Hydrogen from intestinal bacteria is protective for Concanavalin A-induced hepatitis

It is well known that some intestinal bacteria, such as Escherichia coli, can produce a remarkable amount of molecular hydrogen (H₂). Although the antioxidant effects of H₂ are well documented, the present study examined whether H₂ released from intestinally colonized bacteria could affect Concanavalin A (ConA)-induced mouse hepatitis. Systemic antibiotics significantly decreased the level of H₂ in both liver and intestines along with suppression of intestinal bacteria. As determined by the levels of AST, ALT, TNF-α and IFN-γ in serum, suppression of intestinal bacterial flora by antibiotics increased the severity of ConA-induced hepatitis, while reconstitution of intestinal flora with H₂-producing E. coli, but not H₂-deficient mutant E. coli, down-regulated the ConA-induced liver inflammation. Furthermore, in vitro production of both TNF-α and IFN-γ by ConA-stimulated spleen lymphocytes was significantly inhibited by the introduction of H₂. These results indicate that H₂ released from intestinal bacteria can suppress inflammation induced in liver by ConA.     

In Vivo Delivery of Antisense Oligodeoxynucleotides into Rat Kupffer Cells

Abstract

Kupffer cells play a key role in the pathogenesis of liver diseases. Liver injury is believed to result from an excessive release of cytokines and prostanoids from these cells. A targeted delivery of antisense oligonucleotides into Kupffer cells might reduce or prevent liver injury. In this report, we describe a method in which anionic liposome-encapsulated antisense phosphorothioate oligodeoxynucleotides (S-Oligos) are delivered to Kupffer cells in vivo. Delivery was assessed using an antisense S-Oligo (TJU-2749) targeted against the 3’ untranslated region of rat tumor necrosis factor-α mRNA. At 90 min post-intravenous injection, 90% of the S-Oligo was absorbed from circulation. Of this, 40% was found in the liver and 10% in spleen. Other organs, including lungs, kidneys, muscle, stomach, brain, testes and small intestine, showed only minor incorporation (<5%). Greater than 65% of the liver-associated S-Oligo was found in Kupffer cells. Relative accumulation of S-Oligo in Kupffer cells was 200-fold that of the combined body tissues. For an average injected dose of 1.2 mg antisense/Kg body weight, the intracellular concentration of the S-Oligo attained in Kupffer cells was 65 μM. These studies suggest that liposome-encapsulated delivery provides an efficient means of targeting antisense molecules to Kupffer cells in vivo.     

Relationship between CD107a expression and cytotoxic activity

NK cells play important roles in innate immunity against tumors and infections of the host. Studies show that CD107a (LAMP-1) may be a marker for degranulation of NK and activated CD8⁺ T cells. In our study, the relationship between the expression of CD107a, cytokine secretion and cytotoxic activity in CD56⁺ NK, CD8⁺ T cells and lymphocytes has been determined after various stimuli. Effector cells from PBMCs of healthy subjects were isolated and K562 cell line was used as target of cytotoxicity. IL-2 stimulation resulted in a significant increase of CD107a expression in CD56⁺ NK, CD8⁺ T cells and lymphocytes. Increased expression of CD107a after IL-2 stimulation of NK cells was parallel to the increase of cytotoxicity. Our results suggest that CD107a expression may be a sensitive marker for the cytotoxic activity determination.     

细胞因子基因导入人肿瘤细胞的方法及鉴定

从人外周血单核细胞中抽提总RNA为模板,分别用5’含EcoRⅠ、3’含BamHⅠ限制性酶切位点的细胞因子基因特异引物合成合信号肽全长IL－2、γ－IFN及α－TNFcDNA．然后将这些细胞因子cDNA分别重组入含筛选标记基因Neo的逆转录病毒载体LXSN中．采用磷酸钙共沉淀法转染逆转录病毒包装细胞系PA317．分别收集到含IFN－γ、TNF－α和IL－2基因的缺陷型逆转录病毒上清及只具空白载体质粒pLXSN的病毒上清这四种病毒颗粒用于导入人肝癌、胃癌细胞,可获得单克隆化的细胞因子基因修饰株PCR,Southern,RT－PCR及Northern杂交证明,有相应细胞因子及筛选标记基因的转入和表达生物活性分析也证实各基因修饰株细胞培养上清液中有一定活性的相应细胞因子．结果表明逆转录病毒介导的细胞因子基因转移是成功的     

A novel accessory role of neutrophils in concanavalin A-induced hepatitis

Concanavalin A (Con A)-induced hepatitis has been investigated as a model of T cell-mediated liver injury, in which IFN-gamma plays an essential role by inducing apoptosis of liver cells. Since a large number of neutrophils infiltrate into the liver in the model, the role of neutrophils was investigated in this study. Con A hardly caused liver injury in neutrophil-depleted mice, as assessed as to the plasma alanine aminotransferase level as well as histochemistry. Neutrophil-depleted mice also failed to produce IFN-gamma. Intracellular IFN-gamma staining revealed that, among liver leukocytes, T and NK cells but not neutrophils are the main producers of IFN-gamma. Nylon wool-purified "T cells", however, failed to produce IFN-gamma in response to Con A in vitro, while the production was restored by the addition of neutrophils. Overall, this study suggests that neutrophils play a novel accessory role in IFN-gamma production in Con A-induced hepatitis.     

Effects of catalpol on bronchial asthma and its relationship with cytokines

An animal (BALB/c mice) model of catalpol associated with bronchial asthma in vivo was established, and the effects of catalpol and its relationship with cytokines were investigated. A total of 30 adult BALB/c mice were randomly divided into a positive control group, a model group, and a catalpol group, with 10 mice in each group. The lung function of mice, the cell count, and the cytokine concentrations in bronchoalveolar lavage fluid (BALF) were detected. The levels of cytokines [interleukin 4 (IL-4), interleukin 5 (IL5), and interferon gamma (IFN-γ)] in BALF were measured with enzyme-linked immunosorbent assay methods. The total number of cells in the BALF of the group treated with catalpol was significantly lower than the model group. After treatment with catalpol, the eosinophils and neutrophils of the mice were remarkably reduced compared with the model group. The malondialdehyde content in the lung tissue homogenate of the mice was also decreased in the catalpol group. The cytokines IL-5 and IL-4 exhibited a similar tendency: the concentrations of IL-4 and IL-5 for the catalpol group were dramatically decreased compared with the model group. However, the IFN-γ concentration for the catalpol group was higher than the model group. The results indicated that IL-5 may involve in the pathologic process of asthma-like IL-4, and an inflammatory reaction may still exist in the airway during the remission stage of asthma. The imbalances of the cytokine network might be an important molecular basis in the asthma pathogenesis. It is suggested that catalpol may be a potential drug for the clinical treatment of asthma.     

IFN-gamma-induced SOCS-1 regulates STAT6-dependent eotaxin production triggered by IL-4 and TNF-alpha

The production of eotaxin, which is a critical mediator for airway inflammation, is inhibited by IFN-gamma. Here, we investigated the precise mechanisms underlying IFN-gamma-dependent inhibition of eotaxin production using mouse embryonic fibroblasts (MEF). MEF produced high levels of eotaxin in STAT6-dependent manner when they were cultured with both IL-4 and TNF-alpha. However, the eotaxin production by MEF was strongly inhibited by addition of IFN-gamma. Western-blotting analysis demonstrated that IFN-gamma downmodulated STAT6 phosphorylation induced by IL-4 and TNF-alpha. Moreover, IFN-gamma did not exhibit its inhibitory effect on both STAT6-phosphorylation and eotaxin production in MEF obtained from deficient mice in STAT1, a key molecule of IFN-gamma signaling. We also demonstrated that SOCS-1, a potent inhibitory molecule of IL-4 signaling, was induced by IFN-gamma in STAT1-dependent manner. This indicated that SOCS-1 might be involved in IFN-gamma-mediated STAT1-dependent inhibition of eotaxin production. In SOCS-1(-/-) MEF, IFN-gamma inhibited neither STAT6 phosphorylation nor eotaxin production induced by IL-4 and TNF-alpha. Conversely, retroviral transduction of SOCS-1 into MEF inhibited STAT6 phosphorylation and eotaxin production induced by IL-4 and TNF-alpha, in the absence of IFN-gamma. Thus, we demonstrated that IFN-gamma-induced inhibition of STAT6 phosphorylation and eotaxin production were mediated by SOCS-1 induced in STAT1-dependent manner.     

Dietary Effect of Tocopherols and Tocotrienols on the Immune Function of Spleen and Mesenteric Lymph Node Lymphocytes in Brown Norway Rats

The immunoregulatory effects of dietary α-tocopherol (Toc) and tocotrienols (T-3) on humoral and cell-mediated immunity and cytokine productions were examined in Brown Norway rats. We found that the IgA and IgG productivity of spleen and mesenteric lymph node (MLN) lymphocytes was significantly enhanced in the rats fed on Toc or T-3, irrespective of concanavalin A (Con A) stimulation of the lymphocytes. On the contrary, the IgE productivity of lymphocytes from the rats fed on Toc or T-3 was less without Con A stimulation, but was greater in the presence of Con A, especially in the T-3 group. Toc or T-3 feeding significantly decreased the proportion of CD4⁺ T cells and the ratio of CD4⁺/CD8⁺ in both spleen and MLN lymphocytes of the rats fed on Toc or T-3. The interferon-γ productivity of MLN lymphocytes was higher in the rats fed on Toc or T-3 than in those fed on a control diet in the presence of Con A, while that of spleen lymphocytes was lower in the rats fed on Toc or T-3. In addition, T-3 feeding decreased the productivity of tumor necrosis factor-α of spleen lymphocytes, while it enhanced the productivity of MLN lymphocytes. These results suggest that oral administration of Toc and T-3 affects the proliferation and function of spleen and MLN lymphocytes.     

Effect of T helper 1 (Th1)/Th2 cytokine on chemokine-induced dendritic cell functions

Effects of T helper 2 (Th2) and Th1 cytokines, interleukin (IL)-4, and interferon (IFN)-gamma, respectively, on chemokine-induced DC migration and endocytosis are not well understood. We investigated herein the effects of these cytokines on chemokine-induced functions of murine myeloid DCs. As expected, immature DCs markedly migrated to CCL3 but not CCL19, while mature DCs showed vigorous migration in response to CCL19 but not CCL3. Both IL-4 and IFN-gamma significantly decreased CCL3-induced migration of immature DCs. In contrast, these cytokines exerted no significant effects on CCL19-induced migration of mature DCs. Of note, both IL-4 and IFN-gamma markedly enhanced CCL3-induced endocytosis of immature DCs. The messenger RNA level of CCR5, a CCL3 receptor, in immature DCs was slightly increased by IL-4 or IFN-gamma treatment. These results demonstrate that these Th1/Th2 cytokines can act to either inhibit or enhance chemokine-mediated DC functions, and may play a role in increasing antigen uptake by immature DCs at inflammatory sites.     

Comparison of immunomodulatory properties of mesenchymal stem cells derived from adult human tissues

Mesenchymal stem cells (MSCs), which evoke only minimal immune reactivity, may have anti-inflammatory and immunomodulatory effects. In this study, we conducted a comparative analysis of the immunomodulatory properties of MSCs derived from adult human tissues including bone marrow (BM), adipose tissues (AT), umbilical cord blood (CB), and cord Wharton’s jelly (WJ). Using a multiple cytokine detection assay, we showed that there were no significant differences in levels of secreted factors from non-stimulated MSCs. We compared the immunosuppressive effect of BM-MSCs, AT-MSCs, CB-MSCs, and WJ-MSCs on phytohemagglutinin-induced T-cell proliferation. AT-MSCs, CB-MSCs, and WJ-MSCs effectively suppressed mitogen-induced T-cell proliferation as effectively as did BM-MSCs. Levels of interferon (IFN)-γ and tumor necrosis factor (TNF)-α secreted from activated T-cells increased over time, but these levels were significantly reduced when cocultured with each type of MSCs. In addition, the expression of hepatocyte growth factor, IL-10, transforming growth factor-β₁, cyclooxygenase (COX)-1, and COX-2 were unchanged in MSCs treated with IFN-γ and/or TNF-α, while indoleamine 2,3-dioxygenase (IDO) expression increased. IFN-γ and/or TNF-α produced by activated T-cells were correlated with induction of IDO expression by MSCs, which, in turn, suppressed T-cell proliferation. These findings suggest that MSCs derived from AT, CB, or WJ could be substituted for BM-MSCs for treatment of allogeneic conflicts.     

Protective effect of post-ischemic treatment with trans-resveratrol on cytokine production and neutrophil recruitment by rat liver

Oxidative and inflammatory processes are elicited during hepatic post-ischemic reperfusion and generate liver damage. This study investigated the early anti-inflammatory effect of trans-resveratrol (T-res) and its consequences on the late self-aggravating inflammatory process in liver ischemia-reperfusion (I/R). Partial hepatic ischemia was initiated in rats for 1 h and T-res (0.02 and 0.2 mg/kg) was administered intravenously 5 min before starting reperfusion for 3 h. Plasma levels of aminotransferases and cytokines (tumour necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6) and hepatic neutrophil recruitment were assessed. Hepatic expression of stress protein (heat-shock protein (HSP-70), heme oxygenase-1(HO-1)) and cytokine (TNF-α, IL-1β, keratinocyte chemoattractant (KC)) mRNA was investigated. I/R caused an increase in aminotransferase levels and increased polymorphonuclear cell infiltration. Post-ischemic treatment with T-res (0.02 and 0.2 mg/kg) resulted in a significant decrease in aminotransferase, IL-1β and IL-6 plasma levels by about 40%, 60% and 40%, respectively, compared to the vehicle I/R group. Post-ischemic treatment with T-res (0.02 mg/kg) also significantly decreased hepatic neutrophil recruitment. TNF-α, IL-1β, KC and HO-1 hepatic mRNA expression was reduced by T-res without any change in HSP-70 mRNA. This T-res mediated decrease in early release of cytokines and neutrophil recruitment led to a reduction in the late inflammatory process. T-resveratrol might be useful in the prevention of inflammation secondary to hepatic surgery or liver transplantation.     

Trypanosoma cruzi: populations bearing opposite virulence induce differential expansion of circulating CD3+CD4-CD8- T cells and cytokine serum levels in young and adult rats

The JG strain is the least virulent while the CL-Brener clone is one of the most virulent Trypanosoma cruzi populations in young rats. In this study, we determined that the parasitemia peak values in CL-Brener clone-infected adult rats were 50-fold lower than in young rats and that mortality was null as compared to 45% death in young rats. Low parasitemia, milder and sustained myocarditis and myositis characterized JG infections. CL-Brener clone caused a significantly higher production of pro-inflammatory cytokines and higher expansion of CD3⁺CD4⁻CD8⁻, double-negative (DN) T cells, during the acute phase in both adult and young rats. DN T cell frequencies correlated with IFN-γ levels. These findings may explain the higher inflammation and fast acute phase resolution in CL-Brener infection. In young rats, IL-10 levels were similar in both infections. The IL-10/IFN-γ ratio was higher in JG acute infection in accordance with the milder inflammation and parasite persistence leading to a chronic phase. In conclusion, virulence and pathogenicity depend on T. cruzi ability to induce expansion of DN T cells and production of specific cytokines.     

The effect of specific caspase inhibitors on TNF-alpha and butyrate-induced apoptosis of intestinal epithelial cells

Tumour necrosis factor-alpha (TNF-alpha)-induced intestinal epithelial cell apoptosis may contribute to mucosal injury in inflammatory bowel disease. Inhibition of TNF-alpha-induced apoptosis, using specific caspase inhibitors could, therefore, be of benefit in the treatment of disease. In vitro, CaCo-2 colonic epithelial cells are refractory to apoptosis induced by TNF-alpha alone; however, TNF-alpha can act synergistically with the short-chain fatty acid (SCFA) and colonic fermentation product, butyrate, to promote apoptosis. TNF-alpha/butyrate-induced apoptosis was characterised by nuclear condensation and fragmentation and caspase-3 activation. Inhibitors of caspase-8 (z-IETD.fmk) and caspase-10 (z-AEVD.fmk) significantly reduced TNF-alpha/butyrate-induced apoptosis, based on nuclear morphology and terminal deoxynucleotide transferase-mediated dUTP-biotin nick-end labelling (TUNEL), although caspase inhibition was associated with a significant increase in cells demonstrating atypical nuclear condensation. Inclusion of atypical cells in calculations of total cell death, still demonstrated that z-IETD.fmk and z-AEVD.fmk (in combination) significantly reduced cell death. Reduction in cell death was associated with maintenance of viable cell number. Transmembrane resistance was also used a measure of the ability of caspase inhibitors to prevent TNF-alpha/butyrate-mediated damage to epithelial monolayers. TNF-alpha/butyrate resulted in a significant fall in transmembrane resistance, which was prevented by pre-treatment with z-IETD.fmk, but not z-AEVD.fmk. In conclusion, synthetic caspase inhibitors can reduce the apoptotic response of CaCo-2 colonic epithelial cells to TNF-alpha/butyrate, improve the maintenance of viable cell numbers and block loss of transmembrane resistance. We hypothesise that caspase inhibition could be a useful therapeutic goal in the treatment of inflammatory bowel conditions, such as ulcerative colitis.     

手机辐射对孕鼠及子代IFN-γ和IL-4的影响

检测暴露于手机辐射下的孕鼠及其子代的细胞免疫因子含量水平,以探讨手机电磁辐射对孕鼠及其子代免疫功能的影响,为孕妇科学、合理使用移动通信工具提供参考。30只孕鼠随机分为Ⅰ、Ⅱ、Ⅲ、Ⅳ、Ⅴ组,每组6只,对应为空白对照组、待机对照组1、0min低强度组、30min中强度组和60min高强度组。ELISA检测孕鼠分娩24h内孕鼠及新生乳鼠外周血中IFN-γ和IL-4的含量。对Ⅰ、Ⅱ、Ⅲ、Ⅳ、Ⅴ各组母鼠外周血中IFN-γI、L-4含量进行比较,其差异无统计学意义(P>0.05);Ⅳ、Ⅴ组新生小鼠外周血中IFN-γI、L-4含量与Ⅰ、Ⅱ组比较,其差异有统计学意义(P<0.05),而且Ⅴ组新生小鼠外周血中IFN-γI、L-4含量与Ⅲ组比较,其差异也具有统计学意义(P<0.05)。手机辐射降低了胚胎期小鼠的IFN-γ和IL-4含量。     

TNF-alpha and IL-4 regulate expression of IL-13 receptor alpha2 on human fibroblasts

Two interleukin 13 receptors (IL-13Rs) have been identified as IL-13Ralpha1 and IL-13Ralpha2. IL-13Ralpha1 is composed of a heterodimer consisting of IL-13Ralpha1 and IL-4 receptor alpha (IL-4Ralpha) as a signaling subunit. In contrast, IL-13Ralpha2 is known as a decoy receptor for IL-13. In this study, we investigated the expression of IL-13Rs on human fibroblasts. IL-13Ralpha2 was significantly up-regulated after stimulation with tumor necrosis factor-alpha (TNF-alpha) and/or IL-4. In contrast, IL-13Ralpha1 was constitutively detectable and was not up-regulated. After the induction of IL-13alpha2 by IL-4, STAT6 phosphorylation through IL-13Ralpha1 by IL-13 was inhibited. We also detected large intracellular pools of IL-13Ralpha2 in fibroblasts quantitatively. Furthermore, mobilization of the IL-13Ralpha2 protein stores from the cytoplasm to the cell surface was prevented by an inhibitor of protein transport, brefeldin-A. These results indicate that TNF-alpha and IL-4 synergistically up-regulate the expression of IL-13Ralpha2 decoy receptor on human fibroblasts by inducing gene expression and mobilizing intracellular receptors, and thus may down-regulate the IL-13 signaling.     

双歧杆菌对过敏性哮喘儿童树突状细胞分泌IL-10、IL-12等细胞因子的影响

目的探讨双歧杆菌对过敏性哮喘儿童外周血单核细胞（PBMC）来源的树突状细胞（DC）分泌IL-1β、IL-6、IL-10、IL-12、IL-23和IFN-γ的影响。方法从15例过敏性哮喘儿童和15例非哮喘儿童的外周血单个核细胞诱导生成未成熟DC,加入双歧杆菌后继续培养DC2d,用ELISA方法检测培养上清中IL-1β、IL-6、IL-10、IL-12、IL-23和IFN-γ的水平。结果双歧杆菌能明显刺激哮喘儿童DC分泌IL-12、IFN-γ,IL-1β及IL-6和非哮喘儿童DC分泌IL-12、IL-10、IL-1β及IL-23水平增高。结论双歧杆菌能够刺激过敏性哮喘儿童DC分泌IL-12和IFN-γ,可能改变Th2优势分化,纠正Th1／Th2失衡。同时双歧杆菌还能刺激哮喘儿童DC分泌IL-1p及IL-6增高,达到促进,Th17细胞分化的作用。     

Transient infiltration of neutrophils into the thymus following whole-body X-ray irradiation in IL-10 knockout mice

IL-10 is known to suppress the inflammatory responses in a variety of experimental models. Because we previously found that whole-body X-irradiation causes massive apoptosis in the thymus and transient infiltration of neutrophils, in this study, we examined whether or not IL-10 is involved in the regulation of neutrophil infiltration upon whole-body X-ray irradiation using IL-10 knockout mice. Although IL-10 was induced in the thymus on whole-body X-ray irradiation, apoptosis of thymocytes, neutrophil infiltration, and MIP-2 and KC production in the thymus were not affected by an IL-10 deficiency. Coculturing of bone marrow-derived macrophages with late apoptotic cells caused MIP-2 production, which was also not affected by an IL-10 deficiency. These results suggest the uniqueness of the inflammatory response induced by whole-body X-ray irradiation, which does not seem to be regulated by IL-10.     

IFN-gamma acts as anti-angiogenic cytokine in the human cornea by regulating the expression of VEGF-A and sVEGF-R1

Inflammatory processes within the cornea are known to be associated with corneal neovascularization (CN). We examined the effects of inflammatory mediators on the expression of angiogenic factors by corneal cells. TNF-α and IL-1 induced VEGF-A secretion by corneal fibroblasts (HCRF) and this was inhibited significantly by IFN-γ. Constitutively secreted VEGF-A by corneal epithelial cells (HCE) was not affected by these cytokines. Moreover, sVEGF-R1(sFlt-1) secretion by HCRF was stimulated significantly by IFN-γ. JAK-STAT pathway inhibitor reversed the effects of IFN-γ on VEGF-A and sFlt-1 secretion by HCRF. RT-PCR analysis showed that IFN-γ influences the expression of VEGF-A and sFlt-1 by affecting their mRNA level. IFN-γ inhibited TGF-β induced VEGF-A secretion but not sVEGF-R1secretion. This is the first report demonstrating the inhibitory and stimulatory effects of IFN-γ on VEGF-A and sFlt-1 secretion, respectively. Our results suggest that IFN-γ acts as an anti-angiogenic cytokine in the human cornea.     

硝黄散外敷联合加味五虎汤口服治疗痰热闭肺型肺炎支原体肺炎患儿对肺功能和血清TNF-α、IFN-γ、IL-4水平的影响

目的:观察硝黄散外敷联合加味五虎汤口服治疗痰热闭肺型肺炎支原体肺炎(MPP)患儿对肺功能和血清肿瘤坏死因子-α(TNF-α)、γ-干扰素(IFN-γ)、白介素-4(IL-4)水平的影响。方法:研究对象为我院2019年6月~2021年1月期间收治的MPP患儿80例,采用随机数字表法将患儿分为对照组(n=40,阿奇霉素抗感染治疗)和研究组(n=40,对照组基础上加用硝黄散外敷联合加味五虎汤口服治疗),均治疗7 d。比较两组患儿临床疗效,比较两组治疗前、治疗7 d后的中医证候积分、临床症状改善情况、肺功能和血清TNF-α、IFN-γ、IL-4水平。结果:研究组治疗7 d后的临床总有效率为92.50%(37/40),高于对照组的72.50%(29/40),差异有统计学意义(P<0.05)。与对照组相比,研究组的症状(咳嗽憋喘、发热、肺部干湿啰音)消失时间均更短(P<0.05)。与对照组相比,研究组治疗7 d后用力肺活量(FVC)、最高呼气峰流速(PEF)、第1秒最大呼气容积(FEV1)、FEV1/FVC均更高(P<0.05),研究组治疗7 d后的中医证候积分及血清TNF-α、IFN-γ和IL-4水平均更低(P<0.05)。结论:痰热闭肺型MPP患儿采用硝黄散外敷联合加味五虎汤口服治疗,可有效缩短患儿症状消失时间,显著改善其肺功能、血清炎症因子水平,疗效显著。