In order to explore a new way to study allogeneic reactive T lymphocytes, detection of cytokine-secreting T lymphocytes after allogeneic peripheral blood mononuclear cells (PBMNCs) stimulation and investigation of its clinical significance were performed. A novel cytokine secretion assay (CKSA) was first applied to detect T lymphocytes secreting cytokine including IFN-gamma, IL-4 and IL-10 at single cell level in human mixed lymphocytes reaction. IFN-gamma-secreting T cells from PBMNCs were then evaluated in 2 patients with acute graft versus host disease (aGVHD) after allogeneic bone marrow transplantation. The results showed that compared with IL-4 and IL-10 (which were 0.12 +/- 0.03% and 0.10 +/- 0.03% respectively), a sizable proportion of IFN-gamma-secreting T lymphocytes could be detected (1.12 +/- 0.13)% after allogeneic PBMNCs stimulation. Preliminary results indicated that frequency of IFN-gamma-secreting T lymphocytes correlated with the onset and severity of clinical aGVHD. In conclusion, it is feasible to detect IFN-gamma secreting T lymphocytes after allogeneic PBMNCs stimulation and to apply the CKSA technique for clinical identification of aGVHD.
Acute Disease ; Cytokines ; secretion ; Graft vs Host Disease ; etiology ; Humans ; Interferon-gamma ; secretion ; Interleukin-10 ; secretion ; Interleukin-4 ; secretion ; T-Lymphocytes ; immunology ; Transplantation, Homologous ; immunology

OBJECTIVETo investigate the influence of the LPS of Bacteroides fragilis on the secretion of IL-2 and IL-4 from the peripheral blood mononuclear cells of normal individuals, so as to elucidate the mechanism of the infection by Bacteroides fragilis.

METHODSLPS was obtained from both the strains isolated from patients and from standard NCTC9343. Peripheral blood mononuclear cells (PBMCs) were treated with different concentrations of LPS thus obtained. The supernatants from the cell culture of the PBMCs were harvested at 24 PBHs and were subjected to the determination of the IL-2 and IL-4 contents by ELISA method. RESULTS The IL-2 secretion from the PBMCs of normal volunteers was obviously inhibited by the LPS from Bacteroides fragilis (P < 0.01), and the inhibitory effect was dose-dependent. Nevertheless, the IL-4 secretion from the PBMCs of normal volunteers was significantly stimulated by the LPS from Bacteroides Fragilis (P < 0.05), and it was not concentration dependent. There was no difference between the effects of the LPSs from patients and standard strains (P < 0.05).

CONCLUSIONThe LPS from Bacteroides fragilis was inhibitory to the secretion of IL-2 from PBMCs and was stimulative to that of IL-4 from PBMCs of normal human persons.

Bacteroides fragilis ; metabolism ; Cells, Cultured ; Humans ; Interleukin-2 ; immunology ; secretion ; Interleukin-4 ; immunology ; secretion ; Lipopolysaccharides ; pharmacology ; Monocytes ; drug effects ; immunology

This study was aimed to investigate the expression and clinical significance of IL-18, IL-18 binding protein (IL-18BP), IFN-γ and IL-4 secreted from splenocytes of patients with idiopathic thrombocytopenic purpura (ITP) in vitro. Spleen mononuclear cells (MNC) were prepared by using routine sterile method, and were cultured in RPMI 1640 complete medium containing 10 µg/ml PHA, 10% fetal calf serum at 37°C and 5% CO2. The levels of IFN-γ, IL-4, IL-18 and IL-18BP secreted from MNC of ITP patients and normal controls were determined after culture for 48 hours. The results showed that after culture of spleen MNC for 48 hours, the levels of IL-18 and IFN-γ were significantly higher in patients with ITP than that in controls, but the levels of IL-18BP was not significantly elevated in ITP patients. The level of IL-4 was below the detectable limit of the assay used. It is concluded that imbalance between IL-18 and IL-18BP may play an important role in pathogenesis of ITP, and regulation of balance between IL-18 and IL-18BP may be a therapeutic approach against ITP.
Adolescent ; Adult ; Aged ; Aged, 80 and over ; Cells, Cultured ; Female ; Humans ; Intercellular Signaling Peptides and Proteins ; secretion ; Interferon-gamma ; secretion ; Interleukin-18 ; secretion ; Interleukin-4 ; secretion ; Lymphocytes ; cytology ; metabolism ; Male ; Middle Aged ; Purpura, Thrombocytopenic, Idiopathic ; metabolism ; Spleen ; cytology ; metabolism ; Young Adult

OBJECTIVETo investigate the effects of Trichophyton rubrum exposure on the expressions of toll-like receptor-2 (TLR-2), TLR-4 and dendritic cell associated C-type lectin-1 (Dectin-1) and cytokine secretions in human keratinocytes cell line HaCaT.

METHODSThe mRNA of TLR-2,4, and dectin-1 in the HaCaT co-cultured with the conidia of Trichophyton rubrum conidia for 24 h was measured with real-time PCR. The mean fluorescence intensity and the percentage of cells positive for TLR-2, 4, and dectin-1 was detected during the co-culture using flow cytometry. The cytokine secretion profiles in the cell culture supernatant was analyzed using a cytokine antibody array.

RESULTSThe TLR-2,4, and dectin-1 mRNA expressions, mean fluorescence intensity and percentage of positive cells for TLR-2,4, and dectin-1 all increased in HaCaT cells in response to Trichophyton rubrum conidia exposure. The results of cytokine antibody array demonstrated obviously increased secretions of IL-8, I-309, IFN-γ, IL-6, and IL-13 in the culture supernatant of HaCaT cells in response to Trichophyton rubrum exposure.

CONCLUSIONThe immune responses and immunological recognition of human keratinocytes to Trichophyton rubrum conidia are partially mediated by up-regulating the expressions of TLR-2, TLR-4 and dectin-1 and secretions of multiple cytokines.

Cell Line ; Chemokine CCL1 ; secretion ; Coculture Techniques ; Humans ; Interferon-gamma ; secretion ; Interleukin-13 ; secretion ; Interleukin-6 ; secretion ; Interleukin-8 ; secretion ; Keratinocytes ; metabolism ; Lectins, C-Type ; metabolism ; Toll-Like Receptor 2 ; metabolism ; Toll-Like Receptor 4 ; metabolism ; Trichophyton ; Tumor Necrosis Factor-alpha ; secretion

OBJECTIVETo study the effect of a new obesity-related gene NYGGF4 on the insulin sensitivity and secretory function of adipocytes.

METHODS3T3-L1 preadipocytes transfected with either an empty expression vector (pcDNA3.1; control group) or an NYGGF4 expression vector (NYGGF4-pcDNA3.1) were cultured in vitro and differentiated into the matured adipocytes with the standard insulin plus dexamethasone plus 3-isobutyl-methylxanthine (MDI) induction cocktail. 2-deoxy-D-[3H] glucose uptake was determined by liquid scintillation counting. Western blot was performed to detect the protein content and translocation of glucose transporter 4 (GLUT4). The supernatant concentrations of TNF-alpha, IL-6, adiponectin and resistin were measured using ELISA.

RESULTSNYGGF4 over-expression in 3T3-L1 adipocytes reduced insulin-stimulated glucose uptake. NYGGF4 over-expression impaired insulin-stimulated GLUT4 translocation without affecting the total protein content of GLUT4. The concentrations of TNF-alpha, IL-6, adiponectin and resistin in the culture medium of 3T3-L1 transfected with NYGGF4 were not significantly different from those in the control group.

CONCLUSIONSNYGGF4 over-expression impairs the insulin sensitivity of 3T3-L1 adipocytes through decreasing GLUT4 translocation and had no effects on the secretory function of adipocytes.

3T3-L1 Cells ; Adipocytes ; drug effects ; secretion ; Adiponectin ; secretion ; Animals ; Carrier Proteins ; genetics ; physiology ; Glucose ; metabolism ; Glucose Transporter Type 4 ; analysis ; metabolism ; Insulin ; pharmacology ; Interleukin-6 ; secretion ; Mice ; Resistin ; analysis ; Transfection ; Tumor Necrosis Factor-alpha ; secretion

In order to investigate the effect of vitamin A (VA) on the secretion of IFN-gamma and IL-4 in Mycoplasma Pneumoniae (MP)-induced A549 cells, A549 cells were co-cultured with MP for different time lengths and then the levels of IFN-gamma and IL-4 in the cell culture supernatants were detected before and after treatment with different concentrations of VA by using the enzyme-linked immunosorbent assay (ELISA). The results showed that the level of IFN-gamma and IL-4 in the supernatants of MP-induced A549 cells was much higher than that in non-induced cells (P<0.01). After application of VA, IL-4 level was not increased until the concentration of VA was up to 0.5x10(-5) mol/L (P<0.01). However, with concentration of VA increased up to 1x10(-4) mol/L, IL-4 was significantly suppressed (P<0.01). It was concluded that MP could induce the secretion of IFN-gamma and IL-4 in A549 cells. VA could inhibit the secretion of IFN-gamma and increase the IL-4 level in MP-induced A549 cells. However, high concentration of VA had an inhibitory effect on the secretion of IL-4 as well as on the IFN-gamma. These data provided a theoretical basis for the application of VA in MP pneumonia in the clinical practice.
Cell Line, Tumor ; Coculture Techniques ; Culture Techniques ; Interferon-gamma/*secretion ; Interleukin-4/*secretion ; Lung Neoplasms/pathology ; Mycoplasma pneumoniae/growth & development ; Mycoplasma pneumoniae/*physiology ; Vitamin A/*pharmacology

T lymphocytes are an integral part of the effective immune response against various tumors, but they are frequently functionally unresponsive in tumor-bearing patients. This study was aimed to investigate the T lymphocyte function of patients with acute myeloid leukemia (AML) in different status through analyzing intracellular cytokine characteristics of T lymphocytes in AML patients. The T lymphocytes from 18 de nuevo AML patients in different status and 10 healthy controls were stimulated with phorbol 12-myristate 13-acetate (PMA) and ionomycin in the presence of monensin, and were stained with fluorescent McAbs CD4-PITC, CD8-FITC and IFNgamma-PE, ILA-PE, then their T lymphocytes were analyzed by flow cytometry. The results showed that IFN-gamma level in CD4+ and CD8+ T cells of de novo AML patients was significantly lower as compared with healthy controls (p<0.05), while IL-4 level in CD4+ and CD8+ T cells was low too, there was no significant difference between de novo AML patients and healthy controls. In AML patients in clinical remission, IFNgamma level in CD8+ T cells was significantly higher than that in de novo AML patients (p<0.05), but there was no significant difference as compared with healthy controls (p>0.05). In relapsed AML patients, IFNgamma level in CD4+ and CD8+ T cells was significantly lower than that in healthy controls and in AML patients with CR (p<0.05), while IL-4 level was significantly higher than that in healthy controls and de nuevo AML patients (p<0.05). It is concluded that the cytokine secretion in T cell subsets of AML patients in different status is changed. Correspondingly, the Th1/Tc1 level is low after stimulating CD4+ and CD8+ T cells in peripheral blood of de novo AML patients, but not different from healthy controls. Though the Th1 level in T cells of AML patients in complete remission is low, but Tc1 response is even enhanced as high as the healthy controls. The Th2/Tc2-like response of CD4+ and CD8+ T cells in relapsed AML patients obviously increases when compared with Th1/Tc1-like response.
CD4-Positive T-Lymphocytes ; metabolism ; CD8-Positive T-Lymphocytes ; metabolism ; Humans ; Interferon-gamma ; secretion ; Interleukin-4 ; secretion ; Leukemia, Myeloid, Acute ; immunology

This study was purposed to investigate the phenotype, in vitro expansion and cytokine secretion profile of Valpha24(+) NKT cells from cord blood (CB), peripheral blood (PB), and granulocyte colony stimulating factor-mobilized peripheral blood mononuclear cells (G-PBMNCs). Fresh mononuclear cells (MNCs) were isolated by the method of gradient centrifugation and then cultured with alpha-GalCer (100 ng/ml), IL-2 (50 U/ml), IL-15 (50 ng/ml) for 12 days. Valpha24(+) NKT cells were purified by anti-Vbeta11 TCR McAb and goat anti-mouse IgG magnetic beads. The phenotype and purity of Valpha24(+) NKT cells were determined by flow cytometry. Cytokine production was analyzed by ELISA. The results showed that Valpha24(+) NKT cells in CB, PB and G-PBMNCs were expanded by 221.5 (95 - 501), 456.5 (101 - 2207), and 756.38 (82 - 20373)-fold respectively. After stimulation by phorbol-12-myristate-13-acetate (PMA) for 24 hours, IL-4 and IFN-gamma produced by Valpha24(+) NKT cells from CB and PB were 180.33 (144.67 - 2253.48) vs 190.67 (110.07 - 6060.16) ng/ml, 864.33 (401.33 - 3386.67) vs 508.49 (253.82 - 8840.00) ng/ml respectively, with IL-4/IFN-gamma ratio of 0.503 +/- 0.642 vs 0.455 +/- 0.562 respectively. After expansion of Valpha24(+) NKT cells from G-PBMNCs, IL-4 and IFN-gamma produced by Valpha24(+) NKT cells at day 9 and day 12 were 139.08 (7.62 - 606) vs 89.3 (0 - 729.2) ng/ml, 14264.8 (1168 - 18059) vs 14488 (1041 - 18261) ng/ml respectively, with IL-4/IFN-gamma ratio of 0.0531 +/- 0.1081 vs 0.0376 +/- 0.1148 respectively. It is concluded that in presence of IL-2 and IL-15, alpha-GalCer can facilitate the rapid short-term expansion of Valpha24(+) NKT cells from CB, PB, and G-PBMNCs. Valpha24(+) NKT cells from G-PBMNCs show much high potential of expansion in comparison to the counterparts from CB or PB (p < 0.05). The activated Valpha24(+) NKT cells can secrete IFN-gamma and IL-4 in large amounts, with IFN-gamma in particular.
Cell Culture Techniques ; Fetal Blood ; cytology ; Galactosylceramides ; pharmacology ; Humans ; Interferon-gamma ; secretion ; Interleukin-15 ; pharmacology ; Interleukin-2 ; pharmacology ; Interleukin-4 ; secretion ; Leukocytes, Mononuclear ; cytology ; Lymphocyte Activation ; Natural Killer T-Cells ; metabolism

UNLABELLEDDendritic cells (DC) are very potent antigen-presenting cells (APC) with a unique ability to activate naive T cells to induce the differentiation of TH1/TH2. Monocytes can develop into DC in the presence of different cytokines such as granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-4. DCs are thought to play a key role in the initiation and maintenance of T cell immunity to inhaled antigens. While the density of DC within the bronchial mucosa is increased in asthma, there is little information currently available concerning the effects of DC in asthmatic children.

OBJECTIVETo investigate the role of dendritic cells in the pathogenesis of acute attack of asthma in children.

METHODSThomas' method was adopted. The adherent precursors of DC were isolated from peripheral blood of asthmatic children in acute attack stage and healthy controls. The adherent cells were induced with granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-4 (IL-4) and tumor necrosis factor-alpha (TNF-alpha) to DC in vitro. The expression of the surface molecules CD80, CD86, HLA-DR etc. on the DC was examined by fluorescent activated cell sorter (FACS). And the ability to secret IL-10, IL-12 and their potentials to stimulate the proliferative reaction of DC inductive self T- lymphocyte were observed.

RESULTSThe results showed that in asthmatic children's acute attack stage, self T- lymphocyte proliferative reaction induced by DC was remarkably increased compared with normal control subjects (P < 0.01). At the same time, the asthmatic children in acute attack stage had remarkably decreased the ability to secret IL-10 compared with normal control subjects (P < 0.01), while the ability to secret IL-12 remarkably decreased compared with normal control subjects (P < 0.01); meanwhile, the HLA-DR and co-stimulating factor CD86(B(7-2)) expressed by DCs remarkably increased in the asthmatic children in acute attack stage compared with normal control subjects (P < 0.01).

CONCLUSIONDC possibly plays a vital role in the immunological mechanism of asthma by means of inducing the differentiation of TH1/TH2, that is DC may be the key factor in initiating the airway allergic reaction and the possible mechanism may involve interleukins (especially IL-10 and IL-12, etc.) secreted by DCs.

Adolescent ; Antigens, CD ; metabolism ; Asthma ; metabolism ; physiopathology ; Child ; Child, Preschool ; Dendritic Cells ; cytology ; drug effects ; metabolism ; Female ; Flow Cytometry ; Granulocyte-Macrophage Colony-Stimulating Factor ; pharmacology ; HLA-DR Antigens ; metabolism ; Humans ; Interleukin-10 ; secretion ; Interleukin-12 ; secretion ; Interleukin-4 ; pharmacology ; Male

OBJECTIVETo investigate the effects of zinc deficiency on the relevant immune function in rats with LPS-induced sepsis.

METHODSSixty rats were divided into low zinc group (LZ), normal zinc pair-fed group (NP), and normal zinc control group (NC) according to the random number table, with 20 rats in each group. The rats in group LZ were fed with low zinc diet, and the rats in group NP were fed with normal zinc diet, with the same intake as that of group LZ by manual control, and the rats in group NC were fed with normal zinc diet freely. After being fed for 7 d, the rats all fasted and were further divide into the below subgroups named LZ-LPS, LZ-normal saline (NS), NP-LPS, NP-NS, NC-LPS, and NC-NS according to the random number table, with 10 rats in each subgroup. Rats in the LPS subgroups were intraperitoneally injected with 1 mg/mL LPS solution with the dosage of 5 mg/kg, rats in the corresponding NS subgroups were intraperitoneally injected with equivalent NS. The rats were sacrificed at post injection hour 6 to collect blood, spleen, and thymus. The serum level of zinc was detected by inductively coupled plasma mass spectrometry, and the serum alkaline phosphatase (ALP) activity was detected by automatic blood biochemical analyzer. The body weight and weight of spleen and thymus of rats were weighed, and the indices of spleen and thymus were calculated. Six routine blood indices were examined by automatic blood cell analyzer. The serum levels of interferon gamma (IFN-γ), TNF-α, IL-4, and IL-10 were determined with ELISA, and the ratio of IFN-γ to IL-4 was calculated. Data were processed with one-way analysis of variance and SNK test.

RESULTS(1) Serum levels of zinc and ALP activity in the LPS subgroups were significantly lower than those in the corresponding NS subgroups (with P values below 0.05). The two former indices in subgroups NP-NS and NC-NS were significantly higher than those in subgroup LZ-NS (with P values below 0.05). The two former indices in subgroups NP-LPS and NC-LPS were significantly higher than those in subgroup LZ-LPS (with P values below 0.05). (2) Body weight, spleen and thymus weight, indices of spleen and thymus in the LPS subgroups were similar with those in the corresponding NS subgroups (with P values above 0.05). The 4 former indices, except for body weight, in subgroups NP-NS and NC-NS were significantly higher than those in subgroup LZ-NS (with P values below 0.05). The 4 former indices, except for body weight, in subgroups NP-LPS and NC-LPS were significantly higher than those in subgroup LZ-LPS (with P values below 0.05). (3) Levels of leucocyte count in subgroups LZ-LPS and NP-LPS were significantly higher than those in the corresponding NS subgroups (with P values below 0.05). Level of leucocyte count in subgroup NC-NS was significantly higher than that in subgroup LZ-NS (P<0.05). Level of leucocyte count in subgroup NC-LPS was significantly lower than that in subgroup LZ-LPS (P<0.05). Levels of neutrophilic granulocyte count (NGC) and NG in the LPS subgroups were significantly higher than those in the corresponding NS subgroups (with P values below 0.05). The two former indices in subgroup NC-LPS were significantly lower than those in subgroup LZ-LPS (with P values below 0.05). Level of NG in subgroup NC-NS was significantly lower than that in subgroup LZ-NS (P<0.05). Levels of lymphocyte count and lymphocyte in subgroups LZ-NS, LZ-LPS, NP-NS, NP-LPS, NC-NS, and NC-LPS were respectively (1.8 ± 0.4) × 10⁹/L, (1.0 ± 0.3)× 10⁹/L, (2.6 ± 0.7) × 10⁹/L, (1.4 ± 0.4) × 10⁹/L, (3.3 ± 0.6) × 10⁹/L, (1.5 ± 0.5) × 10⁹/L, and 0.39 ± 0.10, 0.11 ± 0.03, 0.47 ± 0.12, 0.14 ± 0.04, 0.50 ± 0.09, 0.24 ± 0.07. The two former indices in the LPS subgroups were significantly lower than those in the corresponding NS subgroups (with P values below 0.05). The two former indices in subgroup NC-NS were significantly higher than those in subgroup LZ-NS (with P values below 0.05). The two former indices in subgroups NP-LPS and NC-LPS were significantly higher than those in subgroup LZ-LPS (with P values below 0.05). Level of lymphocyte count in subgroup NP-NS was significantly higher than that in subgroup LZ-NS (P<0.05). Levels of platelet count (PC) in subgroups NP-LPS and NC-LPS were significantly lower than those in the corresponding NS subgroups (with P values below 0.05). Levels of PC in subgroups NP-NS and NC-NS were significantly higher than those in subgroup LZ-NS (with P values below 0.05). Level of PC in subgroup NC-LPS was significantly higher than that in subgroup LZ-LPS (P<0.05). (4) Serum levels of TNF-α, IL-4, and IL-10 in each subgroup showed no significant differences (with P values above 0.05). Serum levels of IFN-γ and ratios of IFN-γ to IL-4 in subgroups LZ-NS, LZ-LPS, NP-NS, NP-LPS, NC-NS, and NC-LPS were respectively (75 ± 21), (233 ± 40), (80 ± 14), (345 ± 74), (66 ± 7), (821 ± 189) pg/mL, and 3.1 ± 1.0, 6.6 ± 1.7, 3.9 ± 1.7, 20.2 ± 8.3, 3.4 ± 1.5, 45.7 ± 7.6. The two former indices in the LPS subgroups were significantly higher than those in the corresponding NS subgroups (with P values below 0.05). The two former indices in subgroups NP-NS and NC-NS were similar with those in subgroup LZ-NS (with P values above 0.05). The two former indices in subgroups NP-LPS and NC-LPS were significantly higher than those in subgroup LZ-LPS (with P values below 0.05).

CONCLUSIONSZinc deficiency can induce the atrophy of spleen and thymus, and reduction of peripheral blood lymphocyte. In sepsis, zinc deficiency can further decrease the production of IFN-γ, thus making the cytokines of Th1/Th2 shift to Th2 and the immune imbalance worse.

Animals ; Cytokines ; Interferon-gamma ; Interleukin-10 ; Interleukin-4 ; Lipopolysaccharides ; pharmacology ; Rats ; Sepsis ; chemically induced ; Tumor Necrosis Factor-alpha ; metabolism ; secretion ; Zinc ; deficiency