Protective Effects of Lentinan against T Lymphocytes Injury in Mice under Chronic Radiation Stress

Objective To study the effects of lentinan (LTN) on mice exposed to chronic radiation. Methods Animals were divided into three groups (n = 10), they were animals exposed to radiation (Rad), normal control animals (Ctr), and irradiated animals treated with LTN (Rad + LTN). Animal model of chronic radiation stress injury was induced by irradiating mice with 60Co γ-ray for 6 weeks from Monday to Friday consecutively. Before radiation, the mice in Rad + LTN group were ip injected with 0.5 mL LTN (0.01 mg/mL), whereas mice in other groups were injected with 0.9% physiological saline. The effects of LTN treatment on irradiated mice were examined by histological analysis on the spleen. The cell numbers and viability of T lymphocytes, which were isolated from the spleen, were determined by Trypan blue staining. Nitric oxide (NO) production and interleukin-2 (IL-2) secretion in T lymphocytes were also measured. Results Chronic radiation significantly reduced the body weights and the spleen and thymus indexes, associated with reduced T lymphocytes viability and functions, and elevated NO production. Treatment with LTN significantly normalized the elevated NO production, and attenuated the negative outcomes resulting from radiation mentioned above. Conclusion The results suggest that radioprotective effect of LTN may be contributed by improved T lymphocytes viability and functions via regulating the NO and IL-2 production in T lymphocytes.     

Rapamycin Modulates the Maturation of Rat Bone Marrow-derived Dendritic Cells

The purpose of the study was to observe the effect of rapamycin （RAPA） on the differentiation and maturation of rat bone marrow-derived dendritic cells （BMDCs） in vitro. BMDCs from Wistar rats were cultured with granulocyte-macrophage colony-stimulating factor plus interleukin-4 in the presence or absence of RAPA （20 ng/mL）, and stimulated with lipopolysaccharide （LPS） for 24 h before cells and supernatants were collected. Surface phenotype of BMDCs was flow-cytometrically detected to determine the expression of maturation markers, MHC class Ⅱ and CD86. Supernatants were analyzed for the production of IL-12 and IFN-γ cytokines by using ELISA. BMDCs were co-cultured with T cells from Lewis rats and mixed lymphocyte reaction was assessed by MTT method. The morphology of BMDCs stimulated with LPS remained immature after RAPA pretreatment. RAPA significantly decreased the CD86 expression, impaired the IL-12 and IFN-γ production of BMDCs stimulated with LPS, and inhibited the proliferation of allogeneic T cells. In conclusion, RAPA can inhibit the maturation of BMDCs stimulated with LPS in terms of the morphology, surface phenotype, cytokine production, and ability of BMDCs to stimulate the proliferation of allogeneic T cells in vitro.     

Change of Th1/Th2 Cytokine Equilibrium in Rats with Severe Sepsis and Therapeutic Effect of Recombinant Interleukin-12 and Shenmai Injection(参麦注射液)

Objective: To evaluate the dynamic change of Th1/Th2 cytokines in serum, peritoneal lavage fluid (PLF) and splenic macrophages (SM) in rats with severe peritonitis, and to observe the therapeutic preparation. Methods: Severe peritonitis (SP) model was induced by intraperitoneal injection of E. coli and B. frag, and mild peritonitis (MP) model was induced by cecal ligation and punching. Then the following experiments were done: (1) Survival rates of animals after every 6 hrs in the 72 hrs after modeling were recorded, serum and PLF levels of cytokines, including tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ),and interleukin-10 (IL-10), 6 hrs, 12 hrs, 24 hrs and 48 hrs after modeling were measured. (2) Model rats were treated with rlL-12 or SMI, the survival rate was recorded and serum levels of TNF-α, INF-γ, and IL-10before and after treatment were measured, and (3) amount of these cytokines produced by SM were determined 6 hrs, 12 hrs and 24 hrs after treatment. The survival rates and levels of cytokines were then compared between the groups (model group treated with rlL-12 or SMI, untreated model group, and blank group). Results: Serum and PLF levels of IFN-γ, TNF-α at all the time points in SP rats were significantly lower than those in MP rats while those of IL-10 6 hrs and 12 hrs after modeling were significantly higher in the former than that in the latter ( P＜0.05). IFN-γ secretion of SM in SP rats was significantly higher than that in MP rats 6 hrs after modeling (P＜0.05). Administration of rlL-12 or SMI given before modeling could improve the survival rate of the model rats (P＜0.05) and cause significant increase of the serum level and SM secretion of IFN-γ. Conclusion: Imbalance in promoting/antagonizing inflammatory cytokines and Th2 response dominance appear in SP rats early at the initiating stage, and SM secretion of inflammation promoting factor also reduces. Administration in time of rlL-12 and SMI, may increase the survival rate, and its mechanism may be related with their immuno-stimulating action.     

Others

<正>209471 The immunological characteristics of CD8+ CD28-suppressor T cell induced by siRNA technique/Xue Lijun(,Dept Surg Organ Transpalant Centre Affil Ruijin Hosp,Shanghai Jiaotong Univ School Med,Shanghai 200025)…∥Chin J Organ Transplant.-2009,30(5).-295～299Objective To induce CD8+ CD48-suppressor T cells(CD8+ Ts)by DC with MHCⅠ expression by siRNA,under the stimulator of allograft antigen and then investigate the immunological characteristics of Ts.Methods After the isolation and cutivation of DCs from femoral bone of SD rats,the optimal sequence of MHC Ⅰ siRNA was designed and constructed,and the MHC Ⅰ siRNA was transfected into DCs.The DCs were stimulated by mesenteric lymphatic tissue fluid of Wistar rats,and then co-cultured with CD8+ Ts isolated from SD rat spleen.SD rat CD8+T cell were obtained by magnetic separation method.Cell proliferation was assayed after SD rat spleen cells loading Wistar rat mesenteric lymph node tissue antigent amount of Ts.After SD rat spleen cells were stimulated by Wistar rat’s mesenteric lymph node and reacted with OVA respectively,different amounts of Ts were added.Cell proliferation of the spleen lymphocytes in different groups was assayed.rrIL-2 was added to the mixed lymphocyte culture system containing Wistat rat’s mesenteric lymph node tissue antigen,SD rat’s Ts and SD rat spleen lymphocytes,then the influence of the IL-2 on the function of SD rat Ts was observed.The expression of TGF-β and IFN-γ mRNA was examined by Real time PCR,and CD25 was detected by Real time-PCR and flow cytometry in CD8+ CD28-T cell group.Results Ts had inhibitory influence on the mixed lymphocytes proliferation between SD rat’s spleen lymphocytes and Wistar rat’s mesenteric lymph node tissue,but experted no efforts on the mixed lymphocytes proliferation between OVA and SD rat’s spleen lymphocytes.After addition of rrIL-2 into the mixed lymphocyte culture system containing Wistar rat mesenteric lymph node tissue antigen Ts,there was no significant increas     

Apoptosis of matured T lymphocytes induced by mouse sertoli cells in cocultures in vitro

Objective: To study whether mouse sertoli cells can induce the apoptosis of matured T lymphocytes in cocultures in vitro. Methods: With TUNEL, DNA electrophoresis, eleetro-mierography and flow cytometry, we examined the apoptosis and its rates of mouse matured T lymphocytes in control group (T lymphocytes only), group A (T lymphocytes culture medium of sertoli cells), group B (T lymphocytes sertoli cells). Results: Under electro-micrography, chromatin condensation, karyopyknosis, karyorhexis and apoptotic body were observed in some T lymphocytes in 3 groups; some nucleuses were stained dark blue with TUNEL; a typical DNA ladder was found with DNA electrophoresis. The apoptotic rates of T lymphocytes in group A and B were significantly higher than that in control group (P＜0.01). The apoptotic rate of T lymphocytes in group B was significantly higher than that in group A (P＜0.01). Conclusion: In coculture condition in vitro,mouse sertoli cells can induce the apoptosis of matured T lymphocytes.     

Increased Expression of PI-3K in Asthmatic Rat T Lymphocytes

In order to explore the expression of PI-3K in T lymphocytes of asthmatic rats and the relationship between PI-3K and activation of T lymphocytes, 24 Wistar rats were randomly divided into 4 groups: normal control group, asthmatic one-week group, asthmatic two-week group and asth-matic four-week group. T cells were purified from blood of each rat and the expression of PI-3K was observed by immunocytochemical fluorescence staining, the semiquantitative fluorescence intensity was measured by HPIAS-2000 analytic software, and the expression of IL-4 in supernatants was de-tected by ELISA. The results showed that the fluorescence intensity of T lymphocytes in asthmatic groups was significantly higher than that in normal control (P<0.001), indicating that the expression of PI-3K in T lymphocytes of asthmatic rats was significantly higher than that in those of normal controls, and the difference between acute and chronic stage asthmatic groups was significant (P<0.05). The expression levels of IL-4 protein in supernatants of asthmatic T lymphocytes were sig-nificantly higher than those in the normal controls (P<0.05). There was a significant positive correla-tion between the expression of PI-3K in T lymphocytes and the IL-4 protein expression in super-natants (r=0.583, P<0.01). It was suggested that PI-3K signal pathway may participate in the proc-esses of activation and other cytological effects of asthmatic T lymphocytes, thus may play an impor-tant roles in the pathogenesis of asthma.     

Analysis of the T cell receptor Vδ region gene repertoire in bronchoalveolar lavage fluid (BALF) and peripheral blood of asthmatics

Objective To explore the role of γδT cells in the airway of asthmatics and to identify t he forces which induce and maintain the inflammatory process.Methods Peripheral blood (PB) and bronchoalveolar lavage fluid (BALF) were obtained from 7 asthmatic subjects and 7 nonsmoker control subjects. The percentage of γδT cells in the PB and BALF was measured by immunofluorescent staining and flow cy tometry. The frequency of usage and the clonality of Vδ subfamilies (Vδ(1)-V δ(3)) were assessed by RT- PCR and gene scanning. Results A higher proportion of γδT cell was detected in the BALF of asthmatic subjects (7.8%±4.7%) than that from control subjects (3.3%±3.0%, P=0.04). No selective usage for a particular Vδ subfamily was found, but the relative ex pression level of Vδ(1) was significantly higher in the asthmatic airway (44%± 13%) than in the control (19%±5%, P=0.0002). In asthmatic subjects, the m onoclonal or oligoclonal expansion of γδT lymphocytes was predominant in the B ALF, especially Vδ(1)(+) T lymphocytes.Conclusions Antigenic specific γδT cells might play an important role in the inducement an d maintenance of airway inflammation. Persistent antigenic stimulation may be t he key factor that maintains chronic airway inflammation in asthma.     

Effect of Chinese Herbal Medicines for Nourishing Yin, Supplementing Qi, and Activating Blood on Reproductive Endocrine Activity and Immune Functions in Patients with Primary Sjogren's Syndrome

Objective:To investigate the effect of Chinese herbal medicines for nourishing yin,supplementing qi,and activating blood on the reproductive endocrine-immune network and its mechanisms in patients with primary Sjogren's syndrome(pSS).Methods:Seventy pSS patients were randomly assigned to two groups using a randomized digital table:the integrative therapy group(36 cases) and the control group(34 cases).Thirty healthy subjects were taken as a normal group.The control group was treated with hydroxychloroquine sulfate tablets alone,and the integrative therapy group was treated by Chinese herbal medicines for nourishing yin,supplementing qi,and activating blood combined with hydroxychloroquine sulfate tablets.The treatment course was 6 months for both groups.Before and after treatment,serum estradiol(E2),testosterone(T),luteinizing hormone(LH),prolactin(PRL) by radioimmunoassay and immunoglobulin(IgG) by immunodiffusion,erythrocyte sedimentation rate(ESR) by Westergren,interferon-γ(IFN-γ) and interleukin-4(IL-4) by enzyme linked immunosorbent assay were determined.Results:E2 and T levels in all patients were lower than those of normal subjects before treatment(P0.05) and were increased significantly after 6-month treatment(P0.05).ESR,FSH,LH,IgG,IFN-γ,IL- 4 and ratios of E2/T,and IFN- 7/IL in the patients were higher than those of normal subjects before the treatments(P0.05),and were reduced significantly after the treatments(P0.05).The T and IFN-γ levels and E2/T ratio in the patients treated with integrative therapy were reduced significantly compared with the control group(P0.05).However,the PRL levels before and after treatment were not significantly changed in the two groups(P0.05).The ratios of E2/T and IFN-γ/IL-4,and levels of IgG and ESR were positively correlated before and after treatment(P0.05).Conclusions:The ratios of E2/T and IFN- γ /IL-4 might be used as indicators of pSS activity.Chinese herbal medicines for nourishing yin,supplementing qi,and activating blood combined with Western medicine could improve the therapeutic effect by regulating the reproductive endocrine-immune network in pSS patients.     

Effect of IL-10 gene transmission on the PTg-stimulated splenocytes proliferation and Th1 cytokines production from experimental autoimminue thyroiditis rats

Objective: To investigate the effects of gene therapy with IL-10 on PTg-induced proliferation of splenocytes and Th1 cytokine production from PTg-stimulated splenocytes. Methods: EAT rats were divided into four groups:group A (PBS PLL) , group B (pORF PLL), group C (pORFmIL10 PLL), and group D (pORFmIL10 MEM). The substances mixed with lipofectamine were injected into the thyroid tissues of rats on the 18th dday after immunization. The rats were sacrificed at the 8th week. In vitro proliferative responses to ConA and different concentration of PTg were measured by culturing 4×105 splenocytes pulsed with 18.SKBq of [3H] thymidine for the final 12h and then harvested for liquid scintillation counting. In vitro splenocytes were cultured with PTg (25 mg/L). Th1 cytokine IFN-γ,TNF-αand IL-2 were detected by ELISA. Results: The proliferative response to PTg was suppressed in group C, compared with that of group A and B (P＜0.05). The levels of IFN-γ,TNF-oand IL-2 in the supernatant of PTg-stimulated splenocytes were 3548.25 ± 779.47 pg/ml, 27.66±10.50 pg/ml and 3617.73± 609.15 pg/ml, respectively,which were much lower in group C than those in group A and B(P＜0.01, P＜0.05, P＜0.001, respectively). Conclusion: IL-10 gene transmission in thyroid tissues could inhibit PTg specific proliferation of splenocytes from EAT rats and the secretion of Thl cytokines from PTg-stimulated splenocytes.     

Effect of Spleen Lymphocytes on the Splenomegaly in Hepatocellular Carcinoma-bearing Mice

Objective To study the effect of spleen lymphocytes on the splenomegaly by hepatocellular carcinoma-bearing mouse model. Methods Cell counts,cell cycle distribution,the percentage of lymphocytes subsets and the levels of IL-2 were measured,and two-dimensional gel electrophoresis(2-DE) was used to investigate the relationship between spleen lymphocytes and splenomegaly in hepatocellular carcinoma-bearing mice. Results Compared with the normal group,the thymus was obviously atrophied and the spleen was significantly enlarged in the tumor-bearing group. Correlation study showed that the number of whole spleen cells was positively correlated with the splenic index. The cell diameter and cell-cycle phase distribution of splenocytes in the tumor-bearing group showed no significant difference compared to the normal group. The percentage of CD3+ T lymphocytes and CD8+ T lymphocytes in spleen and peripheral blood of tumor-bearing mice were substantially higher than that in the normal mice. Meanwhile,the IL-2 level was also higher in the tumor-bearing group than in the normal group. Furthermore,two dysregulated protein,β-actin and S100-A9 were identi?ed in spleen lymphocytes from H22-bearing mice,which were closely related to cellular motility. Conclusion It is suggested that dysregulated β-actin and S100-A9 can result in recirculating T lymphocytes trapped in the spleen,which may explain the underlying cause of splenomegaly in H22-bearing mice.     

Nuclear factor- κB in signal conduction of protein kinase C in T lymphocytes from an asthmatic guinea pig model

Objective To explore the role of nuclear factor- κB (NF- κB) in the signal conduction of protein kinase C (PKC) regulated proliferation, apoptosis and expression of Th2 cytokines - interleukin- 4 (IL- 4) and interleukin- 5 (IL- 5) of T lymphocytes in the bronchial alveolus lavage fluid (BALF).Methods T lymphocytes were isolated and purified from BALF of asthmatic guinea pigs in normal and asthmatic groups, and were stimulated with PKC agitator phorbol 12- myristate 13- acetate (PMA) and NF- κB inhibitor pyrrolidine dithiocarbamate (PDTC), respectively.The expressions of NF- κB, IL- 4 and IL- 5 mRNA and protein, the proliferation and apoptosis of T lymphocytes were observed by immunohistochemistry, in situ hybridization, ELISA, MTT and TUNEL, respectively. Results The activation of NF- κB, proliferation response, and expression of IL- 4 and IL- 5 mRNA and protein in T lymphocytes stimulated by PMA were significantly higher than those of their blank control (P&lt;0.01), while those indexes of T lymphocytes stimulated by PMA and PDTC simultaneously were significantly lower than those stimulated by PMA alone (P&lt;0.01).The apoptotic index of T lymphocytes stimulated with PMA were significantly lower than that of their blank control (P&lt;0.01), and the apoptotic index of asthmatic guinea pig T lymphocytes stimulated with PMA and PDTC simultaneously were significantly higher than that stimulated by PMA alone (P&lt;0.01).The significant positive correlations were found between the activation of NF- κB and the proliferation (r=0.64, P&lt;0.001), and the expression of IL- 4 and IL- 5 mRNA and protein of T lymphocytes, respectively (r=0.55-0.68, P&lt;0.001).There was also significant negative correlation between the activation of NF- κB and apoptosis of T lymphocytes (r=0.62, P&lt;0.001). Conclusions NF- κB may participate in the signal conduction of PKC regulated proliferation, apoptosis and expression of IL- 4 and IL- 5 of T lymphocytes in asthma.The activation of NF- κB in PKC signal conduction pathway of T lymphocytes may play an important role in the pathogenesis of asthma.     

Carboxymethylpachymaran enhances immunologic function of dendritic cells cultured in two kinds of hepatoma carcinoma cell line’s supernatant via nuclear factor κB/Rel pathway

Objective:To study the immunologic function of dendritic cells(DCs) cultured in two kinds of hepatoma cell line’s supernatant and the enhancing effects of carboxymethylpachymaran(CMP) on DCs.Methods:DCs were harvested after stimulation by granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin(IL)-4 from umbilical cord blood using density-gradient centrifugation method. Cultured supernatant of two hepatoma cell lines(HepG2 and HepG2.2.15) were collected for condition medium(CM) according to a volume ratio of supernatant to incomplete RPMI-1640 medium,which was 3:1.CMP was dissolved in incomplete RPMI-1640 medium.Experimental groups were divided according to the culture medium,either CM or with CMP in it.DCs subsets CD83,CD86,CD1a,and d-related human leukocyte antigens(HLA-DR) were analyzed by flow cytometry.The proliferation ability of allogeneic T cells in mixed lymphocyte reaction(MLR) stimulated by DCs was examined by 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide(MTT) analysis.IL-12p70,interferon-γ(IFN-γ),and nuclear factor k B(NF- k B) were detected by enzyme-linked immunosorbent assay analysis.Results:The proliferation of lymphocytes and secreting level of IL-12 and expression of phenotype of DCs cultured in two kinds of CM were lower than those of normal group(P<0.01).Compared with the normal group,groups treated with CMP showed a higher expression level of DCs subsets,lymphocyte reproductive activity,as well as IL-12 and IFN-γsecretion levels.Groups treated with CMP also demonstrated higher levels of DCs phenotype expression and IL-12 and IFN-γsecretion in supernatant of MLR and higher lymphocyte reproductive activity compared with CM group(P<0.05).Compared with the normal group,the expression level of NF-k B in DCs nuclear was higher in CMP groups but lower in two CM groups(P<0.05).After CMP was added,the NF-κB expression levels of two CM groups were increased compared with levels before CMP was added(P<0.05).However,there was no significant difference between the two CM groups(P>0.05).Conclusions:Two kinds of hepatoma cell line’s supernatant can inhibit the immunologic function of DCs.This suppressive effect may be related to the inhibition of NF-κB/Rel pathway.CMP may up-regulate the DCs function by activating the NF-κB/Rel pathway.     

Effect of Ligustrazine Injection on Levels of Interleukin-4 and Interferon-γ in Patients with Bronchial Asthma

Objective:To explore the effect of ligustrazine injection (LI) on serum levels of interleukin-4 (IL-4) and interferon-γ (IFN-γ) in patients with bronchial asthma and determine the mechanism of action of LI in preventing and treating asthma.Methods:Sixty-eight patients with mild or moderate bronchial asthma were assigned to two groups equally according to their sequence number,odd or even.The conventional treatment was administered to both groups,and LI was given to the treatment group by ultrasonic spray inhalation twice a day but not to the control group.The therapeutic course for all was 2 weeks.Further,30 healthy subjects who had no history of smoking were enrolled as the normal control group.The clinical condition scores,frequency of attacks and dosage of Terbutaline inhaled were scored and recorded on the first day of hospitalization (before treatment) and after treatment.At the same time,the indexes of lung function,including forced expiratory volume in one second (FEV1),forced expiratory volume in one second to forced vital capacity ratio (FEV1%) and the peak expiratory flow (PEF) were determined before treatment.The levels of IL-4 and IFN-γ in peripheral blood were detected by ELISA before and after treatment,and compared with that of the healthy control group.]Results:After treatment,the clinical condition scores were found to be lower,indexes of lung function were elevated,but serum level of IL-4 and ratio of IL-4/IFN-γ were reduced in both groups,showing significant differences as compared to those before treatment (P＜0.05).However,the changes in all the indexes were more significant in the treatment group than in the control group,also showing statistical significance (P＜0.05).No significant difference was revealed when IFN-γ levels were compared before and after treatment in both groups,though a lowering trend could be seen,significant difference could not be found in the comparison of IFN- γ levels between groups after treatment (P＞0.05).Conclusion:LI shows good clinical effect in treating bronchial asthma,and its mechanism might be related to the suppression of the synthesis of IL-4,thus leading to the inhibition of TH2 cell subset preponderant response and immune equilibrium regulation.     

特应性皮炎小鼠中Th1/Th2类细胞因子失衡对神经生长因子表达的影响

目的 初步探讨特应性皮炎(AD)小鼠中Th1/Th2 类细胞因子失衡对脾淋巴细胞神经生长因子(NGF) mRNA 表达的影响.方法 用卵清蛋白经皮肤致敏建立小鼠AD模型, ELISA测定血清总IgE水平.分离脾淋巴细胞并培养.逆转录-聚合酶链反应(RT-PCR)半定量法测定细胞在刀豆素A(ConA)刺激下表达NGF mRNA的基础水平及γ-干扰素(IFN-γ)和白细胞介素-4(IL -4)干预后NGF mRNA表达改变.结果 ①AD小鼠脾淋巴细胞在基础状态下具有表达NGF mRNA的功能,在12、24、36、48 h时基础NGF mRNA表达量随着时间的延长逐渐升高,IL-4组(50 μg/L)随着时间的延长基础NGF mRNA表达量逐渐升高,并高于同时间空白对照组(P均<0.01). 相反, IFN-γ组(25 μg/L)的NGF mRNA水平随时间逐渐下降,并均低于同时间空白对照组(P均<0.01).②干预24 h时,随着IL-4干预浓度的增加,NGF mRNA的表达水平亦升高,并且分别与前一梯度低浓度干预组比较差异有统计学意义(P均<0.01),随着IFN-γ干预浓度的增加, NGF mRNA的表达水平却逐渐减低,分别比前一梯度低浓度干预组有显著降低(P均<0.01).结论 在特应性皮炎中Th2类细胞因子IL-4可上调淋巴细胞NGF mRNA表达,Th1类细胞因子IFN-γ可下调淋巴细胞NGF mRNA表达,两者均呈时间和浓度依赖性,Th1 / Th2类细胞因子免疫失衡可能通过调控NGF mRNA 表达间接促进其诱导神经源性炎症. Abstract： Objective To explore the effect of Th1 / Th2 cytokines on the expression of nerve growth factor (NGF) in splenic lymphocytes in atopic dermatitis mice.Methods Four AD mice were sensitized and challenged with ovalbumin to establish AD model, and serum IgE level was measured by ELISA.The mouse splenic lymphocytes were isolated and cultured with ConA. The expressions of NGF mRNA were detected by RT-PCR, and were observed after the lymphocytes were exogenously added with interferon -γ(IFN-γ) or interleukin-4 (IL-4). Results ①The lymphocytes of the AD model stimulated by ConA in vitro expressed NGF mRNA in a time-dependent manner. After the lymphocytes had been cultured with IL-4 for 12 h, 24 h, 36 h, and 48 h, 50μg/L IL-4 upregulated the expressions of NGF mRNA in a time-dependent manner and all the NGF mRNA expressions were significantly higher than the basal values at the same time (P all<0.01), however,25μg /L IFN-γ downregulated the expressions of NGF mRNA in a time-dependent manner and all the NGF mRNA expressions were significantly lower than the basal values at the same time (P all<0.01).②The lymphocytes of the AD model stimulated by ConA in vitro also expressed NGF mRNA in a dose-dependent manner.After 0,10, 25,50, and 100 μg /L IL-4 had been added for 24 h, IL-4 upregulated the expressions of NGF mRNA in a dose-dependent manner and the NGF mRNA expressions were all significantly higher than the values of the lower dose IL -4 (P all <0.01), however,after 0, 1, 10, 25,and 50μg /L IFN-γ had been added for 24 h, IFN-γ downregulated the expressions of NGFmRNA in a dose-dependent mannerand all the NGF mRNA expressions were significantly lower than the values of the lower IFN-γ dose (P all<0.01).Conclusions In the splenic lymphocytes of atopic dermatitis mice, IL -4, one of the Th2 cytokines, can upregulate the expressions of NGF, IFN-γ, one of the Th1 cytokines, can downregulate the expressions of NGF both in a time-dependent manner and in a dose-dependent manner. Th1 /Th2 cytokine immune imbalance may indirectly induce the skin neurogenic inflammation by regulating the NGF mRNA expression.     

Original article Inhibition of collagen-induced arthritis by DNA vaccines encoding TCR Vβ5.2 and TCR Vβ8.2

Background Arthritogenic T lymphocytes with common T cell receptor （TCR） Vβ clonotypes, infiltrating in the articulars of rheumatoid arthritis （RA） patients, play a central role in the pathogenesis of RA. TCR Vβ5.2 and TCR Vβ8.2 are the main pathogenic T cell clonotypes in the course of collagen-induced arthritis （CIA） progression in Lewis rats. To investigate a TCR-based immunotherapy for RA, we constructed recombinant DNA vaccines encoding TCR Vβ5.2 and TCR Vβ8.2, and evaluated the inhibitive effects of the two vaccines on CIA rats.
Methods Genes encoding TCR Vβ5.2 and TCR Vβ8.2 were amplified by RT-PCR from spleen lymphocytes of Lewis rats and cloned into the eukaryotic expression vector pTargeT. The expression of vaccines was confirmed by RT-PCR and immunohistochemistry. The inhibitive effects of the vaccines on articulars of CIA rats were assessed with arthritis index evaluation and histology. Interferon γ （IFN-γ） and interleukin （IL）-4 production by spleen lymphocytes were tested with enzyme-linked immunospot assay （ELISPOT） technique, the changes in peripheral CD4^＋ and CD8^＋ lymphocyte populations were tested by flow cytometry, and the level of anti-CII antibody in serum was assayed by enzyme-linked immunosorbent assay （ELISA）.
Results Recombinant DNA vaccines pTargeT-TCR Vβ5.2 and pTargeT-pTCR Vβ8.2 were successfully constructed. Both vaccines inhibited CIA, which alleviated the arthritis index score （P 〈0.05）, decreased the level of IFN-γ （P 〈0.05）, and reduced the ratio of CD4^＋/CD8^＋ lymphocytes （P 〈0.05） and the anti-CII antibody in serum （P 〈0.05）. In addition, the histological change in DNA-vaccinated rats was less serious than CIA rats. Compared to pTCR Vβ 8.2 and pTCR Vβ 5.2 groups, the group that was injected with a combination of the two vaccines showed stronger inhibitive effects on CIA than either individual vaccine.
Conclusion The recombinant plasmids pTargeT-TCR Vβ5.2 and pTargeT-TCR Vβ8.2 have obvious inhibatory effects on CIA rats and better effects could be achieved when the vaccines were used in combination.     

Atorvastatin Attenuates TNF-alpha Production via Heme Oxygenase-1 Pathway in LPS-stimulated RAW264.7 Macrophages

Objective To assess the effect of atorvastatin on lipopolysaccharide(LPS)-induced TNF-α production in RAW264.7 macrophages. Methods RAW264.7 macrophages were treated in different LPS concentrations or at different time points with or without atorvastatin. TNF-α level in supernatant was measured. Expressions of TNF-α mRNA and protein and heme oxygenase-1(HO-1) were detected by ELISA, PCR, and Western blot, respectively. HO activity was assayed. Results LPS significantly increased the TNF-α expression and secretion in a dose- and time-dependent manner. The HO-1 activity and HO-1 expression level were significantly higher after atorvastatin treatment than before atorvastatin treatment and attenuated by SB203580 and PD98059 but not by SP600125, suggesting that the ERK and p38 mitogen-activated protein kinase(MAPK) pathways participate in regulating the above-mentioned effects of atorvastatin. Moreover, the HO-1 activity suppressed by SnPP or the HO-1 expression inhibited by siRNA significantly attenuated the effect of atorvastatin on TNF-α expression and production in LPS-stimulated macrophages. Conclusion Atorvastatin can attenuate LPS-induced TNF-α expression and production by activating HO-1 via the ERK and p38 MAPK pathways, suggesting that atorvastatin can be used in treatment of inflammatory diseases such as sepsis, especially in those with atherosclerotic diseases.     

Bone marrow-derived mesenchymal stem cell transplantation for acute lung injury in mice

To investigate the effect of intravenous bone marrow-derived mesenchymal stem cell (MSC)transplantation for early intervention of lipopolysaccharide (LPS)-induced acute lung injury (ALI) in mice. Methods Thirty-sixmice were randomized into control group, PBS-treated ALI group, and MSC-treated ALI group. In the latter two groups,mouse models of ALI were established by intranasal instillation of LPS, and 1 h later, the 4th passage of MSCs isolated fromthe bone marrow of mice or PBS were administered via the tail vein. The histological findings, lung wet/dry (W/D) weightratio, neutrophil count and protein and cytokine contents in the bronchoalveolar lavage fluid (BALF), and myeloperoxidase(MPO) level in the lung tissue were analyzed at 24 h after MSC administration. Engraftment of MSCs in the recipient lung wasdetermined by fluorescent PKH26 staining and flow cytometry. Results Compared with the control group, PBS-treated ALIgroup showed significantly higher levels of protein, tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and neutrophil countin the BALF and MPO content in the lung tissue, with also severe damage of lung histology. MSCs administration significantlyreduced the lung W/D weight ratio, the levels of protein, TNF-α, IL-6 and neutrophil count in the BALF and MPO content inthe lung tissue, and obviously lessened the lung injury 24 h after the transplantation. MSC administration also significantlyincreased the level of IL-10 in the BALF. Conclusion Intravenous MSC transplantation can effectively improve the lunghistology, attenuate the inflammatory response, reduce pulmonary edema in the early stage of LPS-induced ALI in mice, andsuch effects are independent of MSC engraftment in the lungs.