Surface antigen expression on bladder tumor cells induced by bacillus Calmette-Guérin (BCG): A role of BCG internalization into tumor cells

BACKGROUND: The antitumor mechanisms of bacillus Calmette-Guérin (BCG) against bladder cancer is still unclear. We previously reported that BCG was internalized by and survived within murine bladder tumor cells (MBT-2) for at least 40 days. In the present study, we investigated the effect of BCG on the surface antigen expression of bladder tumor cells and the characteristics of these cells as antigen-presenting cells in vitro. METHODS: Surface antigen (major histocompatibility complex (MHC) Class II, CD1, CD80 and intercellular adhesion molecule-1 (ICAM-1)) expression on BCG-treated murine (MBT-2) and human (T-24, J82) bladder tumor cells were analyzed using flow cytometry. The production of interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) from murine lymphocytes sensitized with BCG or BCG-treated tumor cells were also investigated. RESULTS: The expressions of MHC Class II, CD1, CD80 and ICAM-1 were augmented in all of the bladder tumor cell lines used; however, they were augmented to varying degrees among the cell lines that were treated with live BCG. Heat-killed BCG had little or no effect. When murine lymph node cells sensitized with BCG or BCG-treated MBT-2 cells were cocultured with BCG-treated MBT-2 cells, significant amounts of IL-2 and IFN-gamma were produced in the culture medium. CONCLUSIONS: BCG induced the augmented expression of surface antigens, such as MHC Class II, CD1, CD80 and ICAM-1, of bladder tumor cells. Furthermore, BCG-treated MBT-2 cells could stimulate BCG-sensitized lymphocytes to produce IL-2 and IFN-gamma. These results strongly suggest that bladder tumor cells gained the characteristics and functions of antigen-presenting cells (APC).     

Antitumor activity of interleukin-12 against murine bladder cancer

PURPOSE: We investigated the antitumor activity of interleukin-12 (IL-12) against MBT-2, a murine bladder carcinoma, to clarify whether or not IL-12 is effective against urothelial tumors. MATERIALS AND METHODS: MBT-2, a murine carcinogen-induced, poorly differentiated transitional cell carcinoma of C3H/He origin, was used. Three or 10 days after the subcutaneous administration of MBT-2 cells, C3H/He mice were injected intraperitoneally with IL-12 five times per wk. for 2 wk. Tumor growth was measured twice weekly. Spleen cells from the C3H/He mice that had rejected MBT-2 after the IL-12 treatment were examined for MBT-2-specific cytolytic T lymphocytes (CTL) activity and cytokine production. RESULTS: Tumor growth and acceptance was obviously suppressed when C3H/He mice were treated with IL-12 from 3 days after the tumor inoculation. In the spleen cells from the C3H/He mice that had rejected MBT-2, MBT-2-specific CTL activity and secretion of IL-2 and interferon (IFN)-gamma were clearly detected. However, the established MBT-2 tumor cells were not rejected when C3H/He mice were given IL-12 from 10 days after the tumor inoculation, although the tumor growth was transiently suppressed during the IL-12 treatment. CONCLUSION: These data demonstrate that IL-12 is considerably effective against murine bladder cancer and suggest the clinical application of IL-12 against human bladder cancer.     

The essential role of interferon-gamma during interleukin-12 therapy for murine transitional cell carcinoma of the bladder

PURPOSE: Recombinant interleukin (IL)-12 and adenoviral IL-12 gene therapy have been shown to be potent therapeutic interventions for murine transitional cell carcinoma (TCC) of the bladder in vivo. We investigated the mechanisms through which IL-12 induces antibladder cancer immunity. MATERIALS AND METHODS: The ability of IL-12 to enhance interferon-gamma (IFN-gamma) expression, a major T-helper type 1 cytokine, was analyzed in murine serum, urine and splenocyte cultures. MB49, a murine TCC line, was treated with IFN-gamma and evaluated for its proliferation, surface molecule expression and sensitivity to splenocyte mediated cytotoxicity. Neutralizing antiIFN-gamma antibody was applied to test the role of IFN-gamma in the IL-12 therapy of MB49 tumor. RESULTS: IL-12 was observed to significantly increase IFN-gamma concentrations in serum and urine as well as in splenocyte cultures. While IL-12 had no direct activity against TCC in vitro, IFN-gamma showed potent dose dependent antiproliferative and pro-apoptotic activity, which was further enhanced by supplementation of tumor necrosis factor-alpha. In addition, IFN-gamma substantially up-regulated the expression of surface immune molecules on TCC cells, including MHC-I, MHC-II, ICAM-I, B7.1, B7.2 and Fas. Maximum splenocyte mediated cytotoxicity against TCC was enhanced by pretreatment of target bladder cancer cells with IFN-gamma plus tumor necrosis factor-alpha. Furthermore, IL-2 in combination with IL-12 further enhanced splenocyte mediated cytotoxicity. The in vivo antibladder cancer activity of IL-12 was abolished by concurrent treatment with antibodies to IFN-gamma. CONCLUSIONS: This study strongly suggests that IFN-gamma has an essential role in IL-12 induced antibladder tumor immunity. Activation of host effector immune cells by IL-12 is also required for induction of optimal tumor destruction in IL-12 therapy.     

Interleukin-12 immunotherapy of murine transitional cell carcinoma of the bladder: dose dependent tumor eradication and generation of protective immunity

PURPOSE: The antitumor activity of interleukin (IL)-12 has been demonstrated in a number of tumor models but barely tested in bladder cancer models. We evaluated the antibladder cancer activity of this cytokine in syngeneic mice bearing subcutaneous, metastatic and orthotopic tumors. MATERIALS AND METHODS: Mice were implanted subcutaneously, intravenously or orthotopically with syngeneic transitional cell carcinoma (TCC) of the bladder. The tumor bearing mice were then treated with IL-12 locally or systemically and monitored for tumor regression and survival. RESULTS: In the subcutaneous model dose dependent suppression of tumorigenesis was observed when IL-12 was administered subcutaneously at a distal site with the MB49 line being more sensitive than MBT-2. IL-12 (10 days) above 50 ng daily was tumor inhibitory, while doses of 500 or 1000 ng daily prolonged survival and cured 70% and 75% of subjects, respectively. Upon re-challenge with parental tumor cells mice previously cured with IL-12 (1000 vs 500 ng daily) exhibited specific protection (70% vs 35% rejection) that was dependent on the earlier dose of cytokine. IL-12 administered intraperitoneally at a dose of 250 ng daily was more potent than subcutaneous administration and complete regression was observed. Metastatic TCC in the lungs and orthotopic tumors in the bladder also favorably responded to systemic or intravesical IL-12 therapy, respectively. Addition of IL-2 to IL-12 therapy increased tumor regression, long-term survival and rejection of re-challenged parental tumor. CONCLUSIONS: IL-12 is exceptionally effective for treating murine bladder TCC in subcutaneous, metastatic and orthotopic models. The antibladder cancer activity of this cytokine should be tested in human bladder cancer therapy.     

BCG dose reduction by decreasing the instillation frequency: effects on local Th1/Th2 cytokine responses in a mouse model

OBJECTIVES: Based on the requirement of a Th1 immune response for clinical efficacy, and incited by the arbitrary induction scheme, frequent side effects and the empirical approach in improving BCG immunotherapy for superficial bladder cancer, an alternative intravesical BCG treatment schedule for dose reduction was investigated without compromising Th1 cytokine induction in the bladder in a mouse model. METHODS: Mice were submitted to 6 weekly BCG instillations and treatment schedules omitting intermediate instillations during this standard scheme. Th1 (IFN-gamma, IL-2, IL-12p40), and Th2 (IL-10, IL-4) cytokine responses in individual mouse bladders were measured by a semiquantitative RT-PCR based method. RESULTS: A schedule of only two BCG instillations, administered in week 1 and week 6, resulted in induction of at least the same levels of IFN-gamma, IL-2 and IL-12p40 Th1 cytokine mRNA compared to 6 weekly instillations, whereas significantly lower levels of Th2 cytokines IL-10 and IL-4 mRNA were observed. CONCLUSIONS: During the 6-week period the intermediate weekly BCG instillations 2, 3, 4, and 5 do not contribute to Th1 cytokine upregulation in the bladder, provided that the BCG dose is sufficient. Whether such a reduced BCG frequency schedule has immune stimulating capacity and therapeutic efficacy associated with less side effects in patients remains to be investigated.     

Immunological protection induced by bacillus Calmette–Guérin treatment in a murine bladder tumor model

BACKGROUND: It has been previously reported that MBT-2 tumor growth is completely inhibited when mice are inoculated with bacillus Calmette-Guérin (BCG). In this study it was examined whether or not vaccination with a mixture of BCG and MBT-2 cells also induces immunological protection against murine bladder tumors. METHODS: Seven hundred thousand MBT-2 cells and 1 mg of BCG (Tokyo 172 strain) per mouse were injected subcutaneously into female C3H/HeN mice. Four and eight weeks after vaccination with this mixture, animals were reinoculated with MBT-2 cells alone or MBT-2 cells cocultured with BCG. RESULTS: Animals vaccinated with a mixture of BCG and MBT-2 cells showed MBT-2 tumor growth but completely rejected the MBT-2 cells cocultured with BCG. MBT-2 cells cocultured with BCG developed into tumors when they were inoculated into the control animals. Splenocytes prepared from vaccinated animals showed specific cytocidal activity against MBT-2 cells precultured with BCG. CONCLUSIONS: The results suggest that a mixture of BCG and MBT-2 cells induces antitumor immunological protection against BCG- or MBT-2-associated antigens presented on MBT-2 cells precultured with BCG.     

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.     

Combination immunotherapy of murine transitional cell cancer using BCG and an interferon-inducing pyrimidinone

The immunomodulator ABPP (2-amino-5-iodo-6-phenyl-4(3H) pyrimidinone) is an interferon inducer and has been shown to have in vivo activity against the murine bladder tumor MBT-2. Two experiments were performed to determine if ABPP might enhance the in vivo anti-tumor activity of Bacillus Calmette-Guerin (BCG). First, in vivo stimulation of cell-mediated cytotoxicity by BCG and ABPP was measured in C3H mice using a chromium-release assay. An earlier, greater, and longer-lasting increase in cytotoxicity was caused by ABPP than by BCG. Based on the differing times to peak cytotoxic stimulation, groups of 15 mice each were pretreated intraperitoneally at different times prior to inoculation with MBT-2. Compared to saline, ABPP alone did not increase survival, while BCG alone did increase survival (p less than 0.01), and the combination of BCG and ABPP yielded the highest survival (p less than 0.001). These results indicate that 1) ABPP affects the immune system differently than BCG, and 2) while ABPP may have less single-agent activity against MBT-2 than BCG, ABPP may serve to potentiate the activity of BCG.     

重组卡介苗和传统卡介苗免疫原性的比较

目的 观察分泌人白细胞介素(IL)-2的重组卡介苗(rBCG)和传统卡介苗(BCG)对小鼠免疫应答的影响,评价其免疫原性,初步探讨rBCG免疫治疗的作用机制.方法 连续6周对BALB/C小鼠尾静脉注射rBCG和BCG,分别在第2、4和6周后杀死小鼠取腹腔巨噬细胞和脾脏.以一氧化氮(NO)释放量检测巨噬细胞吞噬活性,以淋巴细胞刺激指数(SI)反映细胞增殖能力,以流式细胞仪测定T淋巴细胞CD亚群分化和酶联免疫吸附试验(ELISA)法检测小鼠脾淋巴细胞培养上清细胞因子分泌的变化,比较rBCG治疗组和BCG治疗组之间的差异.结果 在连续6周观察期间,随着免疫时间的延长,rBCG对小鼠脾脏T淋巴细胞的增殖能力逐渐增强,CD4+、CD8+T细胞和CD4+/CD8+的比例逐渐增高,腹腔巨噬细胞的吞噬活性逐渐增高,诱导小鼠脾淋巴细胞分泌的Th1型和Th2型细胞因子逐渐增多.在同一时点上,不同rBCG和BCG的上述作用均高于空白对照组(P均0.01),差异有统计学意义;rBCG治疗组的上述作用均高于BCG治疗组.结论 rBCG具有良好的免疫原性,能明显增强BALB/c小鼠的免疫应答反应.rBCG诱导的T细胞免疫应答主要是CD4+T细胞依赖的,CD4+细胞免疫应答主要是以Th1型细胞因子参与为主,Th2型细胞因子也发挥一定作用.     

Prophylaxis and therapy of an experimental bladder cancer with biological response modifiers

We have previously reported that the intraperitoneal injection of viable bacillus Calmette-Guerin (BCG) reduces the incidence of tumor takes and the rate of tumor growth and also increases tumor regression rate and survival in C3H/HeN mice challenged with the syngeneic MBT-2 bladder cancer. We have now investigated the immunoprophylactic effect of BCG and other biological response modifiers. Groups of 12 to 15 female C3H/HeN mice were challenged with 5 X 10(5) MBT-2 viable cells and treated with BCG, poly I:C, tilorone, levamisole or a combination of these agents. Appropriate controls were included in each experiment. In this study we confirmed previous findings that BCG alone is effective in prophylaxis and can also decrease the rate of tumor growth in those animals not protected against tumor takes. Both i.p. levamisole and oral tilorone lacked activity against the MBT-2 tumor. A single i.p. injection of poly I:C was also ineffective although its repeated administration reduced the rate of tumor growth and induced a significant number of tumor regressions. Combined therapy of BCG with either levamisole or tilorone offered no therapeutic advantage over BCG alone. Heat-inactivated BCG also failed to induce anti-tumor activity in C3H/HeN mice challenged with MBT-2 cells. These results indicate that live BCG remains the most active immunoprophylactic and immunotherapeutic agent against the MBT-2 murine bladder cancer. Furthermore, the anti-tumor effect of viable BCG is not potentiated further by the addition of other immune modulating agents.     

Reduction of bladder cancer growth in mice treated with intravesical Bacillus Calmette Guerin and systemic interleukin 2

The effect of systemic administration of Interleukin 2 (IL2) on intravesical Bacillus Calmette-Guerin (BCG) therapy was studied in an established murine bladder tumor, MBT-2. BCG (100 micrograms.) was administered intravesically on days 7 and 14 after seeding bladders with MBT-2 cells. IL2 (5,000 U/injection) was given intraperitoneally every eight hours for 10 times (days seven through 10 and 14 through 17). BCG or IL2 therapy alone failed to reduce incidence of tumor implantation and tumor weight; whereas, combined treatment with BCG and IL2 reduced tumor weight significantly compared to saline or BCG treated mice. Cytotoxicity was assessed in a four-hour 75Semethionine-release assay. Augmentation of natural killer cell activity was only observed in mice treated with BCG plus IL2. MBT-2 target cells were not lysed by spleen cells from mice treated with BCG or saline. IL2 therapy produced lymphokine-activated killer cell activity, though combining BCG with IL2 suppressed this activity after each course of treatment. The results suggest that combined treatment with IL2 enhances the therapeutic effect of BCG therapy. However, this enhancement of antitumor activity is not clearly explained by augmentation of natural killer or in vivo-generated lymphokine-activated killer cells.     

Endogenously formed nitric oxide modulates cell growth in bladder cancer cell lines

Objectives. Nitric oxide (NO) is formed in many mammalian tissues, and a growing body of evidence suggests that NO is involved in cell growth and cell differentiation. Low concentrations of NO can stimulate cell growth; high concentrations result in cytostatic/cytotoxic effects. It has previously been shown that intravesical treatment with bacille Calmette-Guérin (BCG) for bladder cancer increases NO production in the human urinary bladder and that NO inhibits bladder cancer cell growth in vitro. In this study, we investigated nitric oxide synthase (NOS) activity in different bladder cancer cells and the role of the NO precursor -arginine in cell proliferation.Methods. NOS activity was assessed by citrulline assay in cultured normal human urothelial cells and bladder cancer cell lines T24 and MBT-2 before and after treatment with cytokines. We also measured cell growth at various -arginine concentrations and after addition of the NOS inhibitor N^G-nitro- -arginine ( -NNA) in unstimulated and cytokine-stimulated cells.Results. Normal urothelial cells, as well as T24 and MBT-2 cells, showed calcium-dependent NOS activity under basal conditions. The bladder cancer cell lines also showed calcium-independent NOS activity in contrast to the normal cells. After cytokine treatment, both the normal cells and the cancer cell lines showed a marked increase in calcium-independent NOS activity. There was a dose-dependent stimulation of cell growth in the cancer cell lines after -arginine addition, and this effect could be antagonized by -NNA. Cytokine treatment inhibited cell growth, and this inhibition was partly reversed by -NNA.Conclusions. Normal urothelial cells and bladder cancer cell lines MBT-2 and T24 show NOS activity, and cytokine treatment induces calcium-independent NOS activity. Our results suggest that endogenous activity of the constitutively expressed form of NOS in unstimulated cells promotes cell proliferation, and NO production secondary to increased activity of the inducible form of NOS after cytokine treatment inhibits cell growth.     

重组hIFN-α-2b-BCG对小鼠原位膀胱肿瘤免疫效应的研究

目的 探讨重组hIFN-α-2b-BCG对原位膀胱肿瘤小鼠体内抗肿瘤免疫反应及其作用机制.方法 构建C57BL/6原位膀胱肿瘤小鼠模型,采用流式细胞仪对小鼠膀胱灌注治疗后的淋巴细胞亚群进行分析,并测定小鼠血清中mTNF-α和mIL-12的水平,了解小鼠全身免疫状况.肿瘤组织冰冻切片进行T细胞亚群的免疫组织化学分析,免疫组化检测肿瘤Fas表达,了解膀胱局部免疫反应. 结果 重组BCG灌注治疗后小鼠外周血CD4+ T细胞比例明显增高,CD8+ T细胞比例无明显变化,CD4+ /CD8+比例为2.63,与野生BCG组(2.10)比较差异有统计学意义(P＜0.05).荷瘤小鼠外周血中Th1型细胞因子mTNF-α和mIL-12灌注治疗后比PBS对照组大幅提高,重组BCG组mTNF-α为806 pg/ml,mIL-12为860 pg/ml,与野生BCG组及野生BCG联合IFN组比较差异无统计学意义(P＞0.05).重组BCG组瘤组织内CD3、CD4和CD5检测强阳性,显著高于PBS对照组(P＜0.05),重组BCG组和野生BCG加IFN组瘤组织CD4+、重组BCG组CD8+检测值显著高于野生BCG组(P＜0.05).BCG灌注后荷瘤小鼠膀胱肿瘤表达Fas明显高于PBS对照组(P＜0.05). 结论 重组hIFN-α-2b-BCG具有在小鼠体内调节系统及局部免疫能力的作用,纠正荷瘤小鼠淋巴细胞亚群的比例失调,增强局部淋巴细胞浸润,具有增强Th1型细胞因子产生作用,上调荷瘤小鼠Fas的表达,诱导对膀胱肿瘤细胞的免疫攻击.

Abstract：

Objective To study local and systemic immune response in an animal model treated with recombinant hIFN-α-2b-BCG instillation. Methods The MB49 orthotopic bladder cancer model in C57BL/6 mice was established and treated separately with rBCG, wild BCG, wild BCG combined with IFN-α-2b and PBS as the control. The changes of lymphocyte subgroups in peripheral blood were analyzed with FCM, and mTNF-α and mIL-12 in peripheral blood of mice were detected with ELISA.Immunohistochemistry was carried out to detect the local immune reaction, T cell subsets and FAS, in bladder cancer after being treated with rBCG or wBCG. Results The content of CD4+ T lymphocyte was up-regulated in the rBCG group. The CD4+/CD8+ ratio of 2. 63 was up-regulated than pretreatment, significantly different than that of wBCG group(P＜0.05). ELISA assay showed that BCG significantly up-regulated the level of mTNF-α and mIL-12 in serum of orthotopie murine bladder cancer mice. The mTNF-α 806 pg/ml, mIL-12 860 pg/ml in rBCG group, was not significantly higher than those in wBCG group and combination group. The immunocompetent cell numbers with CD3, CD4,CD8 phenotype increased significantly in the tumor tissue of BCG treated group than the control(P＜0.05). The results of CD4+ in rBCG group and the combination group, and CD8+ in rBCG group were significantly higher than that of the wBCG(P＜0.05). The expression of Fas in tumor tissues treated with intravesical BCG was increased(P＜0. 05). Conclusions The recombinant IFN-α-2b-BCG can retrieve the disproportion of systemic lymphocyte subgroups, and increases Th1-type factors and local Fas expression in orthotopic murine bladder cancer. The recombinant IFN-α-2b-BCG is effective in regulating local and systemic immune reaction in orthotopic murine bladder cancer model.     

人干扰素α-2b基因重组卡介苗的构建与鉴定

目的 构建能自动分泌表达人干扰素α-2b(IFNα-2b)的重组卡介苗(rBCG-IFNα-2b)菌株,并对其进行鉴定.方法 分别从人外周血和BCG基因组中提取DNA,聚合酶联反应(PCR)扩增人IFNα-2b基因和BCGAg85B信号肽基因,将该两片段插入质粒pMV261,构建分泌型卡介苗穿梭表达载体pMV261-Ag85B-IFNα-2b.电穿孔将该载体导入BCG中,构建rBCG-IFNα-2b.分别用PCR扩增和Western blot检测rBCG-IFNα-2b中人IFNα-2b基因和蛋白的表达情况,酶联免疫吸附(ELISA)检测rBCG-IFNα-2b培养上清中IFNα-2b蛋白的表达情况.结果 采用酶切、PCR扩增及DNA测序对pMV261-Ag85B-IFNα-2b进行鉴定,结果 显示BCG Ag85B信号肽片段和人IFNα-2b片段与文献结果 一致,连接方向正确.以rBCG-IFNα-2b DNA为模板、IFNα-2b引物进行PCR扩增后得到与理论上大小相同的扩增片段,Western blot显示rBCG-IFNα-2b的培养上清和菌体中均可检测到IFNα-2b蛋白的表达,且培养上清中蛋白的表达量明显多于菌体,ELISA法可检测到培养上清中有高表达的IFNα-2b蛋白(301.45 pg/ml).结论 成功构建了能自动分泌表达人IFNα-2b的新型重组卡介苗菌株rBCG-IFNα-2b,为进一步研究其免疫活性和抗膀胱肿瘤疗效奠定了基础.     

Urinary cytokines reflecting the immunological response in the urinary bladder to biological response modifiers: their practical use

BACKGROUND: Intravesical bacillus Calmette-Guérin (BCG) immunotherapy is currently the most effective treatment for superficial transitional cell carcinoma (TCC) of the urinary bladder. In recent years, the substantial number of patients not responding to BCG or experiencing considerable toxicities has stimulated studies addressing either the development of improved BCG treatment schedules or the exploration of the therapeutic value of a series of (novel) biological response modifiers, like interferons (IFNs), interleukin (IL) 2 and keyhole limpet hemocyanin. Although the actual mechanism by which BCG exerts its antitumor effect still needs detailed unraveling, current available knowledge suggests the induction of a T helper 1 (Th1) or Th1-like cytokine profile, represented by IL-2, IL-12 and IFN-gamma, as essential in the development of a cell-mediated antitumor activity. CONCLUSIONS: In this review, it is argued that incorporation of urinary cytokine determinations, like IL-2 and possibly IL-12 and IFN-gamma, may represent a valuable approach in the optimization and individualization of the BCG therapy and an early, initial evaluation of the potential efficacy of novel immunomodulating agents in the treatment of superficial TCC.     

Retrovirus type C in the mouse bladder carcinoma cell line MBT-2

PURPOSE: The presence of replicating type C retrovirus in MBT-2 mouse bladder carcinoma cells is reported. This MBT-2 tumor cell line is nowadays globally distributed. The cells have been and are still used to study various aspects of bladder cancer. While studying the phagocytic capacity of MBT-2 cells for BCG organisms by electron microscopic methods, the presence of this retrovirus was noticed. MATERIALS AND METHODS: MBT-2 cells that were cultured in vitro as well as cells from intravesically and intradermally grown MBT-2 tumors from syngeneic mice were investigated using transmission electron microscopy (TEM) and scanning electron microscopy (SEM) techniques. RESULTS: All samples including the earliest generation MBT-2 cells that could be traced from stocks of other research groups contained the C type retrovirus, suggesting a contamination in all available generations of the MBT-2 cell line. CONCLUSIONS: As this tumor cell line is widely used in immunologic studies of the response to bladder cancer, it is important to consider the possible presence of type C viruses and associated antigens, since they could contribute to or interfere with the responses being measured. Studies should be initiated to determine whether viral antigen expression is involved in the immune rejection of MBT-2 bladder cancer. As a consequence, clinical implementation of immunological treatment strategies should not be based on results obtained with the MBT-2 model alone, but preferably should be confirmed with other (bladder) carcinoma models.     

Cytotoxicity in glioma cells due to interleukin-12 and interleukin-18-stimulated macrophages mediated by interferon-gamma-regulated nitric oxide

OBJECT: Interleukin (IL)-12 and IL-18 synergistically mediate antitumor responses through the production of interferon-gamma (IFNgamma) by T and natural killer (NK) cells. Recently, it has been reported that macrophages stimulated with these cytokines also produce IFNgamma, which led the authors to investigate the antiglioma activity of macrophages stimulated by the combination of these cytokines in vitro. METHODS: Dish-adherent peritoneal exudate cells, which had been elicited in thioglycollate broth as a source of macrophages, were used in the experiment. The murine glioma cell lines VM-glioma and 203G were labeled with [3H]thymidine for a cytotoxicity assay of macrophages. In response to the combined stimulation by IL-12 and IL-18, macrophages expressed potent cytotoxic activity against glioma cells in association with increasing production of IFNgamma and nitric oxide (NO). Inhibitors of NO abrogated the cytotoxic activity of the macrophages, which had been induced by IL-12 and IL-18, despite the increase in IFNgamma production. Neutralization of IFNgamma or use of macrophages obtained from IFNgamma gene-knockout mice markedly reduced not only cytotoxic activity, but also NO production. Depletion of T and NK cells from the macrophage population, which was achieved using antibody plus complement treatment, slightly reduced macrophage activities, suggesting that these are the main effector cells, although T and NK cells may partially participate in this cytotoxicity. CONCLUSIONS: Macrophages stimulated with IL-12 and IL-18 produced IFNgamma and NO, which in turn mediated the antiglioma response. Therefore, macrophages as well as T and NK cells play an important role in antitumor responses stimulated by IL-12 and IL-18.     

Intravesical BCG therapy in bladder carcinoma. Effect on cytotoxicity, IL-2 production and phenotype of peripheral blood mononuclear cells.

The purpose of the study was to examine the effects of intravesical BCG treatment on the cytotoxicity, interleukin-2 (IL-2) production and distribution of the subsets of peripheral blood mononuclear cells (PBMC) in patients with carcinoma in situ of the bladder. Treatments were made in 6 patients during a conventional BCG treatment schedule. Four patients showed a complete response, one a partial response and one had a progressive disease after BCG treatment. Intravesical BCG did not induce significant changes in the cytotoxicity of PBMC. The distribution of NK-cells and T-cells also remained unchanged and so did the lectin induced production of IL-2. The results suggest that the effects of intravesical BCG on the immune system should be studied in lymphocytes isolated from the bladder.