Detection and enrichment of T lymphocytes based on cytokine secretion in human mixed lymphocyte reaction

AIM: Detection and enrichment of T lymphocytes after allogeneic PBMNC stimulation according to secreted cytokine were performed in order to explore a new approach for studying allogeneic reactive T lymphocytes. METHODS: The novel cytokine secretion assay (CKSA) was applied to detect T lymphocyte secreting IFN-γ, IL-4 and IL-10 at single cell level in human mixed lymphocyte reaction. IFN-γ secreting T cells were enriched by means of magnetic sorting system. RESULTS: Allogeneic PBMNC stimulation didn't alter the proportion of IL-4 and IL-10 secreting T lymphocytes (which were 0.12%±0.03% and 0.10%±0.03%, respectively), but increased proportion of IFN-γ secreting T lymphocytes (1.12%±0.13%). These IFN-γ- secreting T lymphocytes could be further enriched to 67.3%±10.5% . CONCLUSION: It is feasible to detect significantly increased IFN-γ-secreting T cells after allogeneic PBMNC stimulation based on the novel CKSA technique, and these cells could be efficiently enriched for further use.     

生物炭对砂土水肥保持及苹果生长的影响研究

为解决砂土易漏水漏肥的问题,以富士/平邑甜茶为试材,探讨了生物炭在砂土苹果园中的应用效果。结果表明,生物炭与化肥配合穴施增加了优质果率,提高了单果质量与单株产量,分别达到70.21%、261.92 g和59.62 kg,较传统单独穴施化肥的对照分别增加了43.40%、15.21%和36.06%;可溶性固形物和维生素C含量较对照提高了7.10%和30.30%,果实品质得到改善;土壤速效氮和速效钾含量分别达153.58和142.2 mg·kg~(-1),较对照提升15.69%和33.27%;在降水后第3天生物炭施用土层土壤含水量较上、下土层分别高36.65%和27.92%,1个月后仍然分别高56.65%和45.79%。结果显示,生物炭与化肥配合穴施的方法在砂土苹果园中对其肥水的保持及苹果产量和品质的提高均有显著效果。     

Effects of sterigmatocystin on interferon-γ secretion of human peripheral blood mononuclear cellsin vitro

AIM:To explore the putative effects of sterigmatocystin (ST) on human help T lymphocyte(Th1)function. METHODS:The effects of ST on interferon-γ(IFN-γ)secretion of human peripheral blood mononuclear cells(HPBMc) in vitro were determined with ELISA method. RESULTS:The effects of ST on IFN-γ secretion of HPBMc in vitro were closely dependent on ST concentrations. ST at relatively lower concentrations (0.03125-0.12500 mg/L) showed inhibiting effects on IFN-γ secretion. While, stimulating effects could be found when ST concentration was above 0.25mg/L. The highest level was seen in ST 1 mg/L group (P＜0.05)。At concentration ranging from 0.25 mg/L to 1 mg/L, a positive dose- effects correlation was found between ST concentration and IFN-γ secretion (r=0.492, P＜0.01). Time-effects analysis from 1 h to 64 h after ST treatment (1 mg/L)showed that the effects of ST on IFN-γ secretion varied as the changes of treatment times. An inhibiting effect on IFN-γ secretion of HPBMc 4 h and 8 h after ST treatment was found (8 h, P＜0.05). As the treatment time prolonged from 16 h, IFN-γ level gradually increased (32 h, P<0.05). There was a positive correlation between treatment time of ST and IFN-γ level of HPBMc in vitro from 16 h to 64 h after ST treatment (r=0.736, P＜0.01).CONCLUSION:The effects of ST on IFN-γ secretion of HPBMc in vitro closely depended on concentration and treatment time of ST. Generally, inhibiting effects were found at relatively lower ST concentration and shorter treatment period, while stimulation effects could be seen at relatively higher ST concentration and longer ST treatment time period.     

Influences of protein kinase C inhibitors on the expression of IL-2 and IFN-γ by human T-lymphocytes

AIM:To investigate the influences of protein kinase C(PKC) inhibitors on the expression of interleukin-2(IL-2) and interferon-γ(IFN-γ) byin vitro activated T-lymphocytes. METHODS:Double fluorescent staining together with flow cytometry was adopted to detect intracellular cytokines and to analyze the effects of H7 and gossypol on IL-2 and IFN-γ expression levels of T-lymphocytes stimulated with phorbol ester (PDB)+ionomycin(I) in the presence of monensin.RESULTS:The expression rates of IL-2 and IFN-γ of CD3⁺ T cells stimulated with PDB+I for 4 h were 16.64±2.04 and 25.81±3.53(x±s), respectively, which were significantly higher than that of control (1.06±0.22 and 3.12±0.77)(P＜0.05). Gossypol was able to inhibit the expression of IL-2 and IFN-γ significantly, with the expression rates of 2.08±0.12 and 9.01±1.90, respectively. At the presence of 50 μmol/L H7, the rates of IL-2⁺ and IFN-γ⁺ CD3⁺ T cells were 0.43±0.06 and 2.40±0.27, respectively. The effect of H7 was stronger than that of gossypol. CONCLUSION:PKC plays an important role in the expression of IL-2 and IFN-γ of CD3⁺T cells and its inhibitors H7 and gossypol exert significant inhibitory effect on the expression of these two cytokines. It is suggested that H7 and gossypol may have modulatory effect on T-cell-dependent specific immune responses by inhibiting PKC activity.     

PDL1Ig gene-modified BMSCs induce immune tolerance in rat liver transplantation

AIM: To investigate the effects of bone marrow mesenchymal stem cells(BMSCs) modified by programed death ligand-1 immunoglobulin(PDL₁Ig) gene on immune rejection of orthotopic liver transplantation in rats. METHODS: Rat BMSCs were cultured and identified. The protein expression of PDL₁Ig in the BMSCs 72 h after infection with pAdEasy-1/PDL₁Ig was detected by Western blot. Mixed lymphocyte reaction was used to detect the inhibitory effect of BMSCs on the viability of T-lymphocytes in peripheral blood. The male Wistar rats were used as donors(n=40), and the male SD rats were used as recipients(n=40). The rat model of orthotopic liver transplantation was established by improved cuff method for observing acute rejection. The rats were randomly divided into control group, BMSCs treatment group, BMSCs/GFP treatment group and BMSCs/PDL₁Ig treatment group with 10 pairs each. Five rats were executed at the 7th day and the remains were used for measuring the survival time. RESULTS: The expression of PDL₁Ig in the BMSCs was detected after pAdEasy-1/PDL₁Ig infection. The effect of BMSCs/PDL₁Ig on the viability of the lymphocytes was stronger than that of BMSCs/GFP. The level of IL-4 in BMSCs/PDL₁Ig group was significantly higher than that in the other 3 groups, while the levels of IFN-γ and IL-2 were significantly decreased. The liver function in BMSCs/PDL₁Ig group was significantly improved and the levels of ALT, AST and TBil were almost recovered to normal at the 7th day after transplantation. Severe rejection reaction was observed in control group, and rejection reactions were decreased with different degrees in BMSCs treatment group and BMSCs/GFP treatment group. Much slighter rejection reaction and significantly longer survival time were showed in BMSCs/PDL₁Ig group than those in the other 3 groups. CONCLUSION: PDL₁Ig-modified BMSCs inhibit the rejection of liver transplantation in rats and induce the immune tolerance, and the effect is better than that of BMSCs alone.     

Experi mental study ontreatment of AIU by oral administration of Santigen

AIM: To observe the influence of oral bovine S antigen on antigen-induced uveoretinitis (AIU) in Wistar rat. METHODS: Rat was immunized with purified bovine retinal S antigen plus complete Freund’s adjuvant, and fourteen days later, S antigen was injected into the vitreous cavity, in which AIU was built. AIU rats were fed S antigen or BSA before or after immunization. Clinical and histopathologic evaluation, immunodiffusion, DTH testing and lymphocyte proliferation assays were carried out. RESULTS: Oral administration of BSA before or after immunization did not influence the inflammatory and immune responses of AIU. The AIU clinical scales on the first, third, fifth day, AIU duration, DTH testing and proliferative responses of lymphocytes to S antigen were significantly decreased in rats treated by oral administration of S antigen before immunization when compared with BAS fed group. But after lymphocytes were cultured with IL-2, the proliferative responses to S antigen increased. At the same time, the histopathologic inflammation and the titers of serum specific antibody in tolerized rats were lower than BSA fed group. On the other hand, the AIU clinical scales on the fifth day, AIU duration, DTH testing significantly decreased in rats treated by oral administration of S antigen after immunization when compared with BSA fed group. The titers of serum specific antibody in tolerized rats were lower than BSA fed group. CONCLUSION: The inflammatory responses, cellular and humoral immune responses were suppressed in AIU by oral administration of S antigen.     

Effect of IFN-γ inhalation on the cellular immunity of the lungs

AIM:To study the effect of IFN-γ inhalation on the anti-infection ability of the lungs in the immunocompromised host. METHODS:The immunological factors in the immunocompromised rats and the immunocompromised rats administrated IFN-γ via aerosol were investigated after 1, 3, 7 days when they were injected Candida albicans via tracheal. The Canidda albicans count of the left lung was also determined after 7 days when injecting pathogen. RESULTS:The Canidda albicans count of the left lung in IFN-γ group was significantly less than that of control group. The phagocyting and bactericidal percentages, Ia antigen expression percentages, the levels of TNF-α, IL-1β and IL-6 in the culture supernatant of the AM, the activity of IFN-γ and TNF-α in BALF (except the TNF-α on 7 th day) in IFN-γ group were markedly higher than those in control group. The expression of IFN-γ and IL-1β pulmonary tissues in IFN-γ group was higher than that in control group. The expression of TNF-α in IFN-γ group was less than that in control group. The expression of IL-6 was no changes between two groups. The levels of IFN-γ, IL-1β and IL-6 in the blood (except IL-1β on 3 rd day), and the killing ability of the lymphocytes in blood had no difference between two groups. CONCLUSION:Administration of IFN-γ via aerosol obviously enhanced the anti-infection ability of the lungs in the immunocompromised host, but has no influence on the whole body cellular immunity.     

Effects of interferon-γ, tumor necrosis factor-α and interleukin-1β on apoptosis of human airway smooth muscle cellsin vitro

AIM:To clarify if interferon-γ(IFN-γ), tumor necrosis factor-α(TNF-α)and interleukin-1β(IL-1β)can induce apoptosis of human airway smooth muscle cells (ASMCs) in vitro.METHODS:Human ASMCs were isolated and cultured in DMEM containing 10% fetal bovine serum. Passage 4-6 cell was used in the experiment. IFN-γ,TNF-α and IL-1β, were used separately or together in the treatment of human ASMCs. The effects of IFN-γ,TNF-α and IL-1β on the growth of the cells was detected by MTT method at the hour 0,24,48 and 72. Light microscopy and electron microscopy were used to examine the morphological change. DNA fragmentation was analyzed by agarose gel electrophoresis. SP immunohistological staing method was performed to detect the change of expressions of p 53, bcl- 2 and bax gene. The apoptosis cell percentage were detected by in situ end labeling technique (TUNEL)of fragmental DNA. RESULTS:(1)IFN-γ or IFN-γ together with TNF-α and IL-1β decreased the number of viable cells in a time dependent manner. (2) Light and electron microscopic examination showed cell shrinkage, membrane blebbing, nuclear contraction, chromatin condensation and nuclear fragmentation in human ASMCs. (3) Agarose gel electrophoresis showed a characteristic"ladder"of DNA bands representing integer multiples of the internucleosomal fragments (about 180-200 bp) in cytokine cotreated human ASMCs. (4)The expression of p 53 and bax gene in cytokine cotreated group was significantly higher than in control group, but the expression of bcl-2 gene was lower than in control group. (5)Stimultaneous treatment with IFN-γ(4×10⁵ U/L),TNF-α(4×10⁵ U/L)and /or IL-1β (10×10⁴ U/L) induced apoptosis of human ASMCs. Apoptotic index of human ASMCs in cytokine co-treated group was significantly higher than in control group (P＜0.01).CONCLUSION:Stimultaneous treatment with IFN-γ,TNF-α and /or IL-1β induced apoptosis of human ASMCs. These immune cytokines may play an important role in airway remodeling of asthma and of chronic obstructive pulmonary disease.     

Functional expression of B7H1 on pancreatic carcinoma panc-1 cells

AIM: To observe the effect of B7H1 expression in pancreatic carcinoma cells on the proliferation and activation of co-cultured T lymphocytes. METHODS: B7H1 expression in panc-1 cells before and after interferon-γ(IFN-γ) treatment or B7H1-siRNA transfection was evaluated by RT-PCR and flow cytometry. The influence of B7H1 expression on co-cultured PHA-activated T lymphocytes was determined by the methods of MTT and enzyme-linked immunosorbent assay (ELISA). RESULTS: B7H1 was highly expressed in panc-1 cells and up-regulated after IFN-γ stimulation. Such up-regulation led to the significant inhibition of T cell proliferation and secretion of cytokines such as IFN-γ and interleukin-2(IL-2). However, IL-10 production was enhanced. In contrast, knockdown of B7H1 expression in panc-1 cells by RNA interference resulted in increased T cell proliferation as well as IFN-γ and IL-2 production. Meanwhile, the IL-10 secretion decreased. CONCLUSION: B7H1-expressing panc-1 cells suppress T cell function by inhibiting T cell proliferation and production of Th1 cytokines. Suppression of B7H1 expression through siRNA restores T cell immune functions, indicating a potential strategy for immunotherapy against pancreatic cancer.     

Induction of tumor-specific cytotoxic T lymphocytes in vitro by dendritic cells pulsed with different glioma antigens

AIM: The goal of this study was to compare different methods for tumor antigen preparation, to observe the induction of tumor-specific cytotoxic T lymphocytes in rats by dendritic cells (DCs) pulsed with different tumor antigens. METHODS: The precursors of dendritic cells were isolated from bone marrow of rats, stimulated in vitro with recombinent rat granulocyte-macrophage colony-stimulating factor (rrGM-CSF) and interleukin-4 (rrIL-4). Then rat DCs were pulsed with C6 tumor cell antigens prepared with different methods: freeze-thaw, boiling or total protein extracted from ultrasonic crushed tumor cell. Subsequently primed DCs were cocultured with T lymphocytes isolated from spleen to induce CTL. Lymphocyte chemoattractant factor from DCs and cytokine IFN-γ release were determined by ELISA, the cytotoxicity of CTL was assayed by JAM test. RESULTS: DCs pulsed with boiled tumor cell in vitro induced an enhanced ability of T-cell proliferation and cytotoxic T lymphocyte activity.CONCLUSION: Our results demonstrated that DCs primed with boiled tumor cell may represent a method for inducing immune responses against the entire repertoire of tumor antigens of malignancies.     

Identification of HLA-A2 restricted cytotoxic T lymphocyte epitopes from tumor antigen PIWIL2

AIM: To identify the human leucocyte antigen A2 (HLA-A2) restricted cytotoxic T lymphocyte (CTL) epitopes from tumor antigen PIWIL2. METHODS: RT-PCR and Western blot was used to determine the expression of PIWIL2 in cancer cell lines MCF-7, SW480 and HT-29. HLA-A2 epitopes from PIWIL2 protein were predicted by the software of BIMAS, RankPep, NetMHC, NetCTL1.2 and IEDB. The peptides were synthesized by standard solid-phase methods. The binding affinity of the peptides to HLA-A2 molecules was evaluated by T2 cells binding assay. ELISPOT assay was used to investigate the levels of IFN-γ. The cytotoxicity assay in vitro was also used to determine the ability of inducing T cell response by the peptides. RESULTS: The expression of PIWIL2 was observed in MCF-7, SW480 and HT-29. The candidate peptide P485, P493 and P965 showed moderate affinity toward HLA-A2 molecule. ELISPOT assay showed P485 and P965 induced CTLs of IFN-γ release form CTLs. The CTLs induced by P485 and P965 lysed the MCF-7 cells. CONCLUSION: The peptides P485 and P965 are excellent HLA-A2 restricted cytotoxic T lymphocyte epitopes from the tumor antigen PIWIL2, which could serve as new candidates towards antitumor peptide vaccines.     

Modulatory function of high-dose hepatitis B surface antigen vaccine to cellular immune responses in mice

AIM:To observe the effects of high-dose hepatitis B surface antigen (HBsAg) vaccine on cellular immune response in BALB/C mice.METHODS:The mice were immunilized separately with low-dose and high-dose HBsAg vaccine by intramuscular injection two times. The specific proliferative activities of T lymphocytes were measured by -[H³]TdR incorporation assay. IL-2 as well as IFN-γ levels in the culture supernatant of T cells and anti-HBs IgG2a lever in sera were detected by enzyme-linked immunoabsorbent assay.RESULTS:After first vaccination with high-dose HBsAg, the proliferative activities of T cells in the experimental group were significantly stronger, both levels of IL-2 and IFN-γ were markedly higher than that in the control group and the percentage of mice to produce serum anti-HBs IgG2a was significantly higher compared to that of mice immunilized by low-dose HBsAg. All data in experimental groups were further increased after second dose of vaccine.CONCLUSION:Vaccination of mice with high-dose HBsAg can induce cellular immune responses tended to Th1(T helper 1 subset) response.     

Expression of PI3K in airway smooth muscle cell of asthmatic rats

AIM: To investigate the expression of PI3K in airway smooth muscle cell (ASMC) of asthmatic rats.METHODS: 16 Wistar rats were divided into two groups, asthma and normal control at random. After establishment of asthmatic model, flow cytometry, immunofluorescence and Western blotting were applied to detect the growth fraction of ASMC and the expression of PI3K in cultured ASMC from each rat.RESULTS: It was revealed from flow cytometry that the ratio of S + G2/M to total number of cells in asthma group ［ (27.90±3.44) % ］ was higher than that in normal control group ［ (13.00±1.56) %, P<0.05］. The expression of PI3K was observed in both asthma and normal control group. However, it was much higher in asthma group than that in normal control group. There was a positive correlation between the expression of PI3K and the growth fraction in ASMC. CONCLUSION: The increased expression of PI3K might play an important role in regulating the proliferation of ASMC in asthma.     

Effects of sterigmatocystin on IL-2 and IFN-γ secretion and expression in murine spleen cells in vitro

AIM: To explore the effects of sterigmatocystin (ST) on IL-2 and IFN-γ expression and secretion in murine spleen cells in vitro. METHODS: The secretion and expression of IL-2 and IFN-γ in murine spleen cells after ST pretreatment at five different dosages(0.125 mg/L,0.25 mg/L,0.5mg/L,1 mg/L,2 mg/L) were studied with ELISA and semi-quantitative RT-PCR method, respectively. RESULTS: Pretreatment of murine spleen cells in vitro with ST at five different dosages affected the IL-2 and IFN-γ secretion at protein level and expression at mRNA level of the treated cells. The effects varied dependently to the ST dosage. At relatively lower dosages, ST induced the expression of IL-2 and IFN-γ in murine spleen cells, while at relatively higher dosages, inhibitory effects were found, with the most significant inhibitory effects seen in ST 1 mg/L group. CONCLUSION:ST affected the se-cretion and expression of IL-2 and IFN-γ in treated murine spleen cells.At relatively lower dosage, ST induced IL-2 and IFN-γ secretion and expression, while at relatively higher dosages，inhibitory effects appeared.     

The expression of IFN-γ and IL-4 on T lymphocytes that infiltrate in nasal polyps

AIM:To investigate the expression of Th1-typed cytokine IFN-γ and Th2-typed cytokine IL-4 on T lymphocytes that infiltrate in nasal polyps for searching the pathogenesis of nasal polyps. METHODS:Nasal polyps tissue samples and peripheral blood were obtained from 21 patients. Normal human inferior turbinate mucosa and peripheral blood were obtained as well. Flow cytometry was adopted to detect the expression of IFN-γ and IL-4 of T lymphocytes. RESULTS: Th cytokines were rarely detected in inferior turbinate from normal human. Nasal polyps tissue consisted of abundant T lymphocytes. The expression of IL-4 and IFN-γ increased in peripheral blood from patients compared with normal human (P<0.05). The expression of IL-4 increased but the expression of IFN-γ decreased in nasal polyps compared with that of peripheral blood from the same patient (P＜0.05). CONCLUSION:There were generous of T lymphocytes infiltrating in nasal polyps. There was abnormal immune status in the local nasal mucosa from the patients, and the predomination of Th cytokine secretion changed compared with peripheral blood from the same patients, which resulted in the change of microenvironment of nasal mucosa and possibly close related to the formation of nasal polyps.     

Effect of microRNA-7 knockdown on ConA-induced acute liver injury in mice

AIM: To study the effect of microRNA-7 (miR-7) knockdown (KD) on concanavalin A (ConA)-induced acute liver injury (ALI) in mice.METHODS: Wild type (WT) mice and miR-7KD mice were received ConA (30 mg/kg) to induced acute liver injury model by intraperitoneal injection, and the morphological changes, liver weight and weight index were measured 48 h later. The pathological changes of the liver tissues were observed by HE staining. The levels of serum alanine aminotransferase (ALT), IL-4 and IFN-γ were detected by ELISA. The proportional changes of CD4⁺ T cells and the relative levels of IL-4 and IFN-γ were analyzed by flow cytometry.RESULTS: The color of the liver tissue became lighter, and the weight and weight index were changed significantly in miR-7KD mice compared with control group (P<0.05). HE staining showed that the inflammatory cell infiltration was increased in the liver of miR-7KD mice. Moreover, the level of serum ALT was significantly increased (P<0.05). The serum level of IFN-γ elevated significantly (P<0.01), while the IL-4 levels decreased significantly (P<0.01) in the serum of miR-7KD mice. Furthermore, the proportion of CD4⁺ T cells and relative IFN-γ cells increased obviously (P<0.01).CONCLUSION: miR-7 knockdown promotes the pathogenesis of the ConA-induced acute liver injury in mice.     

Transplantation of human amnion epithelial cells improves learning and memory function in Alzheimer's disease-like pathology rat model

AIM: To observe the treatment effect and its immune regulation of human amnion epithelial cells (hAECs) on Alzheimer's disease (AD)-like pathology rat model. METHODS: The hAECs were isolated from amnion with trypsin digestion, and the phenotype of hAECs was analyzed by flow cytometry. SD rats (n=48) were randomly divided into sham control group, model group, medium group and hAECs group. AD-like pathology rat model was induced by bilateral intraventricular injection of lipopolysaccharide (LPS). hAECs (5×10⁵) were injected into the hippocampus of the AD-like pathology rats. At 2 weeks after transplantation, the animals were tested by Morris water maze to observe the function of learning and memory. The pathological change of the brain was observed by HE staining. The expression of amyloid β-protein 42(Aβ42) and Tau protein and the level of acetylcholine (ACh) in the injury brain were determined by immunohistochemistry. The survival and differentiation of hAECs in the hippocampus were measured by immunofluorescent technique. The percentages of lymphocyte subsets in the peripheral blood mononuclear cells were analyzed by flow cytometry. The contents of serum cytokines were detected by cytometric bead array. RESULTS: Compared with model group and medium group, hAECs group showed shortened escape latency (P<0.01), increased frequency of going through the platform (P<0.05), reduced loss of hippocampal neurons, decreased expression of Tau protein and Aβ42 in the hippocampus (P<0.05), increased ACh level in the hippocampus (P<0.05), decreased percentages of Th1 and Th17 subsets, increased percentages of Th2 and Treg cells (P<0.05), decreased concentrations of IFN-γ and IL-2 in the serum, and increased concentration of IL-4(P<0.05). CONCLUSION: hAECs improve the cognitive learning and memory function and alleviate pathologic damage of hippocampus through immune regulation in AD-like pathology rats.     

The potential role of staphylococcal enterotoxin B in the early intestinal injury in postburn Staphylococcus aureus sepsis

AIM: To investigate the role of staphylococcal enterotoxin B (SEB) in early intestinal injury in scald rats with Staphylococcus aureus sepsis. METHODS: 86 male Wistar rats were randomly divided into four groups as folows: normal controls (n=10), scald control group(n=10), postburn sepsis group (n=50) and SEB monoclonal antibody (MAb) treatment group (n=16). Plasma samples were collected to determine SEB, endotoxin, tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ). RESULTS: After scald injury followed by Staphylococcus aureus challenge, the levels of SEB, TNF-α and IFN-γ in plasma were significantly higher than those of normal controls, peaking at 2-6 h (P<0.05 or P<0.01), while the intestinal diamine oxidase (DAO) activity declined constantly (P<0.05). It was shown that plasma SEB levels were significant negatively correlated with intestinal DAO activity (r=-0.4398, P=0.0170), and SEB MAb pretreatment could ameliorate the intestinal injury to certain extent. Moreover, Staphylococcus aureus challenge could increase the endotoxin levels in plasma and various tissues, which were attenuated by SEB MAb pretreatment. CONCLUSION: In postburn sepsis, SEB might be involved in the development of intestinal barrier dysfunction, which in turn resulting in gut-derived endotoxin translocation and aggravating the pathophysiologic changes caused by Staphylococcus aureus challenge.     

Design and immune activity detection of long peptide vaccines targeting lung cancer antigen COX-2

AIM:To design long peptides based on cytotoxic T-lymphocytes (CTL) epitope prediction for lung cancer antigen cyclo-oxygenase-2 (COX-2) and to study the immune activity of the long peptides. METHODS:HLA-A2 epitopes from COX-2 protein were predicted by NetCTL 1.2, SYFPEITHI and IEDB. The CTL epitope-concentrated area was analyzed, and the appropriate length of long peptides were designed. In vitro activity experiments were used to verify the immune activity of the long peptides. ELISPOT assay and intracellular cytokine staining assay were used to investigate the ability of the peptide to induce specific restricted CTLs and release of interferon-γ (IFN-γ). The ability of the peptides to induce T-cell response was investigated by lactate dehydrogenase (LDH) and CFSE cytotoxicity assay in vitro. RESULTS:ELISPOT and intracellular cytokine staining assay showed P315-338 and P375-401 were able to induce specific CTLs and higher levels of IFN-γ release. The results of LDH and CFSE cytotoxicity assays showed the CTLs induced by P315-338 and P375-401 lysed the target cells. CONCLUSION:Two long peptides pointing to lung cancer antigen COX-2 are successfully identified, which could be used as immunotherapy vaccine in future.