Viable Bacillus Calmette-Guerin Research Articles

1151 DIRECT ANTITUMOR EFFECT OF BACILLUS CALMETTE-GUERIN IN HIGH GRADE UROTHELIAL CANCER CELLS WHICH EXPRESS VLA5 (ALPHA 5 BETA 1 INTEGRIN)

You have accessJournal of UrologyBladder Cancer: Basic Research III1 Apr 20101151 DIRECT ANTITUMOR EFFECT OF BACILLUS CALMETTE-GUERIN IN HIGH GRADE UROTHELIAL CANCER CELLS WHICH EXPRESS VLA5 (ALPHA 5 BETA 1 INTEGRIN) Tomoyuki Kato, Mayu Yagi, Atsushi Yamagishi, Noriyuki Hosoya, Toshihiko Sakurai, Hayato Nishida, Sei Naito, Masaaki Tsukigi, Hisashi Kawazoe, Akinori Muto, Akira Nagaoka, and Yoshihiko Tomita Tomoyuki KatoTomoyuki Kato More articles by this author , Mayu YagiMayu Yagi More articles by this author , Atsushi YamagishiAtsushi Yamagishi More articles by this author , Noriyuki HosoyaNoriyuki Hosoya More articles by this author , Toshihiko SakuraiToshihiko Sakurai More articles by this author , Hayato NishidaHayato Nishida More articles by this author , Sei NaitoSei Naito More articles by this author , Masaaki TsukigiMasaaki Tsukigi More articles by this author , Hisashi KawazoeHisashi Kawazoe More articles by this author , Akinori MutoAkinori Muto More articles by this author , Akira NagaokaAkira Nagaoka More articles by this author , and Yoshihiko TomitaYoshihiko Tomita More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2010.02.650AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Intravesical instillation of Bacillus Calmette-Guerin (BCG) is an effective treatment for superficial high grade bladder cancer. Although both immunological and direct effects on tumor cells have been proposed, the complete mechanisms for its effect are not fully understood. Presence of fibronectin has been shown to be necessary for binding and internalization of BCG into tumor cells to exert direct effect on them. We have previously demonstrated the correlation between high expression of fibronectin receptor VLA5 (alpha 5 beta 1 integrin) and high malignant potential of urothelial cancer (UC). Here, we assessed the direct antitumor effects of BCG for UC cell lines which beard various amount of VLA5 on the cell surface. METHODS The human UC cell lines (T24, HT1376 and RT4) and Tokyo 172 BCG strain (Immunobladder, Japan BCG Laboratory), BCG cell wall skeleton (BCG-CWS SMP105, Dainippon Sumitomo Pharma) were used in this study. Viability of cell lines was evaluated with crystal violet stainig assay. Fluorescence of apoptotic nuclei stained with Propidium Iodide (PI) was measured with a FACScan. DNA synthesis was evaluated with BrDU incorporation assay. RESULTS First, expression of VLA5 on the surface of cell lines was checked. T24 and HT1376, high grade UC, beard large amount of VLA5 on the cell surface and RT4, a low grade UC, had traces of VLA5. Treatment with BCG resulted in dose dependent decrease of relative viability in T24 (p<0.0001) and HT1376 (p<0.0001). This effect was partially reversed by coculturing cells with VLA5 blocking antibody. In RT4, viability was not altered by treatment with BCG. Sub-G1 fraction of PI stained T24 increased from less than 1% (untreated) to more than 4% (1 mg/ml of BCG) after exposure to BCG and it suppressed BrDU incorporation (p=0.004) in T24 cells. Autoclaved killed BCG also demonstrated cell growth retardation effect in T24 (p<0.0001) and HT1376 (p<0.0001). BCG-CWS SMP105 also delayed T24 and HT1376 cell growth (p<0.0001). CONCLUSIONS These findings suggest that BCG exerts direct antitumor effect on UC cells via VLA5 with induction of apoptosis and inhibition of DNA synthesis. Moreover, killed BCG and BCG cell wall skeleton were able to exert similar effect making this approach attractive for clinical application where potentially serious adverse effect of viable BCG can be avoided while maintaining antitumor effect using killed BCG or cell extracts. Yamagata, Japan© 2010 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 183Issue 4SApril 2010Page: e445-e446 Advertisement Copyright & Permissions© 2010 by American Urological Association Education and Research, Inc.MetricsAuthor Information Tomoyuki Kato More articles by this author Mayu Yagi More articles by this author Atsushi Yamagishi More articles by this author Noriyuki Hosoya More articles by this author Toshihiko Sakurai More articles by this author Hayato Nishida More articles by this author Sei Naito More articles by this author Masaaki Tsukigi More articles by this author Hisashi Kawazoe More articles by this author Akinori Muto More articles by this author Akira Nagaoka More articles by this author Yoshihiko Tomita More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Cytokine Gene Expression in a Mouse Model: The First Instillations With Viable bacillus Calmette-Guerin Determine the Succeeding Th1 Response

Bacillus Calmette-Guerin (BCG) therapy for superficial bladder cancer is immune dependent and activation of a Th1 immune response is probably required for clinical efficacy. Given the empirical approach to improving BCG therapy we investigated in a mouse model the consequences of modifications in BCG therapy with regard to Th1 and Th2 cytokine responses in the bladder. These studies may provide a rationale for possible modifications of the established clinical treatment protocol. The dynamics of Th1 (interferon-gamma, interleukin [IL]-2, IL-12p40 and tumor necrosis factor-alpha) and Th2 (IL-10 and IL-4) cytokine responses during and after 6 once weekly intravesical BCG instillations in mice was determined by a semiquantitative reverse transcriptase-polymerase chain reaction based method. During 6 weekly BCG instillations a dose and time dependent induction of the various Th1 as well as Th2 cytokines was observed. The response pattern was comparable to urinary cytokine induction patterns in patients. Electrocauterization prior to BCG instillations led to lower and more variable levels of cytokine polymerase chain reaction products compared with noncauterization. Lowering the dose of BCG seemed to affect the Th1 cytokine response most, whereas the Th2 response was less influenced by dilution of the BCG preparation. Six instillations with nonviable BCG induced Th2 but failed to induce Th1 cytokines, which may explain the necessity of BCG viability for antitumor activity. However, when mice were first treated 3 times with viable BCG and subsequently received 3 instillations with killed BCG, the Th1 and Th2 cytokine pattern was comparable to the standard 6-week regimen with viable BCG. The model seems an appropriate one in which to investigate changes in Th1 and Th2 cytokine gene expression levels in bladders resulting from modifications in intravesical BCG treatment. It was possible to induce a local Th1 cytokine response with nonviable BCG provided that local sensitization to BCG antigens had occurred during preceding instillations with a viable BCG preparation. Whether such an approach could decrease BCG therapy toxicity, while maintaining antitumor efficacy, remains to be further investigated in patients.

THE FATE OF BACILLUS CALMETTE-GUERIN AFTER INTRAVESICAL INSTILLATION

Long-term activation of immunocompetent cells of the bladder wall as well as case reports of systemic infections some months or years after intravesical bacillus Calmette-Guerin (BCG) therapy imply that mycobacteria may persist in the body. Therefore. we investigated the fate of BCG in patients after uncomplicated intravesical instillation therapy. A total of 49 patients were included in the study, from whom various numbers of specimens were used for mycobacterial culture and molecular biological detection techniques. In 23 patients who received a total of 128 instillations urine, sputum, venous blood and bladder biopsies were screened for BCG by acid-fast staining and culture at different times before and after instillation. From 16 of the 23 patients and from an additional 26 a total of 180 bladder biopsies obtained at intervals 3 to 30 months after instillation were screened for mycobacterial 16S ribosomal DNA by a nested polymerase chain reaction protocol. No viable BCG was found in venous blood or in 127 of 128 sputum specimens before and 2 hours after instillation. Two of 56 bladder biopsies were culture positive. In urine BCG was detected in 96.4% of the specimens after 2 hours and in 67.9% after 24 hours after instillation. The number of positive specimens decreased and it was 27.1% on day 7 immediately before the next instillation. In 14 of 44 bladder biopsies (31.8%) mycobacterial ribosomal DNA was found within 1 week after the sixth instillation. A positive polymerase chain reaction was evident up to 24 months in between 4.2% and 37.5% of the investigated biopsies. After 30 months no ribosomal DNA was evident in the 6 samples available for testing. Nontraumatic intravesical instillation of BCG is not accompanied by systemic mycobacterial spread. Local persistence during the instillation course is evident since viable BCG is commonly found in the urine. Long lasting and persistent BCG DNA in the bladder wall may account for long-term immuno-activation. However, the remaining BCG may be a possible source of late systemic infections.

PRIMARY TREATMENT OF CONDYLOMATA ACUMINATA WITH VIABLE BACILLUS CALMETTE-GUERIN

Condylomata acuminata or genital warts are caused by human papillomavirus. Prevalence data show that infection rates are increasing. To our knowledge we report the first successful primary treatment of genital warts with topical bacillus Calmette-Guerin (BCG) and provide long-term followup in a group of adjuvant treated patients with recurrent condylomata acuminata. In 10 consecutive men viable BCG was directly applied to the condylomata acuminata lesions once weekly for 6 weeks. In nonresponding patients another course of 9 applications was administered for 3 weeks. A complete response was achieved in 6 of the 10 men after 1 or 2 treatment cycles. All responding patients are disease-free at a median followup of 9.2 months (range 4 to 12). One patient achieved partial regression of the lesions and in 3 the condylomata did not disappear. Side effects were rare and mild. Long-term followup in 6 adjuvant treated patients with rapidly recurrent condylomata acuminata showed no further recurrence after topical BCG in 5 at a median of 30.8 months (range 29 to 50). Topical application of viable BCG has therapeutic activity as adjuvant and primary treatment for penile condylomata acuminata with negligible side effects. Long-term followup implies the prevention of recurrent disease.

Bacillus Calmette-Guerin Down-Regulates CD1b Induction by Granulocyte-Macrophage Colony Stimulating Factor in Human Peripheral Blood Monocytes

Non-peptide antigens (e.g. glycolipids of microbial origin) presented by monocyte-associated CD1 molecules to T cells appear to play an important role in host immunity against tuberculosis and other pathogenic bacteria. Since vaccination with Bacillus Calmette-Guerin (BCG) has limited efficacy, the influence of viable BCG organisms on the induction of CD1b antigen by granulocyte macrophage-colony stimulating factor (GM-CSF) has been tested in adherent mononuclear cells obtained from peripheral blood of healthy donors. The results indicate that the vaccine reduces substantially CD1b induction by GM-CSF. On the other hand, BCG was found to promote a slight increase in the expression of this molecule on target cells not exposed to GM-CSF. Attempts to reverse the antagonistic effects of BCG on GM-CSF with high concentrations of GM-CSF, alone, or associated with IL-4, were unsuccessful. Moreover, mycobacteria suppression by 10 μmg/ml of rifampin, did not affect BCG influence on CD1b induction. The present results suggest that mycobacterium-induced impairment of the CD1 system could play a role in the unsatisfactory results obtained with BCG vaccination.

EFFECTS OF ACETYLIC SALICYLIC ACID AND PENTOXIFYLLINE ON THE EFFICACY OF INTRAVESICAL BCG THERAPY IN ORTHOTOPIC MURINE BLADDER CANCER (MB49)

Intravesical immunotherapy with bacillus Calmette-Guerin (BCG), which has become the gold standard in the adjuvant treatment of superficial bladder cancer, is hampered by local side effects. Anti-inflammatory drugs may be helpful, but as an undesired side effect, therapeutic efficacy of BCG might be impaired. Therefore, we investigated the effects of anti-inflammatory drugs on the efficacy of intravesical BCG in an animal model. Syngenic tumor cells were implanted into the bladders of 75 mice according to our modification of the method. Mice were randomized to 5 groups with 15 animals each and treated with phosphate buffered saline (PBS), group 1; BCG, group 2; BCG + acetylic salicylic acid (ASA), group 3; BCG + pentoxifylline (POF), group 4; autoclaved BCG (aBCG), group 5. Intravesical instillation of 1.35 mg. BCG was initiated one day after tumor inoculation and repeated in weekly intervals for 4 instillations altogether. ASA and POF in doses of 200 mg./kg. and 150 mg./kg., respectively, were given continuously with the drinking water starting at the first instillation. Autoclaved BCG served as control for the importance of viability and was given at the same dose as viable BCG. Mice were monitored for survival, gross hematuria and body weight and after 28 days evaluated for bladder weight and tumor occurrence. Autoclaved BCG and PBS had no effect on tumor growth, whereas animals treated with viable BCG alone and in combination with POF and ASA, respectively, showed a significant reduction in bladder weight: PBS, 248 mg.; BCG, 140 mg. (p = 0.0009); BCG + ASA, 123 mg. (p = 0.0001); BCG + POF, 145 mg. (p = 0.0004); autoclaved BCG, 283 mg. (p = 0.21). Mice treated with BCG, BCG + ASA and BCG + POF showed a significantly higher proportion of survival until day 28 as compared to PBS alone. Autoclaved BCG had no therapeutic efficacy (Kaplan-Meier method/log rank test: BCG, p = 0.0053; BCG + ASA, p = 0.0044; BCG + POF, p = 0.0027; aBCG, p = 0.33). No significant differences among the 3 groups treated with viable BCG, with or without anti-inflammatory drugs, regarding bladder weight and survival were detectable. The efficacy of BCG therapy in murine orthotopic bladder cancer is dependent on BCG viability and is not compromised by ASA or POF. Clinical trials to evaluate the efficacy of routine ASA or POF to reduce BCG side effects in patients, using self-assessment criteria, should be initiated.

The Effect of Lubricants on Viability of Bacillus Calmette-Guerin for Intravesical Immunotherapy Against Bladder Carcinoma

Purpose The viability of bacillus Calmette-Guerin (BCG) is crucial for induction of a local immune response and for effective therapy of recurrent superficial bladder carcinoma. During intravesical instillation of BCG lubricants are administered to assist catheterization, which contain bacteriostatic components that may interfere with the viability of mycobacteria. To verify this assumption, 5 commercially available lubricants were analyzed with regard to inhibition of viable BCG growth. Materials and Methods Five different lubricants and their components were co-incubated with Connaught strain BCG and the resultant growth of BCG was assessed. To prove the significant passage of lubricants into the bladder, fluid was recovered from the bladder after catheterization, analyzed with regard to the bacteriostatic effect and compared to normal urine of different acidity. Results Significant impairment of BCG viability, dependent on dosage and time of co-incubation, was noted with all lubricants analyzed. Several components, namely lidocaine hydrochloride, glyceryl stearate, propyl-4-hydroxy-benzoate and chlorhexidine digluconate, were identified as responsible for this inhibition. Fluid recovered from the bladder after lubricant assisted catheterization also showed an inhibitory effect, indicating significant mixture of the instillate with lubricants. Conclusions Generous use of lubricants to assist catheterization during intravesical BCG therapy will result in a clinically significant decrease in the number of intravesically instilled viable mycobacteria. For this reason, during intravesical immunotherapy with BCG only small amounts of lubricants should be used for urethral catheterization, and use of catheters not requiring lubricants should be considered.

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 × 105 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.

Immunoprophylaxis of a Murine Bladder Cancer with High Dose Bcg Immunizations

AbstractThe immunoprophylactic effect of Mycobacterium bovis, strain Bacillus Calmette-Guerin (BCG) was studied in a syngeneic bladder tumor system. Injection of a high dose of viable BCG 10 days before tumor cell challenge induced a 46 per cent reduction in tumor take. On the other hand, no protection against tumor take was afforded by the simultaneous administration of BCG and tumor cells. Complete tumor regression, however, was observed in a number of animals of the 2 groups receiving BCG: 90 per cent in the group receiving the vaccine and the tumor simultaneously and 50 per cent in those being immunized 10 days before tumor challenge. These findings indicate that high dose, viable BCG immunization favorably alters the course of this experimental tumor.

Glucan induced inhibition of tumor growth and enhancement of survival in a variety of transplantable and spontaneous murine tumor models.

In recent years, attempts have been made to alter the course of neoplasia by augmenting host defense mechanisms. A number of adjuvants including bacterial vaccines and chemical compounds have been demonstrated to produce a nonspecific stimulation of the reticuloendothelial (RE) system and augment humoral and cellular immune response. The most commonly employed agents are Bacillus Calmette-Guerin (BCG) and Corynebacterium parvum (16,22). The efficacy of such nonspecific immunotherapy, however, varies depending upon the time and mode of injection (4,29,31) as well as the type of malignancy (29,33). In view of the virulent and antigeneic nature of bacterial adjuvants such as BCG, it is not surprising that complications have been observed on occasion following the intratumoral or intradermal injections of these agents (22,32). Furthermore, there is the threat of disseminated infection following inoculation with viable organisms in patients with immunodeficiency states secondary to malignancy (22) or in immunosuppressed animals (25). Indeed, Sparks, et al. (35) reported that rats receiving isonicotinic acid hydrazine and BCG intralesionally had better survival and smaller tumor volumes than BCG alone, possibly by inhibiting diversion of host response to the viable BCG organism.KeywordsMelanoma CellPeritoneal MacrophageTumor WeightAnaplastic CarcinomaSyngeneic TumorThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.