Effects of bacillus Calmette-Guèrin and interferon alpha-2B on cytokine production in human bladder cancer cell lines

PURPOSE: To determine the effects of live BCG, autoclaved BCG and interferon alpha-2b on cytokine production in human bladder cancer cell lines. MATERIALS AND METHODS: The release of nine cytokines from the human bladder cancer cell lines, RT4, RT112, SD, MGH and J82, was measured by ELISA assay. The mRNA level of IL-6 and GM-CSF was determined by RT-PCR. RESULTS: BCG and/or interferon alpha-2b differentially increased IL-1beta, IL-6, IL-8, GM-CSF and TNF-alpha production in the bladder cancer cells. High grade cell lines were more responsive to BCG whereas low grade lines were more sensitive to interferon alpha-2b. This correlated with cytotoxicity and growth inhibition induced by these agents. BCG could also induce low levels of IFN-alpha production in all the cell lines. Compared with live BCG, autoclaved BCG had no antiproliferative effect on MGH cells and was less effective in stimulating the production of IL-6, IL-8 and GM-CSF. However, autoclaved BCG was as effective as live BCG in inhibiting growth and stimulating IL-6 and TNF-alpha production of J82 cells. The combination of BCG and interferon alpha-2b also completely suppressed TGF-beta1 production in the MGH and RT112 cell lines. CONCLUSIONS: The combination of BCG and interferon alpha-2b has additive effects in cytokine production from bladder cancer cells. This correlates with cytotoxicity and growth inhibition induced by these agents.     

卡介苗诱导膀胱癌T24细胞表达钟声蛋白样受体和产生细胞因子的研究

目的探讨卡介苗(BCG)刺激对膀胱癌T24细胞钟声蛋白样受体(TLR)表达和细胞因子产生水平的影响。方法人膀胱癌细胞株T24用含10％FBS的RPMI1640液培养,加入不同剂量(菌数／细胞数比值)梯度的BCG刺激。1h后以RT-PCR检测细胞TLR-2和TLR-4的表达水平,12h后ELISA法测定细胞上清液中IL-4和IL-12浓度。结果BCG刺激后,T24细胞的TLR-2和TLR-4表达水平以及IL-12的分泌量随BCG剂量增加而升高,在菌数／细胞数比值为10时达最高;而IL-4分泌量与未刺激组相比无明显差别。结论BCG刺激可使膀胱癌T24细胞TLR-2和TLR-4表达增强、IL-12水平升高,并呈剂量-效应关系;IL-12水平升高可能与TLR-2和TLR-4表达增强有关。     

The liposome-incorporating cell wall skeleton of Mycobacterium bovis bacillus Calmette-Guéin can directly enhance the susceptibility of cancer cells to lymphokine-activated killer cells through up-regulation of natural-killer group 2, member D ligands

OBJECTIVE

? To conduct a preclinical evaluation of the ability of natural killer cells to cytolyze bladder cancer cells that were modified to show enhanced expression of natural‐killer group 2, member D (NKG2D) ligands by R8‐liposome‐bacillus Calmette‐Guéin (BCG)‐cell wall skeleton (CWS) treatment.

MATERIALS AND METHODS

? The T24 cells and RT‐112 cells were co‐cultured with R8‐liposome‐BCG‐CWS and BCG for 2, 4, or 6 h, and then the surface expression of NKG2D ligands was analyzed using TaqMan real‐time quantitative RT‐PCR. ? Peripheral blood mononuclear cells were obtained with a conventional preparation kit, and then lymphokine‐activated killer (LAK) cells were generated from these purified peripheral blood mononuclear cells via interleukin‐2 stimulation. ? The anti‐tumour effect of LAK cells against untreated and R8‐liposome‐BCG‐CWS co‐cultured with cells of the human bladder cancer cell lines T24 and RT‐112 was analyzed using the cytotoxic WST‐8 assay method at 4 h of culture at various effector/target (E : T) ratios.

RESULTS

? Major histocompatibility complex class I‐related chain B (MICB) expression was increased ≈1.5‐fold on T24 cells and RT‐112 cells with BCG. ? UL‐16‐binding protein (ULBP) 1 expression was also increased ≈1.5‐fold on T24 cells and RT‐112 cells with BCG. R8‐liposome‐BCG‐CWS increased the surface expression of MICB 2.2‐fold on T24 cells but did not increase it significantly on RT‐112 cells. ? ULBP1 expression was increased ≈2.2‐fold on RT‐112 cells, although no differences were observed between the expression of ULBP2 and 3 with R8‐liposome‐BCG‐CWS. ? T24 cells that were co‐cultured with R8‐liposome‐BCG‐CWS showed an ≈1.3‐fold increase in sensitivity to cytolysis by LAK cells at an E : T ratio of 4 and RT‐112 cells showed an ≈1.4‐fold increase at an E : T ratio of 2.

CONCLUSIONS

? In the present study, the induction of surface NKG2D ligands by R8‐liposome‐BCG‐CWS rendered cancer cells more susceptible to cytolysis by LAK cells. ? T24 cells and RT‐112 cells, even when cultured singly in the absence of immune cells, can directly respond to R8‐liposome‐BCG‐CWS. ? The results obtained in the present study may therefore indicate a novel adoptive immunotherapy against bladder cancers.     

N-acetylcysteine augments the cellular redox changes and cytotoxic activity of internalized mycobacterium bovis in human bladder cancer cells

PURPOSE: We determined whether changes in cellular reactive oxygen species correlated with mycobacteria internalization and bladder cancer cell death. MATERIALS AND METHODS: Reactive oxygen species and thiols in RT112 and MGH bladder cancer cells were determined using the fluorescence probes 5-(and 6)-carboxy-2', 7' dichlorodihydrofluorescein diacetate and monobromobimane. Superoxide and nitrite production were measured using bis-N-methylarcridinium nitrate and Griess reagents. Cytotoxicity was determined by the release of 14C-thymidine from cells with 14C labeled DNA. RESULTS: MGH cells that internalize bacillus Calmette-Guerin (BCG) had decreased cellular reactive oxygen species and thiols, although superoxide and nitric oxide production increased. RT112 cells, which do not internalize BCG, did not show a decrease in reactive oxygen species after incubation with BCG. Blocking BCG uptake in MGH cells abrogated reactive oxygen species reduction, confirming that the changes in reactive oxygen species were internalization dependent events. Treating cells with BCG and the antioxidant N-acetylcysteine caused a greater reduction in reactive oxygen species, and induced earlier and greater cytotoxicity in MGH but not in RT112 cells. CONCLUSIONS: The induction of bladder cancer cell killing by BCG parallels the ability of cells to internalize BCG, which in turn indicates that the susceptibility of tumor cells to the cytotoxic effects of BCG may be related to changes in cellular levels of reactive oxygen species and thiols. Supplementation with an antioxidant could enhance the antitumor effect of BCG.     

Lactobacillus species is more cytotoxic to human bladder cancer cells than Mycobacterium Bovis (bacillus Calmette-Guerin)

PURPOSE: We determined if Lactobacillus species has growth inhibitory effects in human bladder cancer cell lines and how this effect compares with the known effects of Mycobacterium bovis, that is bacillus Calmette-Guerin (BCG). MATERIALS AND METHODS: The growth of MGH and RT112 cells were determined by cell counts after 24, 48 and 72 hours of exposure to L. casei strain Shirota (Yakult, Singapore) or L. rhamnosus strain GG (National Collection of Industrial and Marine Bacteria, Ltd., Aberdeen, Scotland) (1 x 10 and 1 x 10 cfu) or BCG (1 x 10 cfu) in the presence and absence of streptomycin. Annexin-V was used to monitor the presence of pre-apoptotic cells. RESULTS: L. rhamnosus GG inhibited MGH proliferation and it was cytotoxic to RT112 cells (p <0.05). L. casei Shirota was cytotoxic to the 2 cell lines (p <0.05). BCG had a similar cytotoxic effect in MGH cells as Lactobacillus species but was not as effective in RT112 cells. Streptomycin abrogated the cytotoxic effect of Lactobacillus species but not that of BCG. Cytotoxic activity was not found in Lactobacilli culture supernates but it was induced in the presence of mammalian cells. L. rhamnosus GG induced apoptosis in RT112 but not in MGH cells. No apoptotic cells were detected after treatment with L. casei Shirota. CONCLUSIONS: Lactobacillus species induced cytotoxic effects in bladder cancer cells. Unlike BCG, it requires bacterial protein synthesis. Like BCG, L. casei Shirota induces cell death primarily via necrosis. The cytoxicity of these lactobacilli in bladder cancer cells raises the possibility of using this species of bacteria as intravesical agents for treating bladder cancer.     

Novel roles of lipopolysaccharide and TLR4/NF-κB signaling pathway in inflammatory response to liver injury in Budd-Chiari syndrome

BACKGROUNDBudd-Chiari syndrome (BCS) is an uncommon disorder characterized by obstruction of hepatic venous outflow. To date, the exact mechanism underlying hepatic injury derived from the hepatic venous outflow obstruction in BCS remains largely unknown.AIMTo assess the role of NF-κB-mediated inflammation in BCS-induced liver injury in humans and rats.METHODSA total of 180 rats were randomly assigned into nine groups, including four BCS model groups (1, 3, 6 and 12 wk), four sham-operated groups (1, 3, 6 and 12 wk), and a control group. Lipopolysaccharide (LPS) levels in each group were detected by the Tachypleus Amebocyte Lysate assay. The mRNA and protein levels of TLR4, NF-κB, tumor necrosis factor (TNF)-α, interleukin (IL)-2 and interferon (IFN)-γ were quantified. In addition, 60 patients with BCS and 30 healthy controls were enrolled, and their blood samples were analyzed.RESULTSHepatic and plasma LPS levels were significantly increased in rats. The mRNA and protein expression levels of TLR4, NF-κB and inflammatory cytokines (TNF-α, IL-2 and IFN-γ) in liver tissues were significantly higher in the BCS model groups compared with the other two groups. In addition, the model groups (1, 3, 6 and 12 wk after BCS induction) showed significant differences in the levels of LPS, TLR4, NF-κB, TNF-α, IL-2 and IFN-γ. Notably, there was a significant correlation between the LPS concentrations and mRNA and protein levels of TLR4, NF-κB and inflammatory cytokines. Importantly, it was revealed that the levels of LPS, TLR4, NF-κB and inflammatory cytokines were significantly greater in chronic BCS patients than healthy controls and acute BCS patients.CONCLUSIONLPS level is markedly elevated in BCS, in turn activating the TLR4/NF-κB signaling pathway, leading to induction of inflammatory cytokines (TNF-α, IL-2 and IFN-γ) in response to BCS-induced liver injury.     

BCG膀胱灌注对局部免疫细胞功能的影响

对２７例表浅膀胱癌术后ＢＣＧ膀胱灌注前后膀胱壁Ｔ＿３、Ｔ＿４、Ｔ＿８、ＩＬ－２Ｒ表达及尿液中肿瘤坏死因子（ＴＮＦ）含量进行了研究。结果表明，ＢＣＧ膀胱灌注后膀胱壁大量淋巴细胞浸润，Ｔ＿４／Ｔ＿８比值由灌注前０．６上升至１．９，２０例患者膀胱壁形成淋巴肉芽肿，粘膜下散在淋巴细胞表面广泛表达ＩＬ－２Ｒ，但肉芽肿淋巴细胞表面几乎不表达ＩＬ－２Ｒ。灌注后２４小时尿液中ＴＮＦ含量显著升高（Ｐ＜０．０１），实验结果提示：Ｔ＿４、巨噬细胞在ＢＣＧ抗肿瘤效应中起着非常重要的作用。     

Toll样受体4信号对结肠癌细胞免疫抑制性细胞因子的调控

目的探讨Toll样受体（TLRs）对原位结肠癌细胞免疫抑制性细胞因子的调控作用及其机制。方法分别采用RT—PCR和蛋白印迹法对HT-29细胞中TLRs mRNA及蛋白质的表达进行检测。ELISA法检测经LPS刺激后及NF—κKB被抑制后,HT-29细胞所分泌的免疫抑制性细胞因子的改变。结果HT-29细胞可表达不同TLRs,以TLR4的表达为最高。经LPS刺激后,HT-29细胞中TLR4的mRNA和蛋白质水平,以及所分泌的转化生长因子（TGF）-β、VEGF、IL-8、CCL20和IL-6均显著升高（P〈0．01）。TGF—β、VEGF、IL-8和CCL20的上调表达不能被NF—κB抑制剂吡咯烷二硫代氨基甲酸盐（PDTC）所抑制,但IL-6的上调表达则依赖于NF—κB的活性。结论结肠癌细胞TLRs通过识别病原体相关模式分子．启动免疫抑制性细胞因子的表达．使肿瘤细胞逃避免疫监视。     

TLR4 influences the humoral and cellular immune response during polymicrobial sepsis

As part of the innate immune system, Toll-like receptors (TLRs) react rapidly on a pathogen challenge without prior exposure. Although it is well known that TLR4 is associated with the receptor for lipopolysaccharide (LPS), its role during sepsis has not yet been clearly defined. To study this, polymicrobial sepsis was induced in male C3H/HeN (TLR4 wild type) and C3H/HeJ (TLR4 mutant) mice by caecal ligation and puncture (CLP).A total of 48 h following the surgical procedure, the mice were sacrificed and plasma was collected. Kupffer cells were isolated and ex vivo cytokine production and plasma levels were determined. Lung neutrophil influx was investigated by myeloperoxidase (MPO) content and immunohistochemistry. T-cell subtypes in blood and spleen were determined by flow cytometry.Mice with intact TLR4 (wild type) had increased Kupffer cell IL-6 production and increased plasma levels as compared with C3H/HeJ mice following sepsis. Furthermore, wild type mice showed increased neutrophil influx in lungs and lower percentages of CD8⁺ splenocytes. This was accompanied with less activity, increased weight loss and decreased core temperature.We conclude that TLR4 influences the humoral and cellular response during the course of sepsis and lack of TLR4 reduces markers of the systemic inflammatory response as well as distant organ damage. Therefore, TLR4 could act as a future therapeutic target modulating the immune response during sepsis.     

The role of cancer stem cells in immunotherapy for bladder cancer: An in vitro study

ObjectiveBladder cancer is characterized by frequent recurrence and progression. CD44+ cancer stem cells (CSCs) might be one of the main reasons for recurrence. Although Bacillus Calmette Guerin (BCG) has become a gold standard immunotherapy, after treatment recurrence frequently occur. Based on this knowledge, the aim of this study was to evaluate the changes in cytokine and chemokine expressions in bladder cancer and CSCs cultures in vitro with BCG only and in combination with IL2 and lymphocyte (MNCs) applications.Material and methodsIn this study, 3 cell lines of human bladder cancer cells with different characteristics (T24, 5637, and JMSU-1) and CD44+ bladder CSCs isolated by magnetic bead isolation (Miltenyl Magtech) were used. Bladder cancer cell lines and bladder CSCs in complete medium were cultured under humidified conditions of 37°C temperature in 5% CO₂. BCG only and its combination with IL2 and MNCs were applied to bladder cancer cell lines and bladder CSCs for 24, 48, and 72 hours. Annexin V-PI was used to detect the percentages of apoptotic and necrotic cells in treatment groups and control groups. After treatments, total RNAs were isolated and converted to cDNA for each group and controls. Quantitative fold changes in terms of gene expression were measured by RT2–PCR array and fold changes for expression levels of genes were compared among groups. Eighty-four genes were analyzed in standard array of chemokines and cytokines (Biorad).ResultsBCG treatment with 7.32 µg/ml dose alone and in combination with IL2 (1000 IU/ml) and MNCs (1000 cells/ml) were found to be most effective on bladder cancer cells. When BCG and its combinations were applied to CSCs of the 3 cell lines, BCG treatment showed cytotoxic effect on CSCs as well as cancer cells. CSCs of 3 cell lines over expressed CXCL5, CCL8, CNTF, and CSF2 compared with cancer cells. Cancer cells over expressed IL6, TNSFF11, FASLG, and CXCL9 compared with CSCs. In all 3 cell lines, BCG application increased expression of CXCL5 and LTB and also decreased CCL20 and IL6. When BCG was combined with IL2 and MNCs, CXCL10, CXCL5, and IFNG were increased and CXCL12, IL6, and TNSF11 were decreased. BCG treatment of CSCs caused increases in ADIPOQ, CXCL10, and XCL1 and a decrease in CCL8. When IL2 and MNCs were combined with BCG, the expression of many cytokines and chemokines decreased.ConclusionBCG treatment changes the expression of many cytokines and chemokines in bladder cancer. The expression differs in 3 different cell lines and their CSCs. Immune modulation of each case differs from each other. The effectivity of BCG-based immunotherapy in bladder cancer on CSCs might decrease in combination with IL2. Our results indicate that recurrence after BCG treatment for bladder cancer may not occur mainly based on the CSCs hypothesis considering bladder cancer occurs at different loci of surface epithelium.     

Innate immune memory is associated with increased disease-free survival in bladder cancer patients treated with bacillus Calmette-Guérin

IntroductionWhile studies suggest that innate immune memory acquired by circulating monocytes may mediate the benefit of bacillus Calmette-Guérin (BCG) in the treatment of patients with high-risk non-muscle-invasive bladder cancer (NMIBC), prospective studies are lacking. Innate immune memory is defined by enhanced release of pro-inflammatory cytokines by innate immune cells following a secondary challenge with pattern recognition receptor (PRR) ligands.MethodsPeripheral blood monocytes isolated from 33 patients with intermediate- or high-risk NMIBC before and after two or five induction BCG instillations were stimulated with the PRR ligand lipopolysaccharide (LPS). Inflammatory cytokine levels in the culture medium were measured. Extent of innate immune memory acquisition was determined by dividing the levels of cytokines released after BCG instillation by the levels released prior to BCG therapy.ResultsMonocytes secreted variable levels of TNFα, IL-1β, IL-6, IFNγ, IL-12, and IL-10. Compared with patients with recurrences, the post-BCG:pre-BCG ratio of IL-12 in monocyte cultures from patients without recurrences after five BCG instillations was significantly increased. Patients with no innate immune memory (based on IL-12 ratios) had significantly shorter time to recurrence than patients with innate immune memory (p<0.001). Eighty-four percent (16/19) of patients with innate immune memory vs. only 22% (2/9) of patients without memory had disease-free survival of over 500 days.ConclusionsResults demonstrate a potential link between BCG-induced innate immune memory peripherally and local anti-tumor responses. Further validation will increase our understanding of the mode of action of BCG and, therefore, will be used to enhance its effectiveness.     

Intravesical bacillus Calmette-Guerin immunotherapy after previous prostate radiotherapy for high-grade non-muscle-invasive bladder cancer

ObjectivesIntravesical bacillus Calmette-Guerin (BCG) immunotherapy is a standard treatment for high-grade non-muscle-invasive bladder cancer (NMIBC). We evaluated outcomes of BCG therapy for NMIBC in patients with a previous history of prostate cancer (CaP) radiotherapy (RT).Materials and methodsA retrospective review of patients with a history of CaP RT who subsequently underwent treatment with intravesical BCG for high-grade NMIBC was performed. Patients were categorized as “BCG success” or “BCG failure” (defined as stage progression or recurrent/persistent disease). We evaluated factors related to the radiotherapy (type, interval to BCG), bladder cancer (clinical stage, immunotherapy type, and course), and patient comorbidities, to identify factors associated with BCG failure.ResultsFrom 1996 to 2008, 26 patients with high-grade NMIBC received intravesical BCG immunotherapy after CaP RT. At a mean follow-up of nearly 5 years, 13 patients (50%) were successfully managed with one or more induction courses of BCG with or without the addition of interferon alpha. Twelve (46%) eventually required cystectomy for disease recurrence or progression, of which half had pathologically advanced disease (≥pT3). Clinical stage was similar between BCG success and failure patients (P = 0.40). Those who failed immunotherapy were more likely to have had a longer interval between RT and BCG induction (5.8 vs. 2.4 years, P = 0.02).ConclusionApproximately 50% of patients with NMIBC who were previously exposed to prostate radiation had a durable response to intravesical BCG. For non-responders, extravesical progression was common.     

Impact of TLR4-siRNA transfection on the expression of TLR4/MyD88 Signaling in rat tubular epithelial cells under high glucose condition

Objective To observe the expression of toll like receptor 4(TLR4) Signaling and the release of inflammation factors in rat tubular epithelial cell(NRK-52E) under high glucose condition after TLR4-siRNA transfection. Methods Three TLR4-siRNA sequences were designed and synthesized. The transfection efficiency was observed by fluorescence microscope after transfection, and the expression of TLR4 mRNA was detected by real time PCR. The most effective siRNA was selected to be used for forward experiments. After transfection for 24 h, cells were stimulated with 25 mmol/L glucose and/or 10-7 mmol/L Angiotension Ⅱ(AngⅡ) for 12 h, 24 h; cells without stimulation were as normal control. Real-time PCR was used to analyze TLR4 and myeloid differentiation factor 88 (MyD88) mRNA expression; Western blot was used to observe TLR4/MyD88 and NF-κB protein expression. ELISA assay was used to detect the concentration of monocyte chemoattractant protein-1(MCP-1), interleukin-6(IL-6) in cell supernatant after cells were stimulated for 24 h. Results TLR4/MyD88 mRNA and TLR4/MyD88/NF-κB protein were highly expressed under high glucose or AngⅡ co-incubated NRK-52E(P＜0.01), the MCP-1 and IL-6 levels were also increased markedly compared with normal control group (P＜0.01). TLR4/MyD88 mRNA and TLR4/MyD88/NF-kB protein expressions were obviously inhibited in cells that were transfected with TLR4-siRNA compared with high glucose group(P＜0.01), MCP-1 and IL-6 production decreased remarkably compared with high glucose or AngⅡ co-stimulated group(P＜0.01). Conclusions High glucose can lead to the activation of TLR4/MyD88/NF-kB signaling and the secretion of inflammation factors in NRK-52E, AngⅡ further augments these effects. The effect can be blocked efficiently by specific siRNA gene silence. TLR4 signaling plays a pivotal role in the innate-immune inflammatory reaction in NRK-52E.     

Localization of IL-1, IL-2, IL-4, IL-8 and TNF in Superficial Bladder Tumors Treated with Intravesical Bacillus Calmette-Guerin

Purpose

Cytokines have been detected in the urine during the first hours after intravesical Bacillus Calmette Guerin (BCG) treatment against superficial bladder cancer. To investigate the long-lasting mucosal inflammatory response, we analyzed intracellular cytokines by immunohistochemistry in biopsies taken 2 weeks after BCG treatment.

Materials and Methods

Tumor biopsies were obtained from 8 patients with noninvasive, papillary transitional cell carcinoma (TCC), and intracellular cytokines were visualized by immunohistochemistry using cytokine-specific monoclonal antibodies.

Results

Interleukin (IL)-1 beta sup + or tumor necrosis factor (TNF)-alpha sup + cells were abundant in tumor and stroma. Interleukin-1 alpha, IL-2, IL-4, IL-8 and TNF-beta were variably expressed, while IL-10 sup + and interferon (IFN)-gamma sup + cells were not detected. Among the few patients studied (5 responders and 3 nonresponders to BCG treatment) no single cytokine or cytokine profile was associated with clinical response to BCG therapy.

Conclusion

We conclude that the in situ cytokine response after BCG treatment is highly complex, since cytokine profiles differed among the 8 patients investigated and between tumor and surrounding tumor-free mucosa. Further studies, investigating larger number of patients, is required to clarify whether cytokine profiles correlate with the clinical response to BCG.     

The therapeutic potential of recombinant BCG expressing the antigen S1PT in the intravesical treatment of bladder cancer

PurposeBacillus Calmette-Guerin (BCG) continues to be employed as the most effective immunotherapy against superficial bladder cancer. We have developed an rBCG-S1PT strain that induces a stronger cellular immune response than BCG. This preclinical study was designed to test the potential of rBCG-S1PT as an immunotherapeutic agent for intravesical bladder cancer therapy.Materials and methodsA tumor was induced in C57BL/6 mice after chemical cauterization of the bladder and inoculation of the tumor cell line MB49. Next, mice were treated by intravesical instillation with BCG, rBCG-S1PT, or PBS once a week for 4 weeks. After 35 days, the bladders were removed and weighed, Th1 (IL-2, IL-12, INOS, INF-γ, TNF-α), and Th2 (IL-5, IL-6, IL-10, TGF-β) cytokine mRNA responses in individual mice bladders were measured by quantitative real time PCR, and the viability of MB49 cells in 18-hour coculture with splenocytes from treated mice was assessed. In an equivalent experiment, animals were observed for 60 days to quantify their survival.ResultsBoth BCG and rBCG-S1PT immunotherapy resulted in bladder weight reduction, and rBCG-S1PT increased survival time compared with the control group. There were increases in TNF-α in the BCG treated group, as well as increases in TNF-α and IL-10 mRNA in the rBCG-S1PT group. The viability of MB49 cells cocultured with splenocytes from rBCG-S1PT-treated mice was lower than in both the BCG and control groups.ConclusionsrBCG-S1PT therapy improved outcomes and lengthened survival times. These results indicate that rBCG could serve as a useful substitute for wild-type BCG.     

TLR4介导大鼠肾脏缺氧复氧炎症反应依赖于MyD88

目的：探讨MyD88是否参与了TLR介导大鼠肾脏缺氧复氧（hypoxia/reoxygenation,H/R）引起的炎症反应.方法：体外分离获得Wistar大鼠原代肾小管上皮细胞（proximal tubule epithelial cells,PTECs）,建立H/R模型.采用TLR4siRNA干扰PTECs,检测白细胞介素8（interleukin-8,IL-8）和肿瘤坏死因子α（tumor necrosis factor α,TNF-α）的表达.之后,加入髓样分化因子88（myeloid differentiation factor 88,MyD88）的抑制剂ST2825后,检测IL-8和TNF-α的表达.结果：TLR4表达沉默后,IL-8、TNF-α和MyD88的表达均显著下降（P〈0.05）;加入ST2825后,IL-8、TNF-α和MyD88的表达均显著下降（P〈0.05）,且抑制MyD88活性后显著降低细胞的凋亡水平（P〈0.05）.结论：MyD88参与了TLR4介导的大鼠肾脏缺氧复氧引起的炎症反应.     

TLR4 Mediates Early Graft Failure After Intraportal Islet Transplantation

We have previously shown that islet emboli in the portal vein block blood flow and induce local inflammatory reaction, resulting in functional loss of islet grafts following intraportal transplantation. This study was designed to test whether Toll‐like receptor (TLR) activation mediates early islet graft failure. Syngeneic islet grafts were transplanted into chemically induced diabetic mice, and TLR deficient mice were used as donors and/or recipients of islet grafts. Islet viability, proinflammatory cytokines, high‐mobility group box‐1 (HMGB1) and NF‐κB activation were analyzed by bioluminesce imaging (BLI), quantitative RT‐PCR (qRT‐PCR) and histology. Early islet graft failure was observed in mice with intraportal islet engrafts with increased proinflammatory cytokines, HMGB1 expression, NF‐κB activation, caspase‐3 and TUNEL positive cells. Deficiency of TLR4 in donor, but not in recipient, inhibited NF‐κB activation, reduced proinflammatory cytokines and improved viability of islet grafts. Blockade of HMGB1 with anti‐HMGB1 monoclonal antibody (mAb, 2g7) inhibited inflammatory reactions, as evidenced by reduced TNFα and IL‐1ß production, and improved islet viability. We conclude that TLR4 activation mediates early graft failure following intraportal islet transplantation. Inhibition of TLR4 activation represents a novel strategy to attenuate early graft failure following intraportal islet transplantation.