Influence of Mycobacterium bovis bacillus Calmette-Guérin on antibody and cytokine responses to human neonatal vaccination.

The immaturity of the immune system increases the susceptibility of young infants to infectious diseases and prevents the induction of protective immune responses by vaccines. We previously reported that Mycobacterium bovis bacillus Calmette-Guérin (BCG) vaccination induces a potent Th1 response to mycobacterial Ags in newborns. In this study, we evaluated the influence of BCG on the response to unrelated vaccines given in early life. Newborns were randomly allocated to one of three study groups receiving BCG at birth, when infants received their first dose of hepatitis B and oral polio vaccines; at 2 mo of age, when infants received their first dose of diphtheria and tetanus vaccines; or at 4.5 mo of age, when immune responses to vaccines were measured. Administration of BCG at the time of priming markedly increased the cellular and Ab responses to the hepatitis B vaccine, but had only a limited influence on the cytokine response to tetanus toxoid and no effect on the Ab responses to tetanus and diphtheria toxoids. Although BCG induced a potent Th1-type response to mycobacterial Ags, it promoted the production of both Th1- and Th2-type cytokines in response to unrelated vaccines. The effect of BCG was apparent at the systemic level, as it increased the Ab response to oral polio vaccine. These results demonstrate that BCG influences the immune response to unrelated Ags in early life, likely through its influence on the maturation of dendritic cells.     

Bacillus Calmette-Guérin vaccination reduces the severity and progression of tuberculosis in badgers

Control of bovine tuberculosis (TB) in cattle has proven particularly challenging where reservoirs of infection exist in wildlife populations. In Britain and Ireland, control is hampered by a reservoir of infection in Eurasian badgers (Meles meles). Badger culling has positive and negative effects on bovine TB in cattle and is difficult, costly and controversial. Here we show that Bacillus Calmette-Guérin (BCG) vaccination of captive badgers reduced the progression, severity and excretion of Mycobacterium bovis infection after experimental challenge. In a clinical field study, BCG vaccination of free-living badgers reduced the incidence of positive serological test results by 73.8 per cent. In common with other species, BCG did not appear to prevent infection of badgers subjected to experimental challenge, but did significantly reduce the overall disease burden. BCG vaccination of badgers could comprise an important component of a comprehensive programme of measures to control bovine TB in cattle.     

Characterization of lung gamma delta T cells following intranasal infection with Mycobacterium bovis bacillus Calmette-Guérin

The lungs are considered to have an impaired capacity to contain infection by pathogenic mycobacteria, even in the presence of effective systemic immunity. In an attempt to understand the underlying cellular mechanisms, we characterized the gammadelta T cell population following intranasal infection with Mycobacterium bovis bacillus Calmette-Guérin (BCG). The peak of gammadelta T cell expansion at 7 days postinfection preceded the 30 day peak of alphabeta T cell expansion and bacterial count. The expanded population of gammadelta T cells in the lungs of BCG-infected mice represents an expansion of the resident Vgamma2 T cell subset as well as an influx of Vgamma1 and of four different Vdelta gene-bearing T cell subsets. The gammadelta T cells in the lungs of BCG-infected mice secreted IFN-gamma following in vitro stimulation with ionomycin and PMA and were cytotoxic against BCG-infected peritoneal macrophages as well as against the uninfected J774 macrophage cell line. The cytotoxicity was selectively blocked by anti-gammadelta TCR mAb and strontium ions, suggesting a granule-exocytosis killing pathway. Depletion of gammadelta T cells by injection of specific mAb had no effect on the subsequent developing CD4 T cell response in the lungs of BCG-infected mice, but significantly reduced cytotoxic activity and IFN-gamma production by lung CD8 T cells. Thus, gammadelta T cells in the lungs might help to control mycobacterial infection in the period between innate and classical adaptive immunity and may also play an important regulatory role in the subsequent onset of alphabeta T lymphocytes.     

Bacillus Calmette-Guérin Failures and Beyond: Contemporary Management of Non-Muscle-Invasive Bladder Cancer

In the United States, bacillus Calmette-Guérin (BCG) is the treatment most used for superficial bladder cancer. Patients with carcinoma in situ (CIS) treated with intravesical BCG plus interferon have a 60% to 70% chance of a complete and durable response if they were never treated with BCG or if they failed only 1 prior induction or relapsed more than a year from induction. Intravesical gemcitabine is safe, but its usefulness for BCG-refractory patients is unclear. Valrubicin, approved for intravesical treatment of BCG-refractory CIS of the bladder, has efficacy and acceptable toxicity. Cystectomy should be considered in high-risk, non-muscle-invasive cancer, particularly if intravesical therapy failed.Key words: Bladder cancer, Bacillus Calmette-Guérin, Cystectomy, Gemcitabine, ValrubicinThe management of superficial bladder cancer requires a clear understanding of diagnostic, prognostic, and treatment parameters. Cystoscopy remains the gold standard for detection, but despite good visualization and resection, bladder cancers recur frequently. Because of this, a variety of drugs has been used intravesically. The most commonly used drug in the United States is bacillus Calmette-Guérin (BCG), both with and without interferon and mitomycin.

Antiproliferative effects of bacillus Calmette-Guérin and interferon α2b on human bladder cancer cells in vitro

Direct inhibitory effects of bacillus Calmette-Guérin (BCG) and interferon 2b (IFN2b) on six human bladder carcinoma cell lines, UCRU-BL-13, UCRU-BL-17, UCRU-BL-28, 5637, T24 and J82, were studied using an in vitro proliferation assay. Effects on proliferation following exposure to BCG or IFN2b were analysed by [³H]thymidine incorporation over 7 days. BCG had an antiproliferative effect on all bladder lines, while sensitivity to IFN2b varied greatly, being as remarkably low as 1 U/ml for some lines. The antiproliferative effect was greatest when cells were exposed continuously to either agent, but was still evident with a limited exposure. When clinical concentrations were simulated in vitro, BCG+IFN2b was more effective than BCG alone and as effective as a double BCG concentration. We conclude that, in addition to their immunomodulatory effects, BCG and IFN2b directly inhibit the proliferation of human bladder cancer cells, and often at extremely low concentrations.     

Association of polymorphisms in oxidative stress genes with clinical outcomes for bladder cancer treated with Bacillus Calmette-Guérin

Genetic polymorphisms in oxidative stress pathway genes may contribute to carcinogenesis, disease recurrence, treatment response, and clinical outcomes. We applied a pathway-based approach to determine the effects of multiple single nucleotide polymorphisms (SNPs) within this pathway on clinical outcomes in non-muscle-invasive bladder cancer (NMIBC) patients treated with Bacillus Calmette-Guérin (BCG). We genotyped 276 SNPs in 38 genes and evaluated their associations with clinical outcomes in 421 NMIBC patients. Twenty-eight SNPs were associated with recurrence in the BCG-treated group (P<0.05). Six SNPs, including five in NEIL2 gene from the overall and BCG group remained significantly associated with recurrence after multiple comparison adjustments (q<0.1). Cumulative unfavorable genotype analysis showed that the risk of recurrence increased with increasing number of unfavorable genotypes. In the analysis of risk factors associated with progression to disease, rs3890995 in UNG, remained significant after adjustment for multiple comparison (q<0.1). These results support the hypothesis that genetic variations in host oxidative stress genes in NMIBC patients may affect response to therapy with BCG.     

A comparative analysis of polyfunctional T cells and secreted cytokines induced by Bacille Calmette-Guérin immunisation in children and adults

BCG vaccine is one of the most commonly-administered vaccines worldwide. Studies suggest the protective efficacy of BCG against TB is better for children than for adults. One potential explanation is that BCG induces a better protective immune response in children. Twenty six children and adults were immunised with BCG. The proportion of Th1-cytokine-producing mycobacterial-specific T cells, and the concentrations of secreted cytokines, were measured before and 10 weeks after BCG immunisation. A significant increase in the proportion of mycobacterial-specific cytokine-producing T cells was observed in both age groups. After BCG immunisation, children and adults had comparable proportions of mycobacterial-specific polyfunctional CD4 T cells when measured relative to the total number of CD4 T cells. However, relative to the subset of Th-1-cytokine-producing CD4 T cells, the proportion of polyfunctional cells was greater in children. Concentrations of secreted cytokines were comparable in children and adults. These findings suggest that the mycobacterial-specific cell-mediated immune response induced by BCG immunisation in children and adults is similar. The implication of a shift to a more polyfunctional immune response within the Th1-cytokine-producing CD4 T cells in children is uncertain as this aspect of the immune response has not been assessed as a potential correlate of protection against TB.     

Effect of Bacillus Calmette-Guérin infection on delayed footpad reaction to Listeria monocytogenes

The role of peritoneal macrophages induced by Bacillus Calmette-Guérin (BCG) in the induction of immune responses to Listeria monocytogenes was studied in mice. The peritoneal macrophages from mice treated with BCG 14 days previously contained a high proportion of Ia-bearing macrophages (approximately 56%) and the cells showed not only a high level of listericidal activity but also a strong ability for presentation of listerial antigen to Listeria-immune T cells. An intraperitoneal inoculation with a low dose of Listeria, which can induce the maximal level of delayed footpad reaction (DFR) and positive migration inhibitory activity of macrophages in untreated mice, did not induce a detectable level of such responses in BCG-treated mice. The bacterial growth at an early stage of infection was suppressed by scavenger macrophages in these mice. On the other hand, BCG-treated mice showed the early development of DFR and macrophage migration inhibitory activity after an inoculation with a high dose of Listeria. It is revealed in transfer experiments that Listeria-pulsed peritoneal exudate cells induced by BCG elicited the highest level of DFR and positive migration inhibition of macrophages in normal mice at the earlier period of injection compared with Listeria-pulsed resident peritoneal cells. These results suggested that the increased activities of macrophages acting as scavenger cells and as antigen-presenting cells play important roles in the modification of immune responses to Listeria in BCG-treated mice.     

Cytokine-mediated antitumor effect of bacillus Calmette-Guérin on tumor cells in vitro

Intravesical instillation therapy of bacillus Calmette-Guérin (BCG) is a useful modality for recurrent superficial transitional-cell carcinoma (TCC) of the urinary bladder. The mechanism of BCG effect has not yet been well characterized. BCG was tested in vitro for cytokine-mediated antiproliferative activity against T24 and KK47 cells (cell lines established from human TCC of the urinary bladder), and ACHN cells (cell line established from human renal cell carcinoma) using a modified human tumor clonogenic assay. Continuous exposure of cells to BCG at concentrations of more than 5 g/ml in the presence of peripheral blood mononuclear cells (PBMC) consisting of a mixture of 5×10⁴ monocytes/dish and 5×10⁵ lymphocytes/dish, obtained from healthy donors, significantly inhibited colony formation of T24 and ACHN cells in comparison with growth inhibition in the absence of PBMC (P<0.05). Slightly inhibited colony formation was observed with KK47 cells under the same conditions. At the same time various cytokines were measured in supernatants when BCG and the same conditioned PBMC were co-cultured. Tumor necrosis factor (TNF) and interleukin-1 (IL-1) were detected at markedly high levels at 24 h, and interferon (IFN) was detected at 120 h. IL-2 and macrophage-colony-stimulating factor were not detected. Neutralizing anti-TNF monoclonal antibody significantly reduced the anti-proliferative activity of ACHN cells, and anti-IFN antibody reduced that of T24 cells. The results obtained suggest that cytokines mediated by BCG play an important role in the antitumor activity of BCG and that the sensitivity of bladder cancer cells to the cytokines induced by BCG may differ considerably.     

Bacillus Calmette-Guérin and TLR4 agonist prevent cardiovascular hypertrophy and fibrosis by regulating immune microenvironment

Hypertension-induced cardiovascular hypertrophy and fibrosis are critical in the development of heart failure. The activity of TLRs has been found to be involved in the development of pressure overload-induced myocardial hypertrophy and cardiac fibrosis. We wondered whether vaccine bacillus Calmette-Guérin (BCG), which activated TLR4 to elicit immune responses, modulated the pressure overload-stimulated cardiovascular hypertrophy and cardiac fibrosis in the murine models of abdominal aortic constriction (AAC)-induced hypertension. Before or after AAC, animals received BCG, TLR4 agonist, IFN-gamma, or TLR4 antagonist i.p. BCG and TLR4 agonist significantly prevented AAC-induced cardiovascular hypertrophy and reactive cardiac fibrosis with no changes in hemodynamics. Moreover, TLR4 antagonist reversed the BCG- and TLR4 agonist-induced actions of anti-cardiovascular hypertrophy and cardiac fibrosis. BCG decreased the expression of TLR2 or TLR4 on the heart tissue but TLR4 agonist increased the expression of TLR2 or TLR4 on the immune cells that infiltrate into the heart tissue. This led to an increased expression ratio of IFN-gamma/TGF-beta in the heart. The cardiac protective effects of BCG and TLR4 agonist are related to their regulation of ERK-Akt and p38-NF-kappaB signal pathways in the heart. In conclusion, the activity of TLR4 plays a critical role in the mediation of pressure overload-induced myocardial hypertrophy and fibrosis. The regulation of immune responses by BCG and TLR4 agonist has a great potential for the prevention and treatment of hypertension-induced myocardial hypertrophy and cardiac fibrosis.     

Genomic Analysis of a Mycobacterium Bovis Bacillus Calmette-Guérin Strain Isolated from an Adult Patient with Pulmonary Tuberculosis

For years, bacillus Calmette-Guérin (BCG) has served as the unique vaccine against tuberculosis and has generally been regarded as safe. However, a clinical strain labeled 3281 that was isolated from a TB patient was identified to be BCG. Via the combination of next-generation sequencing (NGS) and comparative genomic analysis, unique 3281 genetic characteristics were revealed. A region containing the dnaA and dnaN genes that is closely related to the initial chromosome replication was found to repeat three times on the BCG Pasteur-specific tandem duplication region DU1. Due to the minimum number of epitopes in BCG strains, 3281 was inferred to have a high possibility for immune evasion. Additionally, variations in the virulence genes and predictions for potential virulence factors were analyzed. Overall, we report a pathogen that has never previously been thought to be pathogenic and initial insights that are focused on the genetic characteristics of virulent BCG.     

ATP-mediated killing of Mycobacterium bovis bacille Calmette-Guérin within human macrophages is calcium dependent and associated with the acidification of mycobacteria-containing phagosomes

We previously demonstrated that extracellular ATP stimulated macrophage death and mycobacterial killing within Mycobacterium bovis Bacille Calmette-Guérin (BCG)-infected human macrophages. ATP increases the cytosolic Ca(2+) concentration in macrophages by mobilizing intracellular Ca(2+) via G protein-coupled P2Y receptors, or promoting the influx of extracellular Ca(2+) via P2X purinoceptors. The relative contribution of these receptors and Ca(2+) sources to ATP-stimulated macrophage death and mycobacterial killing was investigated. We demonstrate that 1) ATP mobilizes Ca(2+) in UTP-desensitized macrophages (in Ca(2+)-free medium) and 2) UTP but not ATP fails to deplete the intracellular Ca(2+) store, suggesting that the pharmacological properties of ATP and UTP differ, and that a Ca(2+)-mobilizing P2Y purinoceptor in addition to the P2Y(2) subtype is expressed on human macrophages. ATP and the Ca(2+) ionophore, ionomycin, promoted macrophage death and BCG killing, but ionomycin-mediated macrophage death was inhibited whereas BCG killing was largely retained in Ca(2+)-free medium. Pretreatment of cells with thapsigargin (which depletes inositol (1,4,5)-trisphosphate-mobilizable intracellular stores) or 1,2-bis-(2-aminophenoxy)ethane-N, N, N',N'-tetraacetic acid acetoxymethyl ester (an intracellular Ca(2+) chelator) failed to inhibit ATP-stimulated macrophage death but blocked mycobacterial killing. Using the acidotropic molecular probe, 3-(2,4-dinitroanilino)-3'-amino-N-methyl dipropylamine, it was revealed that ATP stimulation promoted the acidification of BCG-containing phagosomes within human macrophages, and this effect was similarly dependent upon Ca(2+) mobilization from intracellular stores. We conclude that the cytotoxic and bactericidal effects of ATP can be uncoupled and that BCG killing is not the inevitable consequence of death of the host macrophage.     

Priming-boosting vaccination with recombinant Mycobacterium bovis bacillus Calmette-Guérin and a nonreplicating vaccinia virus recombinant leads to long-lasting and effective immunity

Virus-specific T-cell responses can limit immunodeficiency virus type 1 (HIV-1) transmission and prevent disease progression and so could serve as the basis for an affordable, safe, and effective vaccine in humans. To assess their potential for a vaccine, we used Mycobacterium bovis bacillus Calmette-Guérin (BCG)-Tokyo and a replication-deficient vaccinia virus strain (DIs) as vectors to express full-length gag from simian immunodeficiency viruses (SIVs) (rBCG-SIVgag and rDIsSIVgag). Cynomolgus macaques were vaccinated with either rBCG-SIVgag dermally as a single modality or in combination with rDIsSIVgag intravenously. When cynomologus macaques were primed with rBCG-SIVgag and then boosted with rDIsSIVgag, high levels of gamma interferon (IFN-gamma) spot-forming cells specific for SIV Gag were induced. This combination regimen elicited effective protective immunity against mucosal challenge with pathogenic simian-human immunodeficiency virus for the 1 year the macaques were under observation. Antigen-specific intracellular IFN-gamma activity was similarly induced in each of the macaques with the priming-boosting regimen. Other groups receiving the opposite combination or the single-modality vaccines were not effectively protected. These results suggest that a recombinant M. bovis BCG-based vector may have potential as an HIV/AIDS vaccine when administered in combination with a replication-deficient vaccinia virus DIs vector in a priming-boosting strategy.     

Is Bladder Tumor Location Associated with Prostate Cancer Detection after Intravesical Bacillus Calmette-Guérin Instillation?

Objectives

The aim of this study was to evaluate the effect of bladder tumor (BT) location on prostate cancer (PCa) detection in patients with elevated PSA levels after intravesical BCG instillation.

Methods

Between February 2004 and January 2013 prostate biopsies were performed in 59 non-muscle invasive bladder cancer (NMIBC) patients whose PSA level were elevated (≥3 ng/ml) after a 6 week course of intravesical BCG (Oncotice, 12.5 mg in 50 ml normal saline). Differences in PCa detection according to the BT location [bladder neck and/or trigone (Group 1, n = 22) vs. other locations (Group 2, n = 37)] were evaluated. The Fisher''s exact test and the Mann-Whitney U test were used to evaluate the association between categorical and continuous variables, respectively.

Results

A total of 14 patients (23.7%) were diagnosed with PCa. The mean ± standard deviation (SD) PSA before intravesical BCG instillation and prostate biopsy were 1.36±1.04 ng/ml in Group 1 and 1.09±1.12 ng/ml in Group 2 (P = 0.633), and 6.05±3.57 ng/ml in Group 1 and 5.13±3.88 ng/ml in Group 2 (P = 0.378), respectively. Interestingly, whereas PCa was detected upon biopsy in only one patient in Group 1 (4.5%), 13 cases were detected in Group 2 (35.1%) (P = 0.009).

Conclusions

PCa detection after intravesical BCG was highly associated with BT location. Prostate biopsy should therefore be considered when PSA level is elevated after BCG instillation and his BT is located far from the bladder neck.     

Effects of BCG (Bacillus Calmette-Guérin) vaccines on immune responses in mice. I. Possible effect of BCG on helper T cells.

The effects of killed and living BCG on antibody production against hamster erythrocytes (HRBC) and the 2, 4, 6-trinitrophenyl (TNP) group were studied in SL mice. Killed and living BCG, each in doses of 0.008 mg, 0.08 mg, 0.8 mg and 8 mg per mouse, were intravenously inoculated 7 days prior to primary immunization with HRBC. Secondary immunization was carried out 28 days later with TNP-HRBC. Anti-HRBC and anti-TNP antibodies were estimated by a hemagglutination test. The results showed that pretreatment with killed or living BCG enhanced the antibody production against both HRBC and TNP. Comparing the effects of these two BCG preparations, it was noted that killed BCG augmented the anti-HRBC antibody production more effectively than living BCG. In regard to the anti-TNP antibody production, living BCG exhibited a greater augmenting effect than killed BCG. This difference in the modes of action of killed and living BCG was remarkable when two groups given 8 mg of killed and living BCG were compared. In addition, it was shown that living BCG at a dose as high as 8 mg was able to augment the anti-TNP antibody production, even in the absence of preceding immunization with HRBC.     

Cytokine gene polymorphisms can alter the effect of Bacillus Calmette-Guérin (BCG) immunotherapy

Various types of cancer are more frequent in men than women, and bladder cancer is one of the most common of these. Intravesical instillation of Bacillus Calmette-Guérin (BCG) after transurethral resection is the most effective treatment for superficial bladder cancers. The main aim of this study was to investigate for possible links between cytokine gene polymorphisms and different outcomes after BCG immunotherapy. Sixty patients who had been diagnosed with transitional cell cancer were investigated. All genotyping experiments were performed using polymerase chain reaction sequence-specific primers and a commercially available kit. The genes investigated were those that code for interleukin (IL)-1alpha, IL-1beta, IL-1R, IL-1RA, IL-4RA, IL-2, IL-4, IL-6, IL-10, IL-12, interferon-gamma (IFN-gamma), transforming growth factor-beta (TGF-beta), and tumor necrosis factor-alpha (TNF-alpha). Analyses of the data identified TGF-beta codon 25 GG (92.85% vs. 64.44%, p=0.04, OR=7.17), IL-4 -1098 GG (16.6% vs. 0.0%, p=0.05, OR=18.33), IL-10 -1082 GG (28.5% vs. 6.8%, p=0.05, OR=5.47), and IL-10 -1082 GCC/GCC (28.57% vs. 4.5%, p=0.025, OR=8.4) polymorphisms as risk factors for progression of bladder cancer.