Escin, as an internally applied anti-inflammatory agent, has been widely used in the treatment of inflammation and edema resulting from trauma or operation in the clinic. However, the effect of its external use on cutaneous inflammation and edema remains unexplored. In the present study, the anti-inflammatory and anti-edematous effects of external use of escin were studied in carrageenan-induced paw edema and histamine-induced capillary permeability in rats, paraxylene-induced ear swelling in mice, and cotton pellet-induced granuloma in rats. Effects of external use of escin gel on prostaglandin E2 (PGE2), tumor necrosis factor-α (TNF-α), and interleukin-1β (IL-1β) were determined by ELISA. The anti-inflammatory mechanism was explored by detecting the expression of glucocorticoid receptor (GR) with Western blotting and Real-time PCR analyses, with further exploration of nuclear factor-κB (NF-κB), p38 mitogen-activated protein kinase (P38MAPK) and activator protein-1 (AP-1) expressions. We demonstrated that external use of escin showed significant anti-inflammatory effects on acute and chronic inflammation in different animal models and its anti-inflammatory effects might be related to down-regulation of PGE2, TNF-α, and IL-1β. The results also showed that escin exerted its anti-inflammatory effects by promoting the expression of GR, with the possible mechanism being inhibition of the expressions of GR-related signaling molecules such as NF-κB and AP-1.
Aesculus ; chemistry ; Animals ; Anti-Inflammatory Agents ; administration & dosage ; Dinoprostone ; immunology ; Edema ; drug therapy ; genetics ; immunology ; Escin ; administration & dosage ; Female ; Humans ; Interleukin-1beta ; genetics ; immunology ; Male ; Mice ; Plant Extracts ; administration & dosage ; Rats ; Rats, Sprague-Dawley ; Receptors, Glucocorticoid ; genetics ; immunology ; Tumor Necrosis Factor-alpha ; genetics ; immunology

BACKGROUNDSepsis is a major cause of mortality in Intensive Care Units. Anesthetic dose isoflurane and 100% oxygen were proved to be beneficial in sepsis; however, their application in septic patients is limited because long-term hyperoxia may induce oxygen toxicity and anesthetic dose isoflurane has potential adverse consequences. This study was scheduled to find the optimal combination of isoflurane and oxygen in protecting experimental sepsis and its mechanisms.

METHODSThe effects of combined therapy with isoflurane and oxygen on lung injury and sepsis were determined in animal models of sepsis induced by cecal ligation and puncture (CLP) or intraperitoneal injection of lipopolysaccharide (LPS) or zymosan. Mouse RAW264.7 cells or human peripheral blood mononuclear cells (PBMCs) were treated by LPS to probe mechanisms. The nuclear factor kappa B (NF-κB) signaling molecules were examined by Western blot and cellular immunohistochemistry.

RESULTSThe 0.5 minimum alveolar concentration (MAC) isoflurane in 60% oxygen was the best combination of oxygen and isoflurane for reducing mortality in experimental sepsis induced by CLP, intraperitoneal injection of LPS, or zymosan. The 0.5 MAC isoflurane in 60% oxygen inhibited proinflammatory cytokines in peritoneal lavage fluids (tumor necrosis factor-alpha [TNF-β]: 149.3 vs. 229.7 pg/ml, interleukin [IL]-1β: 12.5 vs. 20.6 pg/ml, IL-6: 86.1 vs. 116.1 pg/ml, and high-mobility group protein 1 [HMGB1]: 323.7 vs. 449.3 ng/ml; all P< 0.05) and serum (TNF-β: 302.7 vs. 450.7 pg/ml, IL-1β: 51.7 vs. 96.7 pg/ml, IL-6: 390.4 vs. 722.5 pg/ml, and HMGB1: 592.2 vs. 985.4 ng/ml; all P< 0.05) in septic animals. In vitro experiments showed that the 0.5 MAC isoflurane in 60% oxygen reduced inflammatory responses in mouse RAW264.7 cells, after LPS stimulation (all P< 0.05). Suppressed activation of NF-κB pathway was also observed in mouse RAW264.7 macrophages and human PBMCs after LPS stimulation or plasma from septic patients. The 0.5 MAC isoflurane in 60% oxygen also prevented the increases of phospho-IKKβ/β, phospho-IκBβ, and phospho-p65 expressions in RAW264.7 macrophages after LPS stimulation (all P< 0.05).

CONCLUSIONCombined administration of a sedative dose of isoflurane with 60% oxygen improves survival of septic animals through reducing inflammatory responses.

Adult ; Anesthesia ; methods ; Animals ; Blotting, Western ; Bronchoalveolar Lavage Fluid ; Disease Models, Animal ; Female ; Humans ; Inflammation ; drug therapy ; Isoflurane ; therapeutic use ; Leukocytes, Mononuclear ; metabolism ; Lipopolysaccharide Receptors ; metabolism ; Lipopolysaccharides ; pharmacology ; Lung Injury ; drug therapy ; immunology ; metabolism ; Male ; Mice ; Mice, Inbred C57BL ; NF-kappa B ; metabolism ; Oxygen ; therapeutic use ; Peroxidase ; metabolism ; RAW 264.7 Cells ; Rats, Sprague-Dawley ; Sepsis ; drug therapy ; immunology ; Tumor Necrosis Factor-alpha ; metabolism

OBJECTIVETo explore the enhanced sensitivity of leukemia cell line KG-1a to activated immune cell-mediated cytolysis after treated with resveratrol.

METHODSThe value of 50% inhibition concentration (IC₅₀) for KG-1a by resveratrol was analyzed using trypan blue staining. Peripheral blood mononuclear cells were separated, and then activated by interleukin (IL)-2 and IL-15. The sensitivity of KG-1a treated with and without resveratrol to activated immune cell-mediated cytolysis was assayed by lactate dehydrogenase (LDH) -releasing assay. The expression of tumor necrosis factor related apoptosis inducing ligand (TRAIL) on the surface of activated immune cells and its receptors (DR4/5 and DcR1/2) on the surface of KG-1a were detected by flow cytometry.

RESULTSResveratrol could inhibit the proliferation of KG-1a and IC50 at 24 h was 25 mmol/L. At a ratio of 10:1 or 20:1 between effect and target, the cytolytic rates of treated KG-1a by activated immune cells were (55.80 ± 10.88)% and (72.31 ± 13.06)%, significantly higher than (24.96 ± 9.25)% and (37.93 ± 5.21)% of untreated KG-1a (P<0.05). The expression of DR5 on the surface of KG-1a treated with resveratrol was (9.05 ± 3.57)%, significantly higher than (3.11 ± 0.54)% of untreated KG-1a (P<0.05). Conversely, the expression of DcR1 on the surface of treated KG-1a was (13.23 ± 3.56)%, lower than (53.75 ± 10.51)% of KG-1a (P<0.05). When TRAIL pathway on the surface of activated immune cells was blocked, the cytolytic rates of treated KG-1a were (35.97 ± 6.36)% and (49.80 ± 10.68)%, significantly lower than (52.92 ± 6.98)% and (70.73 ± 9.79)% of untreated KG-1a (P<0.05) at the same ratio of effector and target.

CONCLUSIONResveratrol could enhance cytolytic sensitivity of KG-1a by activated immune cells through TRAIL pathway.

Cell Line, Tumor ; Cell Proliferation ; drug effects ; Humans ; Leukemia ; metabolism ; pathology ; Leukocytes, Mononuclear ; drug effects ; immunology ; metabolism ; Male ; Receptors, TNF-Related Apoptosis-Inducing Ligand ; metabolism ; Receptors, Tumor Necrosis Factor, Member 10c ; metabolism ; Stilbenes ; pharmacology ; TNF-Related Apoptosis-Inducing Ligand ; metabolism

OBJECTIVETo investigate the role played by γδ T cells in acute liver injury using the concanavalin A (ConA)-induced liver injury mouse model.

METHODSAcute liver injury was induced by intravenous injection of 10 mug/g of ConA into male C57BL/6J mice with wild-type or T cell receptor-γ knockout (TCR δ-/-) genetic backgrounds. Mice injected with PBS alone served as negative controls. The degree of liver damage was assessed by measuring serum levels of transaminase and cytokines at post-injection hours 3, 6, 12, 24, 48, and 72. The percentage of γδ T cells and proportions of different subsets in liver lymphocytes were measured by flow cytometry.

RESULTSThe TCR δ-/- mice showed significantly higher levels of the inflammatory cytokines IFN-γ, TNFα and IL-4 than the wild-type mice at post-injection hour 3. The percentage of liver γδ T cells increased with increased injury degree, and the extent of increase was significantly higher in the TCR δ-/- mice than the wild-type mice (post-injection hour 6: 6302.61+/-592.06 vs. 1319.26+/-355.48, 12: 6569.44+/-1060.98 vs. 3415.53+/-343.90, 24: 6514.29+/-757.26 vs. 2062.73+/-365.67, 48: 1262.61+/-558.07 vs. 113.66+/-113.26, and 72: 226.54+/-98.20 vs. 42.35+/-21.51 U/L; all P less than 0.05). In addition, compared to the negative control mice, the ConA-induced mice showed a higher proportions of Vγ4 γδ T cells to total γδ T cells (17.78+/-2.95 vs. 25.26+/-2.43) and to total liver lymphocytes (0.47+/-0.07 vs. 0.66+/-0.05). Similarly, compared to the negative control mice, the ConA-induced mice showed a higher proportion of Vγ1 γδ T cells to total γδ T cells (38.37+/-6.10 vs. 50.19+/-5.52) but the proportion to total liver lymphocytes was not significantly different among the groups (0.76+/-0.18 vs. 0.78+/-0.25). Reinfusion of Vγ4 γδ T lymphocytes into TCR δ-/- mice led to lower serum ALT levels than reinfusion of Vγ1 γδ T lymphocytes (5054.10+/-1748.51 vs. 12333.56+/-663.535 U/L).

CONCLUSIONγδ T cells play a protective role in ConA-induced liver injury and this effect maybe mediated by the Vγ4 γδ T cell subset.

Animals ; Chemical and Drug Induced Liver Injury ; immunology ; pathology ; Concanavalin A ; toxicity ; Interferon-gamma ; immunology ; Interleukin-4 ; immunology ; Liver ; drug effects ; immunology ; pathology ; Male ; Mice ; Mice, Inbred C57BL ; Receptors, Antigen, T-Cell, gamma-delta ; metabolism ; T-Lymphocyte Subsets ; immunology ; Tumor Necrosis Factor-alpha ; immunology

As a superantigen protein, Staphylococcal enterotoxin C2 (SEC2) activates the immune system effectively even in extremely low concentrations, and this property could be applied in adjuvant therapy against tumors and infectious diseases. In order to enhance the superantigen activity of SEC2, the residues at position 102-106 of native SEC2 were substituted for WWH, WWT and WWP by over-lap PCR, and three mutants named ST-1, ST-2 and ST-3 were obtained. Stimulating activity to murine lymphocytes proliferation and inhibiting activity to tumor cell growth of the three mutants were significantly improved compared with the native SEC2. Febrile activities of ST-1 and ST-3 were comparable with the native SEC2, but ST-2 showed markedly increased febrile activity than native SEC2. Moreover, the levels of IL-2, IFN-gamma and TNF-alpha secreted by T cells stimulated with the three mutants were significantly improved, which might be the possible reason for enhanced tumor cell growth inhibition activities. Furthermore, mVbeta8.2 gene transcription levels of murine splenocytes stimulated by the three mutants were dramatically increased compared with native SEC2, suggesting their increased affinities to TCR mVbeta8.2 molecular, which might be the main reason for their enhanced superantigen activities.
Animals ; Enterotoxins ; genetics ; immunology ; Female ; Interferon-gamma ; secretion ; Interleukin-2 ; secretion ; Lymphocyte Activation ; Mice ; Mice, Inbred BALB C ; Mutant Proteins ; genetics ; immunology ; Receptors, Antigen, T-Cell ; immunology ; Superantigens ; genetics ; immunology ; Tumor Necrosis Factor-alpha ; secretion

Interleukin-1 receptor antagonist (IL-1ra), tumor necrosis factor soluble receptors (sTNF-R) type I and II, and regulated upon activation, normal T-cell expressed and secreted (RANTES) play an important role in the modulation of primary glomerulonephritis (GN) course. The aim of the study was to assess whether pre-treatment measurements of IL-1ra, sTNF-R, and RANTES assessed conjointly may be useful as predicting factors in patients with GN. In 84 patients (45 males and 39 female) serum concentration (pg/mL) and urinary excretion (pg/mgCr) of cytokines were measured. After 12 months of therapy with steroids and cyclophosphamide the patients were divided into two subgroups: Responders (R) and Non-Responders (NR) according to the treatment results. The urinary IL-1ra, TNF-RI and RII were significantly higher in R than NR (1,732 vs 646 with P < 0.001, 13.1 vs 6.3 with P = 0.005, and 33.6 vs 14.4 with P = 0.012). The urinary RANTES excretion was increased in NR (79.6 vs 28.5; P < 0.001). The multivariable analysis showed that if conjointly assessed, only urinary IL-1ra, TNF-R I and R II, RANTES with 85% probability pointed the feature remission (R). In conclusion, the urinary excretion of IL-1ra, TNF-R I and R II, and RANTES examined conjointly are effective in predicting favorable response to immunosuppressive treatment in patients with GN.
Adult ; Cyclophosphamide/therapeutic use ; Female ; Glomerulonephritis/drug therapy/*metabolism/pathology ; Humans ; Immunosuppressive Agents/therapeutic use ; Interleukin 1 Receptor Antagonist Protein/*analysis/blood/urine ; Lymphocyte Activation ; Male ; Middle Aged ; Multivariate Analysis ; Predictive Value of Tests ; Receptors, Tumor Necrosis Factor, Type I/*analysis/blood/urine ; Receptors, Tumor Necrosis Factor, Type II/*analysis/blood/urine ; Steroids/therapeutic use ; T-Lymphocytes/immunology/metabolism

In order to assess the number and function of macrophages in the placenta of pregnancy complicated with gestational diabetes mellitus (GDM) as well as those of normal pregnancies, placenta samples were collected from 15 GDM patients (GDM group) and 10 normal pregnant women (control group). The expression levels of macrophage markers (CD68/CD14) and inflammatory cytokines (IL-6/TNF-α) in placenta were detected using immunohistochemistry and PCR. The results showed that the number of CD68+ or CD14+ cells in the GMD group was remarkably higher than that in the control group (P<0.05), indicating that the number of macrophages in the GDM group was significantly greater than that in the control group. The mRNA expression levels of CD68+, IL-6 and TNF-α were higher in the GMD group than in the control group. In conclusion, more macrophages accumulate in placenta of pregnancy complicated with GDM, and the expression levels of pro-inflammation factors are also increased in GDM pregnancies, suggesting that macrophages and inflammatory mediators (IL-6 and TNF-α) may play an important role in GDM.
Adult ; Antigens, CD ; Antigens, Differentiation, Myelomonocytic ; Cell Count ; Cytokines ; genetics ; immunology ; metabolism ; Diabetes, Gestational ; genetics ; immunology ; metabolism ; Female ; Humans ; Immunohistochemistry ; Inflammation Mediators ; immunology ; metabolism ; Interleukin-6 ; genetics ; immunology ; metabolism ; Lipopolysaccharide Receptors ; genetics ; metabolism ; Macrophages ; immunology ; metabolism ; pathology ; Placenta ; immunology ; metabolism ; Pregnancy ; Reverse Transcriptase Polymerase Chain Reaction ; Tumor Necrosis Factor-alpha ; genetics ; immunology ; metabolism

OBJECTIVETo investigate the changes of serum soluble CD 163 (sCD 163) level, to assess the severity of critical illness and to evaluate the immune status of sepsis or severe sepsis in children.

METHODA prospective study was conducted. The sCD 163 was determined in 50 cases with sepsis or severe sepsis in pediatric intensive care unit (PICU) and 23 cases of age- and gender-matched healthy children were enrolled as control during the period from April 2010 to March 2011. Double-antibody sandwich ELISA was used for sCD 163 measurement. The relationship with sCD 163 level and disease severity score (pediatric critical illness score, PCIS; and pediatric risk of mortality III, PRISM III), lymphocyte subsets, C-reactive protein (CRP), tumor necrosis factor α (TNFα) were analyzed.

RESULTThe sCD 163 in sepsis/severe sepsis groups (171.04 ± 177.85) mg/L was significantly higher than that in control group (44.19 ± 86.48) mg/L (P < 0.01).sCD 163 in sepsis group [(105.32 ± 145.87) mg/L] was significantly lower than that of severe sepsis group [(233.32 ± 171.78) mg/L] (P < 0.05). sCD 163 level was significantly higher in lower PCIS score patients. (P < 0.01). The sCD 163 levels was higher in PRISM III ≥ 10 than the PRISM III < 10 group. The sCD 163 levels were higher in death group than the survival group. The sCD 163 was negatively correlated with CD4 +, CD4 +/CD8 + (R = -0.820, P < 0.05; R = -0.839, P < 0.01).

CONCLUSIONDetection of sCD 163 was helpful in predicting the severity of sepsis and severe sepsis, and sCD 163 may reflect the immune status of critically ill children with sepsis.

Adolescent ; Antigens, CD ; blood ; Antigens, Differentiation, Myelomonocytic ; blood ; Biomarkers ; blood ; C-Reactive Protein ; analysis ; Case-Control Studies ; Child ; Child, Preschool ; Critical Illness ; Enzyme-Linked Immunosorbent Assay ; Female ; Humans ; Infant ; Intensive Care Units, Pediatric ; Lymphocyte Subsets ; immunology ; Male ; Prognosis ; Prospective Studies ; Receptors, Cell Surface ; blood ; Sepsis ; blood ; immunology ; mortality ; Severity of Illness Index ; Tumor Necrosis Factor-alpha ; blood

OBJECTIVETo investigate the effect of immature dendritic cells (inDC) genetically modified to express sTNFR I on acute graft-versus-host disease (aGVHD) and the graft-versus-leukemia (GVL) effect ofter allogeneic bone marrow transplantation (allo-BMT) in leukemic mice and its mechanism.

METHODSAn EL4 leukemia allo-BMT model was established with the BALB/c (H-2d) donor mice (DM)and C57BL/6 (H-2b) recipient mice (RM). The RM received DM bone marrow (BM) cells at a 1:1 ratio with spleen cells intravenously via tail vein at 4 h after TBI. Fifty DM were separated randomly into five groups: (1) Group A: total body irradiation (TBI) group, (2) Group B: lymphoma cell leukemia group, (3) Group C: allo-BMT group, (4) Group D: pXZ9-DC group, (5) Group E: sTNFR I-DC group. Acute GVHD scores, incidence of leukemic cell infiltration, histopathological analysis, survival rate, and survival rate of the recipients were estimated after allo-BMT. Enzyme-linked immunosorbent assay (ELISA) method was used to detect cytokines (INF-gamma and IL-4 ) production. Flow cytometry (FCM) analysis was used to detect allogeneic chimerism.

RESULTS(1) The mice in group A and group B all died of the BM failure and lymphoma cell leukemia, respectively. The mice in group C developed typical clinical signs of a GVHD after BMT with an average survival time(AST) of (11.50 +/- 3.50) d. The signs of aGVHD were less evident in the group D and E, and their AST (21.70 +/- 5.80 and 25.80 +/- 5.20 days, respectively) were all longer than that in group C (P < 0.05). AST of group E was the longest (P < 0.05). The mice in group B all died of leukemia within 18 days after engraftment of EL4 cells. There was was no significant difference in groups C, D and E in the incidence of leukemia (P > 0.05). (2) Serum IFN-gamma level reached peak value. At + 12 d, then decreased gradually in group C, D, and E, and then reached the nadir at +18 d post-BMT, with the lowest in group E (P < 0.05), and the level was significantly lower in group D than in group C (P < 0.05). After BMT, serum IL-4 level slightly decreased in group C, but gradually elevated in group D and E and reached their peak at +12 d, and even more significantly increased in group E (P < 0.05). There was no statistical significance in the pair wise comparison among three group (P < 0.05). (3) The average proportion of H-2d positive cells in RM was 95%-100% on day 30 post-BMT, with complete donor-type implantation.

CONCLUSIONImmature DC can induce immuno tolerance. Immature DC genetically modified to express sTNFR I has been shown to prevent acute GVHD in lethally irradiated mice reconstituted with allogeneic bone marrow grafts while maintaining the GVL response.

Animals ; Bone Marrow Transplantation ; adverse effects ; methods ; Dendritic Cells ; immunology ; Female ; Graft vs Host Disease ; prevention & control ; Graft vs Leukemia Effect ; Immune Tolerance ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Receptors, Tumor Necrosis Factor, Type I ; genetics ; Transplantation, Homologous

Establishing Epstein-Barr virus(EBV)-specific cytolytic T lymphocytes(EBV-CTLs) from peripheral blood mononuclear cells(PBMCs) for adoptive immunotherapy has been reported in EBV-associated malignancies including Hodgkin's lymphoma and nasopharyngeal carcinoma(NPC). In the current study, we performed ex vivo expansion of tumor-infiltrating lymphocytes(TILs) obtained from NPC biopsy specimens with a rapid expansion protocol using anti-CD3 monoclonal antibody(OKT3), recombinant human interleukin(IL)-2, and irradiated PBMCs from healthy donors to initiate the growth of TILs. Young TIL cultures comprised of more than 90% of CD3+ T cells, a variable percentage of CD3+CD8+ and CD3+CD4+ T cells, and less than 10% of CD3-CD16+ natural killer cells, a similar phenotype of EBV-CTL cultures from PBMCs. Interestingly, TIL cultures secreted high levels of the Th1 cytokines, interferon gamma (IFNγ) and tumor necrosis factor-alpha (TNF-α), and low levels of the Th2 cytokines, IL-4 and IL-10. Moreover, young TILs could recognize autologous EBV-transformed B lymphoblast cell lines, but not autologous EBV-negative blast cells or allogeneic EBV-negative tumor cells. Taken together, these data suggest that ex vivo expansion of TILs from NPC biopsy tissue is an appealing alternative method to establish T cell-based immunotherapy for NPC.
Biopsy ; CD3 Complex ; analysis ; CD4 Antigens ; analysis ; CD8 Antigens ; analysis ; Cells, Cultured ; Herpesvirus 4, Human ; immunology ; Humans ; Immunotherapy, Adoptive ; Interferon-gamma ; metabolism ; Interleukin-10 ; metabolism ; Interleukin-2 ; pharmacology ; Interleukin-4 ; metabolism ; Lymphocytes, Tumor-Infiltrating ; immunology ; virology ; Monocytes ; pathology ; Muromonab-CD3 ; pharmacology ; Nasopharyngeal Neoplasms ; immunology ; pathology ; therapy ; virology ; Receptors, IgG ; analysis ; T-Lymphocytes, Cytotoxic ; immunology ; virology ; Tumor Necrosis Factor-alpha ; metabolism