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

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

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

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

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

OBJECTIVETo assess the association of tumour necrosis factor-α (TNF-α) gene polymorphisms at positions -863C/A, -857C/T, -238G/A and Graves disease (GD) susceptibility in Chinese Han population in Anhui region.

METHODSThe polymorphisms of TNF-α gene were determined by polymerase chain reaction with specific primers in 254 patients affected with GD and 212 healthy controls. Allelic and genotypic frequencies in GD group and normal controls as well as in different genders were compared. The allelic and genotypic frequencies for different thyroid stimulating hormone receptor antibody (TRAb) levels (TRAb > 12 U/L; ≤12 U/L) were also compared among patients with earlier onset GD.

RESULTS(1) The A allele at -863C/A locus in GD group (16.73%) was significantly greater than that of the control group (11.79%) (P< 0.05, OR = 1.503); the frequency of AA+CA genotype of -863C/A locus in GD group (32.68%) was significantly greater than that of control group (23.58%) (P< 0.05, OR = 1.573). There was no significant difference (P> 0.05) in the allelic and genotypic frequencies of -857C/T, -238G/A loci between the two groups. (2) There was no significant difference (P> 0.05) in the allelic and genotypic frequencies of -863C/A, -857C/T, -238G/A loci between patients of different genders. (3) There was no significant difference (P>0.05) in such frequencies between patients with earlier onset GD and different TRAb levels (TRAb > 12 U/L; ≤12 U/L).

CONCLUSION(1) The -863 A allele of TNF-α gene may contribute to the development of GD in Chinese Han population in Anhui, whilst -857C/T, -238G/A alleles may not. (2) There is no association between TNF-α gene -863C/A, -857C/T, -238G/A polymorphisms and development of GD in different genders. (3) There was no association between above polymorphisms and TRAb levels in patients with earlier onset GD.

Adolescent ; Adult ; Aged ; Asian Continental Ancestry Group ; genetics ; Female ; Genetic Predisposition to Disease ; Graves Disease ; genetics ; immunology ; Humans ; Immunoglobulins, Thyroid-Stimulating ; genetics ; immunology ; Male ; Middle Aged ; Polymorphism, Genetic ; Receptors, Thyrotropin ; genetics ; immunology ; Tumor Necrosis Factor-alpha ; genetics ; immunology ; Young Adult

BACKGROUNDThe Fc receptor associated pathway might improve the immune responses against hepatitis B virus (HBV) as previously described by us. In addition, the Flt3 ligand (FL) has been reported to potentiate antigen presenting cells in vivo and may act as a potential adjuvant to boost antigen-specific immune responses. In this study, the immune efficacies of a set of fusion proteins of HBsAg and Fc and/or FL were evaluated in HBsAg transgenic mice.

METHODSThe fusion proteins composed of HBsAg and the Fc domain of murine IgG1 (HBsAg-Fc) and/or the Flt3 ligand, and yeast-derived recombinant HBsAg were used as immunogen to immunize HBsAg transgenic mice, respectively. Serum and liver HBsAg levels, serum anti-HBsAg and cytokine profile, and the activities of alanine aminotransferase (ALT)/AST were investigated after immunization.

RESULTSAfter six injections, the most pronounced decrease in serum and liver HBsAg levels was observed in the HBsAg-Fc immunized group. In addition, serum Th1 cytokines and ALT/AST activities were highest in this group, indicating an effective induction of a favorable cellular immune response. Interestingly, the fusion protein containing HBsAg-Fc and the Flt3 ligand stimulated an alternative Th1-type immune response featured with high level productions of tumor necrosis factor α (TNF-α) and monocyte chemoabstractant protein 1 (MCP-1), causing a more severe cytotoxicity in hepatocytes while showed less effective in reducing serum HBsAg level.

CONCLUSIONHBsAg-Fc is effective in eliciting both the humoral and cellular immune responses against HBsAg in HBsAg transgenic mice, which makes it a potential immunogen for the immunotherapy of chronic hepatitis B.

Animals ; Chemokine CCL2 ; metabolism ; Cytokines ; metabolism ; Enzyme-Linked Immunosorbent Assay ; Female ; Hepatitis B Surface Antigens ; genetics ; immunology ; metabolism ; Immunity, Cellular ; immunology ; Immunity, Humoral ; immunology ; Male ; Mice ; Mice, Inbred C57BL ; Mice, Transgenic ; Receptors, Fc ; genetics ; immunology ; metabolism ; Recombinant Fusion Proteins ; genetics ; immunology ; metabolism ; Tumor Necrosis Factor-alpha ; metabolism