due to the application of betaine, while verapamil, mycifradin, heparin, and procaine could block such increase.

CONCLUSIONBetaine facilitates the entry of mouse spleen lymphocytes from the G0/G1 into the S phase by raising the intracellular [Ca2+]i in these cells, thus promoting their proliferation. Intracellular [Ca2+]i increases mainly in two ways: (1) By affecting the alpha1 subunit of the L-type voltage-gated calcium channel with mediation by G proteins and thus leading to an efflux of intracellular calcium: (2) By affecting the IP3R and RyR calcium channels of the intracellular calcium stores and thus leading to the release of intracellular calcium.

Animals ; Betaine ; pharmacology ; Calcium ; metabolism ; Calcium Channels ; metabolism ; Cell Cycle ; drug effects ; Cell Proliferation ; drug effects ; Female ; Lymphocytes ; cytology ; drug effects ; metabolism ; Male ; Mice ; Mice, Inbred BALB C

The effects of granulocyte colony-stimulation-factor (G-CSF) on stem cell mobilization and its impact on the amplification of myeloid-derived suppressor cells (MDSCs) of donor mice were examined. A mouse model of stem cell mobilization was established by consecutive subcutaneous injection of 100 μg/kg G-CSF for 5 days. The blood from the donor mice was routinely examined during mobilization. Stem cells and MDSCs were analyzed by flow cytometry. The immunosuppressive molecules derived from MDSCs in serum and spleen, including hydrogen dioxide (H2O2) and nitric oxide (NO), and the activity of nitric oxide synthase (NOS) were determined during the mobilization. Apoptosis of T lymphocytes was assessed by using Annexin-V/PI. During stem cell mobilization, the number of lymphocytes and white blood cells in the peripheral blood was increased, and peaked on the 4th day. The number of stem cells in G-CSF-treated mice was significantly greater than that in controls (P<0.01). The expansions of MSDCs were also observed after G-CSF mobilization, with a more notable rate of growth in the peripheral blood than in the spleen. The activity of NOS and the production of NO were increased in the donor mice, and the serum H2O2 levels were approximately 4-fold greater than the controls. Consequently, apoptosis of T lymphocytes was increased and showed a positive correlation with the elevated percentage of MDSCs. It was concluded that G-CSF could provide sufficient peripheral blood stem cells for transplantation. Exogenous administration of G-CSF caused the accumulation of MDSCs in the peripheral blood and the spleen, which could lead to apoptosis of T lymphocytes and may offer a new strategy for the prevention and treatment of graft versus host disease.
Animals ; Apoptosis ; drug effects ; Granulocyte Colony-Stimulating Factor ; pharmacology ; Hematopoietic Stem Cell Mobilization ; Hydrogen Peroxide ; metabolism ; Mice ; Mice, Inbred C57BL ; Myeloid Progenitor Cells ; cytology ; drug effects ; metabolism ; Nitric Oxide ; metabolism ; Nitric Oxide Synthase ; metabolism ; T-Lymphocytes ; cytology ; drug effects ; metabolism

OBJECTIVEDendritic cells play an important role in the pathogenesis of atherosclerosis. To explore the effects of hyperglycemia on the maturation and immune function of human monocyte derived dendritic cells (MDCs).

METHODSImmature MDCs were cultured in RPMI1640 medium with either 5.5 mmol/L D-glucose (NG), 25 mmol/L D-glucose (HG) or 5.5 mmol/L D-glucose + 19.5 mmol/L mannitol (HM) in the absence or presence of 30 mmol/L N-acetylcysteine [NAC, a reactive oxygen species inhibitor (ROS)] for 48 hours. FACS was used to investigate the MDCs immunophenotypic expression. Immune function was evaluated by allogeneic mixed T lymphocyte reaction and measurement of cytokine levels from culture supernatants. Intracellular ROS production in MDCs was also measured by 2', 7'-dichlorodihydrofluorescein (DCF, 10 micromol/L) fluorescence using confocal laser-scanning microscopy techniques.

RESULTSCompared with NG and HM treated MDCs, the expression of maturation markers such as CD1a, HLA-DR, CD83, CD86 were significantly upregulated, allogeneic T cells proliferation as well as the cytokines secretions (IL-2, IL-12, IL-10 and IFN-gamma) significantly increased in HG treated MDCs. Intracellular ROS production in MDCs was also significantly increased and all these stimulatory effects of HG could be partially attenuated by NAC.

CONCLUSIONHigh glucose promote the maturation of MDCs and augment their capacity to stimulate T-cell proliferation and cytokine secretions at least in part through enhancing intracellular ROS generation. These stimulating effects of high glucose on MDCs maturation may be one of the mechanisms of accelerated atherosclerosis found in patients with diabetes.

Cell Differentiation ; drug effects ; Cell Proliferation ; drug effects ; Cells, Cultured ; Culture Media ; Cytokines ; biosynthesis ; Dendritic Cells ; drug effects ; immunology ; metabolism ; Glucose ; adverse effects ; pharmacology ; Humans ; Immunophenotyping ; Monocytes ; cytology ; Reactive Oxygen Species ; metabolism ; T-Lymphocytes ; cytology

AIMTo investigate effects of hsBAFF synthesized in Escherichia coli on spleen B lymphocyte immune response and its intracellular free Ca2+ ([Ca2]i]) signaling in mice.

METHODSTwenty ICR mice, half males-half females, were chosen and randomly divided into a normal control group (n=10) and a hsBAFF treatment group (n-10). The mice in hsBAFF treatment group were given abdominal cavity injection of hsBAFF solution which was diluted with phosphate buffered saline (PBS) at dosage of 0.1 mg/kg body weight once each day for over eight days. The mice in control group were received abdominal injection of PBS at the same dose and frequency. Spleen B lymphocyte proliferation and its immune response to LPS stimulation in mice were evaluated using an MTT assay, and change of spleen B lymphocyte [Ca2+]i was assayed under a laser scanning confocal microscope.

RESULTSB lymphocyte proliferation and its immune response to LPS stimulation were significantly higher in hsBAFF-treated mice than in control mice (P < 0.05). The B lymphocyte [Ca2+]i fluorescence intensity in hsBAFF-treated mice maintained at a relatively high level fluctuation, and its average intensity was significantly higher to that of control mice (P < 0.01), but change rate of the intensity was lower compared to that of control group.

CONCLUSIONhsBAFF synthesized in Escherichia coli can enhance immune function in the body by increasing B lymphocyte proliferation and its immune response. hsBAFF-activated B lymphocyte function may be associated with increasing B lymphocytes [Ca2+]i.

Animals ; B-Cell Activating Factor ; immunology ; pharmacology ; B-Lymphocytes ; cytology ; drug effects ; immunology ; Calcium ; metabolism ; Calcium Signaling ; drug effects ; Cell Proliferation ; drug effects ; Female ; Male ; Mice ; Mice, Inbred ICR ; Spleen ; cytology ; drug effects ; immunology

The aim of this study was to compare cell proliferation and function of the T cells acquired under various culture conditions for establishing a simple, safe and efficient cell expansion protocol in vitro. The peripheral blood mononuclear cells (PBMNC) were isolated and stimulated with autologous dendritic cells (DC) and EBV-transformed B lymphoblastoid cell line (BLCL) weekly. The cell proliferation test, flow cytometry with PI and Annexin V double staining, Cr release test and ELISPOT test were used to detect the cell expansion level, frequency of IFN-γ producing T cells, killing activity of antigen-specific T cells, cell apoptotic status and cell differentiation potential, respectively. The results indicated that use of IL-2 combined with IL-7 and IL-15 resulted in the highest cell expansion comparing to the use of IL-2 alone and the use of CD3/28 Microbeads. Also the cells obtained under cultivating with IL-2, IL-7 and IL-15 together showed high frequency of IFN-γ producing cells, strong killing activity, high viability and high differentiation potential with large portion of CD3(+)CD8(+) population among the T cells. It is concluded that a protocol is established in which the use of IL-2 combined with IL-7 and IL-15 induces the biggest cell expansion, expanded cells show high viability, strong differentiation potential, high frequency of IFN-γ producing cells and strong killing activity.
Cell Line, Transformed ; Cell Proliferation ; Cell Separation ; Dendritic Cells ; cytology ; metabolism ; Humans ; Interleukin-15 ; pharmacology ; Interleukin-2 ; pharmacology ; Interleukin-7 ; pharmacology ; T-Lymphocytes ; cytology ; drug effects ; metabolism

We evaluated the effectiveness of rhamnogalacturonan II (RG-II)-stimulated bone marrow-derived dendritic cells (BMDCs) vaccination on the induction of antitumor immunity in a mouse lymphoma model using EG7-lymphoma cells expressing ovalbumin (OVA). BMDCs treated with RG-II had an activated phenotype. RG-II induced interleukin (IL)-12, IL-1beta, tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) production during dendritic cell (DC) maturation. BMDCs stimulated with RG-II facilitate the proliferation of CD8+ T cells. Using BMDCs from the mice deficient in Toll-like receptors (TLRs), we revealed that RG-II activity is dependent on TLR4. RG-II showed a preventive effect of immunization with OVA-pulsed BMDCs against EG7 lymphoma. These results suggested that RG-II expedites the DC-based immune response through the TLR4 signaling pathway.
Acute-Phase Proteins/metabolism ; Adaptor Proteins, Vesicular Transport/metabolism ; Animals ; Antigens, CD14/metabolism ; Bone Marrow Cells/cytology/drug effects ; CD8-Positive T-Lymphocytes/*immunology ; Carrier Proteins/metabolism ; Cell Differentiation/drug effects ; Cell Nucleus/drug effects/metabolism ; Cell Proliferation/drug effects ; Cytokines/biosynthesis ; Dendritic Cells/cytology/drug effects/enzymology/*immunology ; Enzyme Activation/drug effects ; Lymphocyte Activation/*drug effects ; Membrane Glycoproteins/metabolism ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; Mitogen-Activated Protein Kinases/metabolism ; Myeloid Differentiation Factor 88/metabolism ; NF-kappa B/metabolism ; Neoplasms/immunology/*pathology ; Pectins/*pharmacology ; Phenotype ; Protein Transport/drug effects ; Receptors, Chemokine/metabolism ; Signal Transduction/drug effects ; T-Lymphocytes, Cytotoxic/cytology/drug effects ; Toll-Like Receptor 4/*agonists/metabolism

4-1BB, a member of the tumor necrosis factor receptor (TNFR) superfamily, is a major costimulatory receptor that is rapidly expressed on the surface of CD4+ and CD8+ T cells after antigen- or mitogen-induced activation. The interaction of 4-1BB with 4-1BBL regulates immunity and promotes the survival and expansion of activated T cells. In this study, the expression of 4-1BB and 4-1BBL was examined during regeneration of the murine thymus following acute cyclophosphamide-induced involution. Four-color flow cytometry showed that 4-1BB and 4-1BBL were present in the normal thymus and were preferentially expressed in the regenerating thymus, mainly in CD4+CD8+ double-positive (DP) thymocytes. Furthermore, the CD4loCD8lo, CD4+CD8lo and CD4loCD8+ thymocyte subsets, representing stages of thymocyte differentiation intermediate between DP and single-positive (SP) thymocytes, also expressed 4-1BB and 4-1BBL during thymus regeneration but to a lesser degree. Interestingly, the 4-1BB and 4-1BBL positive cells among the CD4+CD8+ DP thymocytes present during thymus regeneration were TCR(hi) and CD69+ unlike the corresponding controls. Moreover, the 4-1BB and 4-1BBL positive cells among the intermediate subsets present during thymus regeneration also exhibited TCRhi/int and CD69+/int phenotypes, indicating that 4-1BB and 4-1BBL are predominantly expressed by the positively selected population of the CD4+CD8+ DP and the intermediate thymocytes during thymus regeneration. RT-PCR and Western blot analyses confirmed the presence and elevated levels of 4-1BB and 4-1BBL mRNA and protein in thymocytes during thymus regeneration. We also found that the interaction of 4-1BB with 4-1BBL promoted thymocyte adhesion to thymic epithelial cells. Our results suggest that 4-1BB and 4-1BBL participate in T lymphopoiesis associated with positive selection during recovery from acute thymic involution.
4-1BB Ligand/genetics/*metabolism ; Animals ; Antigens, CD137/genetics/*metabolism ; CD4-Positive T-Lymphocytes/cytology/metabolism ; CD8-Positive T-Lymphocytes/cytology/metabolism ; Cell Adhesion ; Cell Differentiation ; Cell Line ; Cells, Cultured ; Cyclophosphamide/pharmacology ; Epithelial Cells/cytology ; Gene Expression Regulation ; Immunosuppressive Agents/pharmacology ; Male ; Mice ; Mice, Inbred C57BL ; RNA, Messenger/genetics ; *Regeneration ; T-Lymphocytes/*cytology/metabolism ; Thymus Gland/*cytology/drug effects/*physiology

OBJECTIVETo investigate the effect of spearmint oil on emphysema-like changes and the expression of tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta(IL-1beta), matrix metalloproteinase-9 (MMP-9) and tissue inhibitor of metalloproteinase-1 (TIMP-9) in lipopolysaccharide (LPS) treated rats.

METHODEmphysematous changes model was induced by intratracheal instillation of LPS once a week for up to 8 weeks in rats. Rats were divided into control, dexamethasone (0.3 mg x kg(-1)), and spearmint oil (10, 30,100 mg x kg(-1)) groups. Each group was treated with saline, dexamethasone, and spearmint of oil respectively for 4 weeks. Then total and different white blood cell counts in bronchoalveolar lavage fluid(BALF) were carried out. The pathologic changes of lung tissue such as alveolar structure, airway inflammation, and goblet cell metaplasia were observed by HE and AB-PAS staining. Expression of TNF-alpha, IL-1beta, TIMP-1 and MMP-9 were measured.

RESULTBoth spearmint and dexamethasone decreased the destruction of pulmonary alveolus. The total and different white blood cell counts in BALF including neutrophile and lymphocyte of spearmint oil 100 mg x kg(-1) and dexamethasone group were significantly reduced, and the goblet cell metaplasia was also inhibited. Dexamethasone had inhibitory effect on the expression of TNF-alpha, IL-1beta, TIMP-1 and MMP-9. Spearmint oil 30, 100 mg x kg(-1) significantly reduced TNF-alpha and IL-1beta respectively. Spearmint oil 10, 30 and 100 mg x kg(-1) had no effect on the expression of TIMP-1, but could decrease the expression of MMP-9 significantly in lung tissues.

CONCLUSIONSpearmint oil has protective effect on rats with emphysematous changes, since it improves alveolar destruction, pulmonary inflammation, and goblet cell metaplasia. The mechanism may include reducing TNF-alpha, IL-1beta content and inhibiting overexpression of matrix metalloproteinase-9 in lung tissues.

Animals ; Azo Compounds ; pharmacology ; Bronchoalveolar Lavage Fluid ; cytology ; Goblet Cells ; drug effects ; Interleukin-1beta ; drug effects ; metabolism ; Leukocytes ; drug effects ; metabolism ; Lipopolysaccharides ; Lymphocytes ; drug effects ; metabolism ; Matrix Metalloproteinase 9 ; drug effects ; metabolism ; Mentha spicata ; chemistry ; Metaplasia ; Monocytes ; drug effects ; metabolism ; Neutrophils ; drug effects ; metabolism ; Phytotherapy ; Plant Oils ; therapeutic use ; Pulmonary Emphysema ; chemically induced ; drug therapy ; enzymology ; pathology ; Rats ; Respiratory System ; drug effects ; pathology ; Tissue Inhibitor of Metalloproteinase-1 ; drug effects ; metabolism ; Tumor Necrosis Factor-alpha ; drug effects ; metabolism