Combination Of Bacillus Calmette-Guerin Research Articles

EXPRESSION OF IMMUNOGLOBULIN, GRANZYME-B AND PERFORIN AGAINST Ag85A AND Ag85B PROTEINS OF MYCOBACTERIUM TUBERCULOSIS IN BALB/C MICE.

Background:Ag85 is a protein that may maintain survival of M. tuberculosis in intracellular parts of host cells and is considered as a virulence factor. The expression of Ag85 protein can stimulate proliferation and differentiation of B- cells and T-cells in patients with tuberculosis. This research aimed to determine the ability of Ag85A and Ag85B proteins in activating the response of antibodies, granzyme-B and perforin in Balb/c mice.Materials and Methods:Twenty-five male Balb/c mice were assigned into five groups. Group I was treated with adjuvant, group II with Bacillus Calmette-Guerin (BCG) vaccine, group III with a combination of BCG and Ag85A, group IV with a combination of BCG and Ag85B and group V with a combination of BCG, Ag85A and Ag85B. Concentrations of immunoglobulin G, granzyme-B and perforin were examined using ELISA and the number of CD8+ T-cells and NK T-cells were checked by flow cytometry.Results:The highest concentration of immunoglobulin G was found in group V with 62.49±5.4327 ng/ml. The highest mean number of CD8+ T-cells, NK T-cells, granzyme-B and perforin was found in group IV with 4.32%, 1.03%, 35.11±1.7789 pg/ml and 6.19±0.2235 pg/ml, respectively. The results of One-Way ANOVA test showed that there were significant differences in immunoglobulin responses, with p<0.05. The expressions of granzyme-B and perforin were higher in mice treated with combination of BCG and recombinant proteins.Conclusions:Ag85 protein can be combined with the BCG vaccine to improve protection against M. tuberculosis infection.

The regulatory effects of polyporus polysaccharide on the nuclear factor kappa B signal pathway of bladder cancer cells stimulated by Bacillus Calmette-Guerin

To detect the effects of Polyporus polysaccharide (PPS), Bacillus Calmette-Guerin (BCG), and their combination on the nuclear factor kappa B (NF-κB) signaling pathway associated-gene expression and investigate the molecular mechanisms of the toxic-reducing effect of PPS in coordination with BCG against bladder cancer. After T739 cells were treated with PPS, BCG and their combination, the changes in mRNA and protein expression of inhibitor of kappa B kinase beta (IKKβ), NF-κB subunit p65 (NF-κB p65), intracellular adhesion molecule 1 (ICAM1) and chemokine (C-c motif) ligand 2 (CCL2) in bladder cancer cell line T739 were determined by relative quantitative real-time PCR, Western blot, and flow cytometry (FCM). NF-κB p65 DNA-binding activity in T739 cell was detected by biotinylated probe-ELISA, and NF-κB p65 nuclear expression in T739 cell was observed by immunohistochemistry. Compared with the T739 control group, the mRNA expression of IKBKB (IKKβ), Rel A (NF-κB p65), ICAM1 and CCL2 in T739 cells treated with BCG were increased obviously (Ratio>2.0), as well as the expression of IKKβ, CCL2 and ICAM1 proteins. Meanwhile, NF-κB p65 DNA-binding activity and NF-κB p65 nuclear expression in T739 cells treated with BCG were up-regulated significantly (P<0.05). Compared with the control, the increased expression in T739 cells were simultaneously down-regulated after PPS treatment, except for ICAM1 protein expression. With cells treated with a combination of BCG and PPS, the expression of genes associated with the NF-κB signaling pathway, such as IKBKB, ICAM1 and CCL2, were all down-regulated compared to the BCG group, as well as Rel A mRNA expression, NF-κB p65 DNA-binding activity and NF-κB p65 nuclear expression. PPS could inhibit the over-activation of the NF-κB signaling pathway induced by BCG in bladder cancer cells and accordingly attenuate the adverse reactions to BCG therapy.

Sunitinib can enhance BCG mediated cytotoxicity to transitional cell carcinoma through apoptosis pathway

Bacillus Calmette-Guerin (BCG)-refractory generated a high risk to patients with bladder cancer during treatment. Tyrosine kinase receptor (TKR) and TKR-mediated signal transduction pathways play an important role in tumor initiation, maintenance, angiogenesis, and vascular proliferation. Theoretically, it is helpful in adjuvant treatment for transitional cell carcinoma (TCC). Hence, we proposed that sunitinib, a endothelial growth factor receptor (VEGFR) inhibitor, may have a synergistic effect with BCG in enhancing its cytotoxicity to bladder cancer. The level of VEGF in various TCC cell lines was quantified by real time PCR. High grade TCC-T24 cell line with high level of VEGF expression was selected as representative tumor cells for further study. The single drug and combined inhibitory effects of BCG and sunitinib in T24 cells were determined by MTT method. The drug mediated cell apoptosis in T24 cells was characterized by flow cytometry with PI and annexin V stain. Bcl-2 apoptotic pathway induction by BCG and sunitinib treatment was evaluated by Western blotting method. Inhibitory ability of sunitinib in BCG induced cell migration was verified by cell migration assay. The results shown that expression level of VEGF mRNA in high grade T24 cells was higher than low grade J82, TSGH 8301, and TCC 9202 cell lines. Both BCG and sunitinib treatment presented cytotoxic effect to T24 cells in a dose-dependent manner. Combination of BCG and sunitinib revealed superior cytotoxicity effect than single agent when cells were pretreated with low dosage BCG before sunitinib treatment. By Annexin V analysis it was observed that cell death associated with increased early and late apoptosis process individually. Furthermore, the bcl-2 expression was significant reduced in T24 cells in metachronous BCG and sunitinib combination treatment than single agent. Tumor cell migration activity was also markedly inhibited with BCG and sunitinib combination treatment. In conclusion, these results suggested that during BCG and sunitinib combination treatment both reagents interacted with each other and caused TCC cells apoptosis in addition to direct cytotoxicity. This combination therapeutic model may have the potential for future clinical application to bladder cancer treatment.

818 HUMAN GENE POLYMORPHISM AND RESPONSE TO BACILLUS CALMETTE-GUERIN IMMUNOTHERAPY FOR SUPERFICIAL BLADDER CANCER

AACR Centennial Conference: Translational Cancer Medicine-- Nov 4-8, 2007; Singapore C61 Objectives: Superficial bladder cancers (SBC) have a 60-70% recurrence rate after transurethral resection, and 10-20% of recurrences progress to invasive cancer. Post-transurethral resection intravesical Bacillus Calmette-Guerin (BCG) instillation has been the ‘gold-standard’ treatment for high risk SBC, for which 20-40% of patients do not respond. There are also frequent, potentially serious side effects to BCG therapy. Although clinical and pathologic variables to predict BCG response and cancer recurrence have been extensively studied, little is known of the role of underlying host genetic susceptibility factors, such as genetic polymorphisms. The NRAMP1 gene has been implicated in susceptibility to tuberculosis and to BCG response in murine models. The Human glutathione peroxidase 1 (hGPX1) is a selenium-dependent enzyme that participates in detoxification of hydrogen peroxide, organic peroxides, and possibly cigarette smoke-derived oxidative radicals. An association between hGPX1 and NRAMP1, and bladder cancer recurrence has been suggested. We aim to determine the predictive value of NRAMP1 and hGPX1 gene polymorphisms in superficial bladder cancer recurrence and response to BCG therapy. Methods: Peripheral blood DNA was prospectively obtained from 99 high risk superficial bladder cancer patients, who underwent post-resection intravesical regimes of BCG (81mg, n=50 or 27mg, n=19) or BCG (27mg) with interferon alpha (IFNa) (n=30), and followed-up for a mean of 4.5 years. The (GT)n and D534N polymorphisms in the NRAMP1 gene, and the Pro198Leu polymorphism in the hGPX1 gene were tested with restriction fragment length polymorphisms and DNA sequencing following PCR amplification. Data was analyzed using Chi-square analysis, multiple logistic regression and Kaplan-Meier curves. Results: The D534N G:A genotype was found to be protective towards cancer recurrence (p=0.016) and cancer-specific death (p=0.036). The presence of allele 3 in the (GT)n gene polymorphism was found to correlate with higher recurrence in BCG treated patients (p=0.003), and had shorter recurrence-free survival (p=0.024) and progression-free survival (p=0.005) in patients treated with a combination of BCG and IFNa. The hGPX1 CC genotype was shown to be protective (p=0.014), while the variant CT genotype (Pro/Leu) was associated with increased bladder cancer recurrence and decreased time to recurrence (p=0.03) after BCG therapy. Conclusion: Our findings suggest that polymorpisms in the NRAMP1 and hGPX1 genes correlate with response to BCG therapy in bladder cancer patients. They may serve as molecular markers to predict BCG failure and cancer recurrence.

HUMAN GENE POLYMORPHISM AND RESPONSE TO BACILLUS CALMETTE-GUERIN IMMUNOTHERAPY FOR SUPERFICIAL BLADDER CANCER

AACR Centennial Conference: Translational Cancer Medicine-- Nov 4-8, 2007; Singapore C61 Objectives: Superficial bladder cancers (SBC) have a 60-70% recurrence rate after transurethral resection, and 10-20% of recurrences progress to invasive cancer. Post-transurethral resection intravesical Bacillus Calmette-Guerin (BCG) instillation has been the ‘gold-standard’ treatment for high risk SBC, for which 20-40% of patients do not respond. There are also frequent, potentially serious side effects to BCG therapy. Although clinical and pathologic variables to predict BCG response and cancer recurrence have been extensively studied, little is known of the role of underlying host genetic susceptibility factors, such as genetic polymorphisms. The NRAMP1 gene has been implicated in susceptibility to tuberculosis and to BCG response in murine models. The Human glutathione peroxidase 1 (hGPX1) is a selenium-dependent enzyme that participates in detoxification of hydrogen peroxide, organic peroxides, and possibly cigarette smoke-derived oxidative radicals. An association between hGPX1 and NRAMP1, and bladder cancer recurrence has been suggested. We aim to determine the predictive value of NRAMP1 and hGPX1 gene polymorphisms in superficial bladder cancer recurrence and response to BCG therapy. Methods: Peripheral blood DNA was prospectively obtained from 99 high risk superficial bladder cancer patients, who underwent post-resection intravesical regimes of BCG (81mg, n=50 or 27mg, n=19) or BCG (27mg) with interferon alpha (IFNa) (n=30), and followed-up for a mean of 4.5 years. The (GT)n and D534N polymorphisms in the NRAMP1 gene, and the Pro198Leu polymorphism in the hGPX1 gene were tested with restriction fragment length polymorphisms and DNA sequencing following PCR amplification. Data was analyzed using Chi-square analysis, multiple logistic regression and Kaplan-Meier curves. Results: The D534N G:A genotype was found to be protective towards cancer recurrence (p=0.016) and cancer-specific death (p=0.036). The presence of allele 3 in the (GT)n gene polymorphism was found to correlate with higher recurrence in BCG treated patients (p=0.003), and had shorter recurrence-free survival (p=0.024) and progression-free survival (p=0.005) in patients treated with a combination of BCG and IFNa. The hGPX1 CC genotype was shown to be protective (p=0.014), while the variant CT genotype (Pro/Leu) was associated with increased bladder cancer recurrence and decreased time to recurrence (p=0.03) after BCG therapy. Conclusion: Our findings suggest that polymorpisms in the NRAMP1 and hGPX1 genes correlate with response to BCG therapy in bladder cancer patients. They may serve as molecular markers to predict BCG failure and cancer recurrence.

Antitumour immunity of bacillus Calmette–Guerin and interferon alpha in murine bladder cancer

Intravesical Bacillus Calmette–Guerin (BCG) immunotherapy is currently the optimal choice for aggressive superficial bladder cancer, with a 70% response rate. This study investigated whether the antitumour response elicited by BCG could be improved by the addition of recombinant interferon alpha (IFNα) in the subcutaneous murine MB49 bladder tumour model. The combination of BCG and IFNα had superior and earlier antitumour activity than BCG alone for MB49 cells in culture. A total of 14/15 BCG plus interferon-treated mice and 8/16 BCG-treated mice became tumour free after treatment. BCG or the combination treatment significantly raised the T-helper 1 (Th1) cytokine IFNγ levels compared with levels in all other groups. Whilst BCG therapy alone increased CD4 + and CD8 + populations in spleens, the combination of BCG and IFNα also increased αβ + T cells significantly. Our results suggest that the combination of BCG and IFNα may represent a more efficacious therapeutic than BCG alone for superficial bladder cancer. ©

Effects of bacillus Calmette-Guerin and interferon-alpha-2B on human bladder cancer in vitro.

The cytolytic and anti-proliferative effects of bacillus Calmette-Guerin (BCG) and/or interferon-alpha-2b (IFN-alpha-2b) on 5 human bladder carcinoma cell lines, RT4, RT112, MGH, SD and J82, were determined. The cell lines showed different sensitivities to BCG and IFN-alpha-2b. BCG had direct dose-dependent cytolytic and anti-proliferative effects on MGH, J82 and SD (grade 3 cell lines), whereas RT4 and RT112 (grades 1 and 2, respectively) were less sensitive. Surprisingly, higher concentrations of BCG enhanced cell growth of RT4. IFN-alpha-2b also had cytolytic and anti-proliferative effects on all 5 cell lines. Thus, the RT4 and RT112 cell lines that were not sensitive to BCG were highly sensitive to IFN-alpha-2b. A combination of BCG and IFN-alpha-2b had additive anti-proliferative effects on MGH, J82 and RT112. Interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha) production by these 5 cell lines was measured after stimulation with BCG and/or IFN-alpha-2b, by ELISA immunoassays. Production of IL-6 and TNF-alpha was significantly increased in MGH and J82 cell lines by the combination of BCG and IFN alpha-2b. The enhanced cytolytic and anti-proliferative effects of the combination of BCG and IFN-alpha-2b may be related to the induction of cytokines.

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 < 0.01), and the combination of BCG and ABPP yielded the highest survival (p < 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. (J. Urol., 142: 1376–1379, 1989)

Results of randomized trials of immunotherapy for acute leukemia

For more than a decade clinical trials have attempted to define the role of immunotherapy in the treatment of patients with acute leukemia. Based on animal studies which indicated that non-specific immune stimulation had an antitumor effect if the tumor burden was small, the use of immunotherapy during remission in patients with acute leukemia seemed appropriate following the initial report of the success of bacillus Calmette-Guerin (BCG) in prolonging remission duration and survival in acute lymphoblastic leukemia. Therefore a series of randomized clinical trials was initiated to confirm these original observations. In four studies comparing BCG inoculations, with or without allogeneic leukemia cells, and chemotherapy or no therapy, no advantage of immunotherapy was noted. Immunotherapy appeared to be equally as good as chemotherapy. A combination of BCG and chemotherapy showed some advantage in one study, but no advantage was noted in two other studies. In acute myeloblastic leukemia several randomized trials suggested that BCG or one of its derivatives when given alone, in combination with allogeneic cells, or with chemotherapy had a marginal effect in prolonging remission duration and survival when compared to chemotherapy or rno therapy. In conclusion, immunotherapy during remission has marginal activity in acute leukemia.

Therapeutic application of various immunostimulators on the L2C guinea-pig leukemia

Immunostimulators such as Corynebacterium parvum (C. parvum), Bacillus Calmette-Guerin (BCG), pyran copolymer, and glucan were examined in the guinea pig L 2 C lymphoblastic leukemia model to determine their capacity for therapeutic modulation of the immune response of the host toward controlling leukemic cell proliferation. The dose, route, and frequency of administration of the stimulators were also evaluated as a function of time in order to obtain an optimal antileukemic effect. Results indicated that only C. parvum and BCG were capable of significantly increasing host survival when given 1 day after an inoculation of 1.5×10 4 viable leukemic cells. Administration of BCG or C. parvum, alone or in combination with irradiated blast cells on either days 4 or 7, was totally ineffective in prolonging survival. In the majority of cases, enhanced leukemic growth was observed on these days. The combination of BCG and/or C. parvum with irradiated syngeneic blast cells given 24 h after leukemia inoculation promoted a synergistic response with a significant increase in median survival time and a number of long-term survivors.