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

OBJECTIVES: We established a Wistar rat model of asthma caused by toluene diisocyanate (TDI) exposure, and investigated the relationship between TDI exposure concentrations and respiratory hypersensitivity, airway inflammation, and cytokine secretions in animals, to better understand the mechanism of TDI induced occupational asthma. METHODS: Wistar rats were exposed to two different concentrations of TDI vapor four hours a day for five consecutive days. Bronchoalveolar lavage (BAL) was performed, and differential leucocytes from the BAL fluid were analyzed. Lung histopathological examination was carried out to investigate the inflammatory status in the airways. Production of cytokines interleukin (IL)-4 and IL-5 productions in the BAL fluid in vivo was determined with enzyme-linked immunosorbent assay kits. RESULTS: The TDI-exposed rats exhibited greater airway hypersensitivity symptoms than the control rats. The BAL differential cell count and lung histopathological examination demonstrated that inflammation reactions were present in both the central and peripheral airways, characterized with marked infiltration of eosinophils in the TDI-exposed rats. The cytokine assay showed that IL-4 and IL-5 were predominantly produced in the BAL fluid in vivo. CONCLUSIONS: These findings imply that TDI exposure concentrations may greatly affect the occurrence and extent of inflammatory events and that Th2 type cytokines may play an important role in the immunopathogenesis of TDI-induced occupational respiratory hypersensitivity.
Animals ; Bronchoalveolar Lavage Fluid/chemistry/cytology ; Enzyme-Linked Immunosorbent Assay ; Eosinophils/cytology/immunology ; Female ; Gases/chemistry ; Hypersensitivity/pathology ; Interleukin-4/*analysis ; Interleukin-5/*analysis ; Lung/*drug effects/pathology/secretion ; Rats ; Rats, Wistar ; Toluene 2,4-Diisocyanate/*toxicity

OBJECTIVETo confirm the efficacy and safety of Jitongning Capsule in the treatment of ankylosing spondylitis (AS).

METHODSA total of 120 AS patients with early-intermediate were randomly and equally assigned to Jitongning Capsule group and sulfasalazine group. Jitongning Capsule was orally taken 4.5 g per day and sulfasalazine was orally taken 2 g daily for 12 months. The primary endpoint was the proportion of patients achieving the Assessment in Ankylosing Spondylitis 20 (ASAS 20), secondary end points included Bath Ankylosing Spondylitis Disease Activity Index (BASDAI), Bath Ankylosing Spondylitis Functional Index (BASFI), Bath Ankylosing Spondylitis Metrology Index (BASMI), patient's global assessment by VAS rating, spinal pain, general pain and night pain, erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP). Tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ) and interleukin-4 (IL-4) in the peripheral blood mononuclear cells (PBMC) of AS patients were measured.

RESULTSA total of 111 patients completed the study. There were 58 patients in Jitongning group and 53 patients in sulfasalazine group. Both drugs showed mild and occasional side effects. After treated by Jitongning Capsule and sulfasalazine, the proportion of ASAS20 responders at 12 month was 72.41% (42/58) and 67.92% (36/53) respectively. Both Jitongning Capsule and sulfasalazine treatment induced significant decrease in the proportion of CD4(+)T cell and CD8(+)T cell expressing TNF-α and IFN-γ at 12-month of treatment compared with baseline values (P<0.05).

CONCLUSIONJitongning Capsule are effective in a setting close to real-life medical care with a sustained improvement in signs and symptoms of AS, and reduce cytokine levels in PBMC. It showed comparable effects to sulfasalazine.

Adult ; Blood Sedimentation ; C-Reactive Protein ; metabolism ; CD4-Positive T-Lymphocytes ; drug effects ; secretion ; CD8-Positive T-Lymphocytes ; drug effects ; secretion ; Drugs, Chinese Herbal ; adverse effects ; therapeutic use ; Endpoint Determination ; Female ; Flow Cytometry ; Humans ; Interferon-gamma ; secretion ; Interleukin-4 ; secretion ; Male ; Middle Aged ; Spondylitis, Ankylosing ; blood ; drug therapy ; immunology ; Treatment Outcome ; Tumor Necrosis Factor-alpha ; secretion ; Young Adult

OBJECTIVETo analyze in vitro the effect of mesenchymal stem cells (MSCs) on secreting cytokines by T lymphocytes and ratio of CD4⁺CD25⁺ T cells from patients with immune thrombocytopenia (ITP).

METHODSHuman bone marrow-derived MSCs were isolated by Ficoll Hypaque and cultured for proliferating to passage cells. Allogeneic T lymphocytes of health adults and ITP patients were isolated from peripheral blood by Ficoll Hypaque and nylon cotton column, and the ratio of CD4⁺CD25⁺ T cells was detected by flow cytometry. Then the different amounts of 1 × 10⁴, 5 × 10⁴, 2 × 10⁵ MSCs per well treated with mitomycin as stromal feeder layers were co-cultured with above-mentioned T lymphocytes, 5 days after cocultivation, the ratio of CD4⁺CD25⁺ T cells was detected by flow cytometry and the levels of IL-2, IFN-γ, IL-4, IL-10 were measured by enzyme- linked immune sorbent assay (ELISA).

RESULTSAfter co-cultured with 2 × 10⁵ MSCs for 5 days, the ratio of CD4⁺CD25⁺ T cells and CD4⁺CD25⁺/CD4⁺ were significantly higher than of separate T lymphocytes in ITP patients [(4.56 ± 0.70)% vs (2.24 ± 0.81)%, (9.91 ± 1.18)% vs (4.08 ± 1.17)%, respectively] (P<0.05). To compare with separate T lymphocytes in ITP patients, the cytokine concentrations of IL-2 and IFN-γ from the culture supernatants significantly reduced from (280.47 ± 17.33) pg/ml to (97.21 ± 12.07) pg/ml and from (129.33 ± 16.34) pg/ml to (72.75 ± 7.81) pg/ml, respectively. In contrast, the cytokine concentrations of IL-4 and IL-10 increased from (16.34 ± 2.60) pg/ml to (37.98 ± 4.05) pg/ml and from (54.78 ± 5.62) pg/ml to (113.77 ± 5.68) pg/ml, respectively.

CONCLUSIONMSCs significantly inhibited the cytokine levels of IL-2 and IFN-γ secreted by Th1 cells and promoted the releases of IL-4 and IL-10 by Th2 cells in ITP , thereby regulating the balance between Th1 and Th2 reaction, as well as up-regulating the expression of CD4⁺CD25⁺ T cells in vitro,then induced the immunologic tolerance of ITP.

Adolescent ; Adult ; CD4-Positive T-Lymphocytes ; secretion ; Cells, Cultured ; Female ; Flow Cytometry ; Humans ; Interferon-gamma ; metabolism ; Interleukin-10 ; metabolism ; Interleukin-2 ; metabolism ; Interleukin-4 ; metabolism ; Male ; Mesenchymal Stromal Cells ; cytology ; Middle Aged ; Thrombocytopenia ; metabolism ; Young Adult

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

The aim of this study was to investigate the effect of brucine on secretion of TNF-α, IFN-γ, IL-4 and proliferation of T lymphocytes in patients with aplastic anemia (AA), and to explore its mechanism. Peripheral blood T lymphocytes from 10 patients with AA and 10 healthy volunteers were isolated, purified and cultured. T lymphocytes from the patients were divided into 0, 100, 200 and 400 µg/ml brucine-treated groups. T lymphocytes from healthy volunteers were used as control group. After being cultured for 72 hours, the levels of TNF-α, IFN-γ, IL-4 in the supernatant of cultured T lymphocytes from AA patients were detected by ELISA, and the proliferation of T lymphocytes from AA patients was detected by MTT. The results showed that compared with the normal control group, the levels of TNF-α and IFN-γ in the culture supernatant significantly increased, and IL-4 was significantly decreased. The levels of TNF-α and IFN-γ in the culture supernatant of brucine treated groups were lower, and were dependent on the concentration of brucine. However, the levels of IL-4 were found to be not obviously changed. The inhibition rate of T lymphocytes in 100, 200 and 400µg/ml brucine-treated groups were (13.61 ± 4.31)%, (14.28 ± 4.31)% and (15.12 ± 4.56)% respectively, among which the differences were not statistically significant. It is concluded that the brucine can reduce the levels of TNF-α and IFN-γ through inhibiting the proliferation of T lymphocytes in AA patients, which provides experimental basis for therapy of AA patients.
Adolescent ; Adult ; Anemia, Aplastic ; immunology ; metabolism ; Case-Control Studies ; Cell Proliferation ; Cells, Cultured ; Child ; Female ; Humans ; Interferon-gamma ; metabolism ; Interleukin-4 ; metabolism ; Male ; Middle Aged ; Strychnine ; analogs & derivatives ; pharmacology ; T-Lymphocytes ; cytology ; secretion ; Tumor Necrosis Factor-alpha ; metabolism ; Young Adult

OBJECTIVETo investigate the effects of polyinosinic-polycytidylic acid (polyI:C) on the production of thymic stromal lymphopoietin (TSLP) and airway inflammation in mice with exacerbated asthma induced by respiratory syncytial virus (RSV).

METHODSThirty-two female BALB/c mice were randomly divided into 4 groups, namely the PBS control group, OVA group, OVA/RSV group, and OVA/RSV/polyI:C group. In the latter 3 groups, the mice were sensitized by OVA and stimulated with nebulized OVA. RSV was inoculated into the nasal cavity of the sensitized mice and polyI:C (1 mg/kg) was intramuscularly administered. The airway response to metacholine was examined, and the serum levels of IL-4, IL-5, IL-13, and IFN-γ and TSLP in the supernatants of bronchoalveolar lavage fluid (BALF) were detected using ELISA. The total BALF cells, eosinophils, lymphocytes and neutrophils were counted. The lung specimens were collected to observe the inflammation with HE staining, and immunohistochemistry was employed to determine TSLP production in the airway epithelial cells.

RESULTSThe mice in RSV/OVA/polyI:C group showed a significantly lower airway responsiveness to metacholine than those in OVA/RSV group (P<0.01). Compared with OVA/RSV group, RSV/OVA/polyI:C group showed significantly lower serum levels of IL-4, IL-5, IL-13 and TSLP in BALF (P<0.05), with also lower total BALF cells, eosinophils and lymphocytes (P<0.05) and lessened infiltration of the airway inflammatory cells. Immunohistochemistry of TSLP also demonstrated a lower production of TSLP in the airway epithelial cells in RSV/OVA/polyI:C group than in OVA/RSV group.

CONCLUSIONSpolyI:C can inhibit the increase in TSLP production in the airway epithelial cells after RSV infection and relieve airway inflammation in mice with RSV-induced asthma exacerbation.

Animals ; Asthma ; blood ; metabolism ; virology ; Bronchoalveolar Lavage Fluid ; Cytokines ; secretion ; Female ; Inflammation ; pathology ; Interleukin-13 ; blood ; Interleukin-4 ; blood ; Interleukin-5 ; blood ; Mice ; Mice, Inbred BALB C ; Poly I-C ; pharmacology ; Respiratory Syncytial Virus Infections ; blood ; metabolism ; Respiratory Syncytial Viruses

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

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

BACKGROUND AND OBJECTIVECytokine-induced killer (CIK) cells and autologous dendritic cells-CIK (DC-CIK) cells co-cultured with autologous dendritic cells (DCs) and CIK cells are commonly used for immunotherapy recently. We compared the anti-tumor immune response of CIK cells, autologous DC-CIK cells, and semi-allogeneic DC-CIK cells to explore a more effective anti-tumor adoptive immunotherapy approach.

METHODSPeripheral monocytes were isolated from patients with renal carcinoma, lung cancer, or maxillary squamous cell carcinoma and their healthy adult children. Isolated cells were cultured and induced as DCs and CIK cells in vitro. CIK cells from patients were co-cultured with autologous DCs and DCs from their children respectively, generating DC-CIK cells and semi-allogeneic DC-CIK cells. The anti-tumor activities of autologous CIK cells, autologous DC-CIK cells, and semi-allogeneic DC-CIK cells were measured by LDH assay. Intracellular staining was used to test the secretion of cytokines. Flow cytometry was applied for detecting the phonotype changes of these three types of cells. Cell proliferation and cell apoptosis were detected by 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE) and Annexin V/PI respectively.

RESULTSCompared with autologous CIK cells and DC-CIK cells, semi-allogeneic DC-CIK cells significantly enhanced the anti-tumor activity and IFN-gamma secretion, reduced IL-4 secretion, increased the ratio of CD3(+)CD56(+) cells and CD3(+)CD8(+) cells, decreased the number of CD4(+)CD25(+) cells, promoted cell proliferation, and lessened cell apoptosis.

CONCLUSIONSSemi-allogeneic DC-CIK cells had a stronger anti-tumor effect than did autologous CIK cells and DC-CIK cells. Our results provided experimental evidence for clinical application of DC-CIK cells.

Apoptosis ; Carcinoma, Squamous Cell ; metabolism ; pathology ; Cell Line, Tumor ; Cell Proliferation ; Cells, Cultured ; Coculture Techniques ; Cytokine-Induced Killer Cells ; cytology ; immunology ; metabolism ; Cytokines ; metabolism ; Cytotoxicity, Immunologic ; Dendritic Cells ; cytology ; immunology ; metabolism ; Hep G2 Cells ; Humans ; Immunotherapy, Adoptive ; Interferon-gamma ; secretion ; Interleukin-4 ; secretion ; K562 Cells ; Kidney Neoplasms ; metabolism ; pathology ; L-Lactate Dehydrogenase ; metabolism ; Lung Neoplasms ; metabolism ; pathology ; Maxillary Neoplasms ; metabolism ; pathology