Presence of activated lymphocytes in the urine of patients with superficial bladder cancer after intravesical immunotherapy with bacillus Calmette-Guérin

Summary To study the mode of action of intravesical bacillus Calmette-Guérin (BCG) immunotherapy in the prevention and cure of superficial bladder cancer, flow-cytofluorometric analysis of the cellular immunological reaction in the urine of patients was performed. Fresh urinederived leucocytes were obtained from eight patients before (t ₀) and 24 h (t ₂₄) and 48 h (t ₄₈) after repeated intravesical BCG instillations (at least 5 instillations). For two patients urine-derived leucocytes were investigated at the first BCG instillation. The number of leucocytes in the urine was markedly increased 24 h after repeated BCG instillations, indicating a local cellular immunological reaction induced by BCG. The mean number of cells per milliliter of urine at that time was 2.9×10⁶±3.6×10⁶ (n = 8). These leucocytes consisted mainly of granulocytes (75±11%,n = 8). In addition monocytes/macrophages (4±2%,n = 8) and T lymphocytes were present (1±1%,n = 5). The relative increase of monocytes/macrophages in the urine after BCG application tended to be higher compared to the other leucocyte subtypes. As T lymphocytes may play an important role in the BCG-mediated antitumour activity, subsets of lymphocytes were further characterized att ₀,t ₂₄, andt ₄₈ after repeated BCG instillations. The lymphocyte population consisted mainly of T cells (86% CD3⁺,t ₀). Most of the T cells were CD4⁺ (helper/inducer) and were significantly decreased at 48 h (62±9% att ₀ vs 49±6% att ₄₈). Lymphocytes partly expressed HLA-DR antigens (44%,t ₀). The percentage of lymphocytes with interleukin-2 (IL-2) receptors (CD25⁺) was significantly increased at 24 h and 48 h, compared to pre-instillation values (19±11% and 10±4% vs 3±3% respectively). Natural killer cells (CD 16⁺ and/or CD56⁺) and B cells (CD 19⁺) were less numerous (10% and 19% att ₀ respectively). After the first BCG instillation the increase in the number of leucocytes in urine seemed to be less compared to the numbers after repeated BCG instillations. Lymphocytes could not be detected in the urine collected before or after the first BCG instillation. In conclusion, we demonstrated the presence of considerable numbers of leucocytes in the urine 24 h after repeated BCG instillations, i.e. shortly after immunological activation. The antigen expression of the lymphocytes suggested that they may represent the lymphocytes in the bladder wall. Expression of HLA-DR and IL-2 receptors on lymphocytes indicated activation of T cells by the intravesical BCG treatment. These leucocytes may be useful for functional studies, which are essential to elucidate the actual effector mechanism(s) in the mode of action of BCG against superficial bladder cancer in man.     

Immune reactions in patients with superficial bladder cancer after intradermal and intravesical treatment with bacillus Calmette-Guérin

Summary The immune reactivity of patients with strongly recurrent superficial bladder cancer was followed after combined intravesical and intradermal bacillus Calmette-Guérin (BCG) immunotherapy. All patients in this study were previously treated without success with intravesical chemotherapy. The BCG treatment regimen consisted of weekly administrations with BCG (RIVM) for six consecutive weeks, both intravesically and intradermally. In this study, sera and peripheral blood leukocytes (PBL) of patients were tested serially. Besides BCG-antigen-specific reactions, e.g. skin reactivity to purified protein derivatives of Mycobacterium tuberculosis (PPD), antibody formation and antigen stimulation of PBL in vitro, non-antigen-specific immune reactivities were also measured, e.g. mitogen response and spontaneous cytotoxic activity of PBL. In addition the antibody response to bladder carcinoma antigens and the cytotoxic activity of PBL for the bladder carcinoma cell line T24 and the natural-killer-sensitive K562 cell line were investigated. The results obtained from the various assays were evaluated for their prognostic value in relation to the length of the tumor-free interval after the BCG treatment. Because sera and PBL were only obtained during the first 6 months after the BCG treatment, the immune reactivity was compared to the clinical results at that same time. At 6 months after therapy 12 out of 40 BCG-treated patients were tumor-free whereas 28 out of 40 showed a recurrence. Skin reactivity to tuberculin PPD was measured in 40 patients during a period of 3–6 months after therapy. Of patients who showed a recurrence of the tumor within 6 months, 48% of them showed a transient response or developed no response at all to PPD. In the group of patients with a longer tumor-free period (n=10), only one patient lost the response to tuberculin PPD. Although PBL of a limited number of patients were tested, it was observed that the cytotoxicity to the bladder carcinoma cell line T24, and the natural-killer-sensitive K562 cell line increased in a number of the patients (7 out of 14, and 9 out of 14 respectively). Reactivity of PBL to mitogens and subset distribution (ratio T-helper: T-suppressor/cytotoxic) were not influenced by the BCG treatment. Antibody response to mycobacterial antigen was detected in 9 out of 23 patients investigated. Of these 9 patients, 8 belonged to the group with a recurrence of the tumor within 6 months (n=17). There was no correlation between the skin reactivity and the antibody response to tuberculin PPD. Furthermore, none of the 25 patients showed an antibody response to bladder carcinoma antigens. Sera of bladder carcinoma patients (n=19) reduced the mitogen-induced proliferation of lymphocytes, compared to sera of healthy controls (n=13), indicating the presence of circulating suppressor factor(s). Our results indicate that the absence of a Mantoux conversion or the presence of transient reaction to tuberculin PPD were highly related (91%) to a relapse of the disease. On the other hand, the cytotoxic activity of PBL to T24 and K562 cell lines, or their reactivity to tuberculin PPD or mitogens, gives no predictive information about the clinical results (tumor-free interval) of the BCG therapy. Abbreviations used: BCG, bacillus Calmette-Guérin; NK, natural killer; PBL, peripheral blood leukocytes; PPD, purified protein derivative of Mycobacterium tuberculosis; ELISA, enzyme-linked immunosorbent assay     

DNA ploidy and immunomarking of bladder urothelial tumors before and after intravesical bacillus Calmette-Guérin treatment

OBJECTIVE: To investigate DNA ploidy and immunoexpression of Ki-67 and p53 as predictivefactors in cases of superficial urothelial cell carcinoma (UCC) treated with bacillus Calmette-Guérin (BCG). STUDY DESIGN: Samples were obtained from 66 patients with UCC (pTa grade 3 or high grade and pT1 independent of grade or with concomitant carcinoma in situ) before and after intravesical BCG treatment. DNA ploidy analysis (ploidy balance, degree of hyperploidy and aneuploidy, proliferation index) was done by static cytometry. Ki-67 and p53 were analyzed immunohistochemically in paraffin-embedded tissue, and their quantification was carried out using an image analysis system. RESULTS: During a mean follow-up of 63.8 months, 31 of the 66 patients developed recurrent tumors (46.9%). DNA ploidy analysis showed that ploidy balance as well as degree of hyperploidy and aneuploidy were not statistically different between recurrent and nonrecurrent tumors. Only proliferation index was statistically significant between recurrent and nonrecurrent tumors. No statistically significant difference was observed in the percentage of Ki-67- and p53-positive cells between primary tumors that recurred and those that did not. CONCLUSION: These findings suggest that only proliferation index has predictive value for recurrence and progression in UCC treated with BCG.     

Urinary interleukin-2 may predict clinical outcome of intravesical bacillus Calmette-Guérin immunotherapy for carcinoma in situ of the bladder

PURPOSE: The mechanism by which bacillus Calmette-Guérin (BCG) mediates antitumor activity has not been clearly established. Specific cytokines in the urine after BCG intravesical instillation therapy may serve as a prognostic factor of treatment response. In this study, various urinary cytokines such as interleukin-1beta (IL-1beta), IL-2, IL-6, IL-8. IL-10, IL-12, interferon-gamma (IFN-gamma), and tumor necrosis factor-alpha (TNF-alpha) were measured. MATERIALS AND METHODS: In total 20 patients were treated with BCG intravesical instillation therapy for carcinoma in situ of the bladder. At the completion of the first and eighth instillations, spontaneously voided urine specimens were collected before BCG instillation, every 2 h until 12 h, and thereafter until 24 h. All specimens were ultrafiltrated using an ADVANTEC UK-10 membrane. The cytokines were measured using ELISA and RIA techniques. RESULTS: Significantly higher levels of IL-2, IL-6, IL-8, IL-10, IFN-gamma, and TNF-alpha were detected in the eighth instillation as compared to the first instillation ( p<0.001). After BCG intravesical instillation therapy, treatment failure occurred in 6 of the 20 patients (30%), including primary failure (persistence of CIS) in 3, and de novo failure (tumor recurrence) in 3 with a median follow-up of 46.9 months. Significantly higher production of IL-2, IL-6, IL-8, IL-10, and TNF-alpha was observed in the responder group than in the non-responder group ( p<0.05). Multivariate analysis revealed IL-2 as an independent prognostic cytokine of responder status. CONCLUSIONS: This study indicates that urinary IL-2 at the eighth instillation of BCG may serve as a valuable prognostic factor of treatment efficacy as well as tumor recurrence after treatment.     

Induction of urinary interleukin-1 (IL-1), IL-2, IL-6, and tumour necrosis factor during intravesical immunotherapy with bacillus Calmette-Guérin in superficial bladder cancer

Summary To study the local immunological effects of intravesical bacillus Calmette-Guérin (BCG) therapy in superficial bladder cancer patients, the production of interleukin-1 (IL-1), IL-2, IL-6, tumour necrosis factor (TNF), and interferon (IFN) was investigated in the urine. Urine specimens were collected during the six weekly BCG instillations, before instillation, and 2, 4, 6, 8, and 24 h thereafter. Results were standardized to urine creatinine. In general, the concentration of IL-1 increased markedly during the first three BCG instillations, reaching a plateau from instillations 3 to 6. IL-2 was not detected after the first BCG instillation, but from the second instillation onwards the mean IL-2 concentration increased rapidly. With respect to IL-6, patients had relatively high levels in the urine after the first BCG instillation. A relatively moderate increase of the IL-6 concentration was observed during the following weeks. Like IL-2, TNF was only detected after repeated BCG instillations. Generally the highest TNF levels were found after BCG instillation 5. The presence of IFN could not be demonstrated. With respect to the occurrence of the cytokines during the first 24 h after the BCG instillation, TNF, IL-2, and IL-6 were detectable 2 h after the instillation. In contrast, IL-1 seemed to appear later, i.e. from 4 h onwards. TNF decreased most rapidly; it was nearly absent in 6-h samples. Generally IL-2 was not detectable in the 8-h samples, whereas IL-1 and IL-6 were present up to 8 h after instillation of BCG. The presence of TNF was found less frequently than the presence of IL-1, IL-2, and IL-6. Neutralization experiments indicated that most of the IL-1 present in the urine after BCG treatment was IL-1. In conclusion, activation of BCG-specific T cells was indicated by the detection of IL-2. The presence of IL-1, IL-6, and TNF might suggest activation of macrophages by intravesically administered BCG, although production by other cell types cannot be excluded. It is suggested that these cytokines, in combination with the leucocytes that are known to be recruited to the bladder in reaction to the BCG treatment, may play an important role in the antitumour activity of BCG against bladder cancer. For monitoring purposes, collection of urine might be performed during the first 6 h after BCG instillations 4–6. A correlation between the presence of cytokines in the urine and the clinical response has yet to be evaluated.     

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.     

Bacillus Calmette-Guérin potentiates monocyte responses to lipopolysaccharide-induced tumor necrosis factor and interleukin-1, but not interleukin-6 in bladder cancer patients

During the past decade, particular attention has been focused on treatment of bladder cancer patients with the bacterial agent bacillus Calmette-Guérin (BCG). In these studies, bladder cancer patients were instilled with BCG (75mg/50ml) once per week for 6 weeks, 1–2 weeks following trans-urethreal resection of the bladder. Cystoscopy was performed after 6 weeks and, unless tumor progression was present, monthly treatments were given for 1 year. Blood was drawn 2 h after the last instillation, and monocytes were isolated (5×10⁶ cells/ml) and treated, or not, with lipopolysaccharide (LPS) (20 g/ml) for tumor necrosis factor (TNF), interleukin-1 (IL-1) and interleukin-6 (IL-6) release. The levels of monokines were determined by a monokine-specific enzyme-linked immunosorbent assay. Out results clearly show that, after 18 h incubation, macrophages from BCG-treated bladder cancer patients produced from 2.8- to 1.9-fold and from 2.0- to 1.3-fold greater amounts of TNF and IL-1 respectively, compared to macrophages from healthy controls, 5-fold higher than bladder cancer patients not treated with BCG. IL-6 was not affected. In another set of experiments macrophages (5×10⁶ cells/ml) from healthy subjects were pretreated, or not, with BCG (100 g/ml) overnight and treated, or not, with LPS 20 g/ml alone and in combination with interleukin-1 receptor antagonist (IL-1ra) 250 ng/ml. Macrophages treated with BCG had a strong stimulatory effect on IL-1 release (9.45 ng/ml) while LPS was less effective (3.59 ng/ml). The combination of BCG plus LPS produced an additive effect on IL-1 release (13.71 ng/ml) compared to the effect of the compound alone. The addition of IL-1ra (250 ng/ml) to BCG was not effective, while when IL-1ra was added to BCG plus LPS only a partial inhibition of IL-1 release was found (9.83 ng/ml), compared to BCG plus LPS without IL-1ra (13.71 ng/ml). These effects seem to be related to the inhibition of IL-1 stimulated with LPS, but not BCG. The priming effect of BCG exerted on LPS-stimulated monocyte production of TNF and IL-1 from bladder cancer patients led us to study the possible modulation of fibrinogen and C-reactive protein in the serum of BCG-treated cancer patients. The plasma levels of fibrinogen and C-reactive protein were higher (approximately twice) in BCG-treated patients compared to values obtained in untreated patients or healthy controls. We conclude that the beneficial immunotherapeutic effects of BCG in bladder cancer patients are related to its capacity to prime macrophages to enhance the release of TNF and IL-1, but not IL-6 in response to physiological secondary stimuli, or through the direct stimulation of BCG on IL-1 or TNF, which are directly involved in the killing of cancer cells. Moreover, the increase of IL-1 or TNF in BCG bladder cancer patients may lead to high plasma levels of fibrinogen and C-reactive protein, two proteins responsible for the acute-phase response.     

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.     

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.     

Membrane subproteomic analysis of Mycobacterium bovis bacillus Calmette-Guérin

Mycobacterium bovis bacillus Calmette-Guérin (BCG) vaccine has been known for a long time to prevent tuberculosis (TB) worldwide since 1921. Nonetheless, we know little about BCG membrane proteome. In the present study, we utilized alkaline incubation and Triton X-114-based methods to enrich BCG membrane proteins and subsequently digested them using proteolytic enzyme. The recovered peptides were further separated by 2-D LC and identified by ESI-MS/MS. As a result, total 474 proteins were identified, including 78 integral membrane proteins (IMPs). Notably, 18 BCG IMPs were described for the first time in mycobacterium. Further analysis of the 78 IMPs indicated that the theoretical molecular mass distribution of them ranged from 8.06 to 167.86 kDa and pI scores ranged from 4.40 to 11.60. Functional classification revealed that a large proportion of the identified IMPs (67.9%, 53 out of 78) were involved in cell wall and cell processes functional group. In conclusion, here we reported a comprehensive profile of the BCG membrane subproteome. The present investigation may allow the identification of some valuable vaccine and drug target candidates and thus provide basement for future designing of preventive, diagnostic, and therapeutic strategies against TB.     

Functional transitions in macrophages during in vivo infection with Mycobacterium bovis bacillus Calmette-Guérin

Macrophage activation during the immune response to intracellular bacteria is critical for resolution of the infection. We have investigated the pathway of macrophage activation during murine Mycobacterium bovis bacillus Calmette-Guérin (BCG) infection. Three distinct phenotypes of macrophages were identified and compared: resident peritoneal macrophages, day 2 postinfection macrophages, and 12-day postinfection macrophages. Compared with resident peritoneal macrophages, day 2 BCG macrophages expressed intermediate levels of the cell surface receptors Mac1 and F4/80 and low levels of MHC class II molecules. These cells were highly phagocytic and produced large amounts of mRNA encoding the chemokine IP-10. In addition, day 2 BCG macrophages did not generate reactive nitrogen intermediates, though they were primed to do so, and did not have increased levels of TNF-alpha mRNA. Blockade of monocyte influx into the peritoneal cavity using Abs to platelet endothelial cell adhesion molecule 1 had no effect on the appearance of day 2 BCG macrophages, suggesting this cell can differentiate from resident peritoneal macrophages. In contrast to day 2 BCG macrophages, day 12 BCG macrophages were poorly phagocytic, but produced high levels of reactive nitrogen intermediates, IP-10 and TNF-alpha mRNA, and class II MHC molecules. We propose that day 2 BCG macrophages are specialized for phagocytic uptake of pathogens from the extracellular space, whereas day 12 BCG macrophages are specialized for killing of the internalized pathogens. This functional transition during activation is reminiscent of that seen during maturation/activation of the related dendritic cell lineage induced by bacterial or inflammatory stimuli.     

Recombinant bacillus Calmette-Guérin (BCG) expressing interferon-alpha 2B enhances human mononuclear cell cytotoxicity against bladder cancer cell lines in vitro

Purpose  The proper induction of cellular immunity is required for effective bacillus Calmette-Guérin (BCG) immunotherapy of bladder cancer. It has been known that BCG stimulation of human peripheral blood mononuclear cells (PBMC) leads to the generation of effector cells cytotoxic to bladder cancer cells in vitro. To improve BCG therapy, we previously developed human interferon (IFN)-α 2B secreting recombinant (r) BCG (rBCG-IFN-α). We demonstrated that rBCG-IFN-α augmented T helper type 1 (Th1) cytokine IFN-γ production by PBMC. In this study, we further investigated whether rBCG-IFN-α could also enhance PBMC cytotoxicity toward bladder cancer cells. Materials and methods  PBMC were prepared from healthy individuals, left alone or stimulated with rBCG-IFN-α or control MV261 BCG, and used as effector cells in ⁵¹Cr-release assays. Human bladder cancer cell lines T24, J82, 5637, TCCSUP, and UMUC-3 were used as target cells. To determine the role of secreted rIFN-α as well as endogenously expressed IFN-γ and IL-2 in inducing the cytotoxicity, PBMC were stimulated with rBCG-IFN-α in the presence of neutralizing antibodies to IFN-α, IFN-γ or IL-2. To determine the role of natural killer (NK) and CD8⁺ T cells in inducing the cytotoxicity, both cell types were isolated after BCG stimulation of PBMC and used as effector cells in ⁵¹Cr-release assays. Results  Non-stimulated PBMC showed basal levels of cytotoxicity against all target cell lines tested. MV261 BCG increased the PBMC cytotoxicity by 1.8- to 4.2-fold. rBCG-IFN-α further increased the PBMC cytotoxicity by up to 2-fold. Elevated production of IFN-γ and IL-2 by PBMC was observed after rBCG-IFN-α stimulation. Blockage of IFN-α, IFN-γ or IL-2 by neutralizing antibodies during rBCG-IFN-α stimulation reduced or abolished the induction of PBMC cytotoxicity. Both NK and CD8⁺ T cells were found to be responsible for the enhanced PBMC cytotoxicity induced by rBCG-IFN-α with the former cell type being more predominant. Conclusions  rBCG-IFN-α is an improved BCG agent that induces enhanced PBMC cytotoxicity against bladder cancer cells in vitro. This rBCG strain may serve as an alternative to BCG for the treatment of superficial bladder cancer.     

Identification of a novel DNA-binding protein from Mycobacterium bovis bacillus Calmette-Guérin

A novel DNA-binding protein expressed (8-10% in total protein) in Mycobacterium bovis bacillus Calmette-Guérin was observed. This protein was designated mycobacterial DNA-binding protein 1 (MDP1). MDP1 recognized bases, sugar moieties, phosphate-backbone on DNA and preferentially bound to DNA guanine and cytosine. In the gel retardation assay, MDP1 preferentially bound to closed circular plasmid DNA than open circular and linear form plasmid DNA and also bound to RNA. MDP1 formed a highly polymerized structure and localized not only in the nucleoid but also at the 50S ribosomal subunits and cell surface. MDP1 was conserved in Mycobacterium thus far examined and the expression was enhanced in stationary growth phases. These results will provide a reasonable basis for further study of the function of MDP1 in living mycobacteria.     

The significance of conversion of skin reactivity to efficacy of bacillus Calmette-Guérin (BCG) vaccinations given immediately after radical surgery in stage II melanoma patients

Summary A group of 668 stage II melanoma patients was entered into a randomized prospective study aimed at evaluating the efficacy of adjuvant BCG, 5-(dimethyltriazeno)imidazole-4-carboxamide (DTIC), or a combination of the two, given immediately after radical lymph node dissection. Of these, 176 patients received BCG and 164 BCG plus DTIC. These 340 patients had histologically proven metastatic nodes and 156 had a negative skin reactivity to BCG at the beginning of treatment. The distribution of known prognostic factors (sex, age, number of positive nodes, extracapsular invasion) was balanced in the groups of patients either with initially negative or with positive skin reactivity. All patients who were initially non-reactive to BCG developed skin reactivity after 6.7±9 BCG vaccinations. Disease-free and overall survival of patients receiving BCG or BCG + DTIC with an initially negative skin reactivity to BCG was significantly (P=7×10^–3) better than that observed in patients with an initial positive skin reactivity. This finding was still evident after adjustment for other known prognostic criteria (P=0.02). It seems likely that the initial BCG skin reactivity as such marks the prognosis; however, some therapeutic effect of BCG treatment in patients having initially no skin reactivity to BCG, can not be ruled out.This study has been partially funded by the Associazione Italiana per la Ricerca sul Cancro, and by a grant (NO1-CM-43726) from the National Cancer Institute of Bethesda, USAWriting committee of the W. H. O. Melanoma Programme (Chairman: U. Veronesi). The following researchers also took part in the study and are co-authors of this paper: J. Adamus, Oncological Institute, Gliwice (Poland); C. Aubert, Institut J. Paoli Calmettes, Marseille (France); E. Bajetta, G. Beretta, Istituto Nazionale per lo Studio e la Cura dei Tumori, Milan (Italy); G. Cocconi, Centro Medico Oncologico, Ospedali Riuniti, Parma (Italy); J. Durand, Fondation Curie, Paris (France); J. De Marsillac, Instituto Nacional de Cancer, Rio de Janeiro (Brasil); R. L. Ikonopisov, Dr. T. Tsanov, Oncological Research Institute, Sofia (Bulgaria); B. Kiss, State Institute of Oncology, Budapest (Hungary); F. Lejeune, Institut Jules Bordet, Brussels (Belgium); G. Madej, Oncological Institute, Warsaw (Poland); H. Mulder, Rotterdamsch Radiotherapeutisch Instituut, Rotterdam (The Netherlands); Z. Mechl, Oncological Institute, Brno (Czechoslovakia); G. W. Milton, Sydney Hospital, Sydney (Australia); H. Peter, P. von Wussow, Abteilung für Klinische Immunologie, Medizinische Hochschule, Hannover (FRG); J. Priario, Hospital de Clinicas M. Quintela, Montevideo (Uruguay); E. Paul, Abteilung für Klinische und Experimentelle Dermatologie, Giessen (FRG); R. Sertoli, Istituto di Oncologia, Genoa (Italy); R. Tomin, Institute of Oncology, Belgrade (Yugoslavia)     

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.     

Role of apoptosis-regulating signal kinase 1 in innate immune responses by Mycobacterium bovis bacillus Calmette-Guérin

Mycobacterium bovis bacillus Calmette-Guérin (BCG) induces innate immune responses through Toll-like receptor (TLR) 2 and TLR4. We investigated the role of apoptosis-regulating signal kinase (ASK) 1 in reactive oxygen species (ROS)-mediated innate immune responses induced by BCG mycobacterial infection. In macrophages, M. bovis BCG stimulation resulted in rapid activation of mitogen-activated protein kinases (MAPKs), secretion of inflammatory cytokines, such as tumor necrosis factor (TNF)-alpha and interleukin (IL)-6, and ROS generation in a TLR2- and TLR4-dependent manner. M. bovis BCG-induced ROS production led to robust activation of ASK1 upstream of the c-jun-N-terminal kinase and p38 MAPK, but not extracellular-regulated kinase 1/2. Blocking ASK1 activity markedly attenuated M. bovis BCG-induced TNF-alpha and IL-6 production by macrophages. Both TLR2 and TLR4 were required for optimal activation of ASK1 in response to M. bovis BCG. Furthermore, we present evidence that TNF receptor-associated factor (TRAF) 6 activities were essential for ROS-mediated ASK1 activation by M. bovis BCG. Finally, ASK1 activities were required for effective control of intracellular mycobacterial survival. Thus, the results of this study suggest a novel role of the TLR-ROS-TRAF6-ASK1 axis in the innate immune response to mycobacteria as a signaling intermediate.     

Major histocompatibility complex class II antigen expression during potentiation of line-10 tumor immunity after intralesional administration of bacillus Calmette-Guérin

Summary Intralesional injection of BCG into an established line-10 hepatocellular carcinoma in the strain-2 guinea pig causes regression of the tumor and induction of line-10 immunity. We found that the animals were already protected for a second challenge with line-10 tumor cells 7 days after BCG treatment. We studied whether this early induction of immunity was correlated with the expression of MHC class II antigens on line-10 tumor cells and was correlated with an increased expression of MHC class II antigens on leukocytes in the primary tumor and in the regional lymph node (Ln. axillaris accessorius). The MHC class II antigens and the leukocyte subpopulations were measured with monoclonal antibodies and flow cytofluorometry. In the draining lymph node the number of nucleated cells increased about 10-fold during the first 5 days after intralesional injection of BCG. At this time the MHC class II antigen expression of these cells was increased from 21%–32% in the naive controls to 39%–53% in animals with BCG-treated tumors. This implies that the number of MHC-class-II-positive cells increased about 20-fold in the draining lymph node. Surprisingly, the increase in percentage of MHC-class-II-antigen-positive cells was mainly due to an increase of IgM-positive B cells from 8%–11% to 22%–41% and an increase of IgG-positive B cells from 7%–27% to 25%–44%. In the tumor, BCG treatment induced a small increase of MHC-class-II-antigen-positive cells from 11%–12% to 15%–20%. Probably this increase came not from tumor cells but mainly from a BCG-induced infiltration of mononuclear cells, as an increase of T cells from 14% to 20%, an increase of macrophages from 8% to 18%, and an increase of B cells from 0 to 6% was observed. We conclude that the potentiation of anti-(line-10 tumor cell) immunity correlated with a 20-fold increase of MHC-class-II-antigen-positive cells in the lymph nodes and a small increase in the number of MHC-class-II-antigen-positive tumor-infiltrating cells.     

Immunological properties of recombinant Mycobacterium bovis bacillus Calmette-Guérin strain expressing fusion protein IL-2-ESAT-6

The live vaccine Mycobacterium bovis bacillus Calmette-Guérin (BCG) provides variable efficacy against adult pulmonary tuberculosis (TB). Recombinant BCG, expressing either immunodominant antigens or Th1 cytokines, is a promising strategy for developing a new TB vaccine. However, not much is known about whether the introduction of cytokine and specific antigen genes concurrently into the BCG strain could improve the immunogenicity of BCG. In this study, a recombinant BCG strain (rBCG) expressing the fusion protein human interleukin (IL)-2 and ESAT-6 (early secreted antigenic target-6 kDa) antigen of Mycobacterium tuberculosis was constructed. Six weeks after BALB/c mice (H-2d) were immunized with 106 colony forming units (CFUs) BCG or rBCG, splenocyte proliferation was determined with MTT [3-(4,5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide] assay, IL-4 and interferon (IFN)-gamma produced by splenocytes were tested by enzyme linked immunosorbent assay (ELISA,) and the cytotoxicity of splenocytes from immunized mice to P815 cells (H-2d) expressing ESAT-6 protein was measured using CytoTox 96 Non-Radioactive Cytotoxicity Assay. Compared with native BCG-vaccinated mice, rBCG induced stronger Th1 responses that were confirmed by high lymphoproliferative responses and IFN-gamma production to culture filtrate protein (CFP) or ESAT-6 protein. Moreover, rBCG induced significant enhanced CTL responses against P815-ESAT-6 cells. Results from rBCG-immunized mice demonstrated that introducing the il-2 and esat-6 genes into BCG could enhance Th1 type immune responses to ESAT-6. Further investigation is needed by introducing other Th1 cytokines and antigens into BCG to optimize the protective efficacy against TB.     

Intravesical treatment of bladder cancer with recombinant human interferon-β

Summary In order to examine its clinical efficacy, recombinant human interferon- (rIFN-) was instilled intravesically into 51 patients with superficial bladder cancer. Ten patients, who received intermittent intravesical instillation at a dose of (3–36) × 10⁶ U rIFN- on days 1–3 every week, showed no response. Thirty-two patients received intravesical instillation at a dose of (3–36) × 10⁶ U every day for 10–20 days. Eight patients showed partial response, indicating an efficacy rate of 25%. Nine patients received divided doses of 18 × 10⁶ U twice a day every day for 10–20 days. Six patients showed partial response, indicating an efficacy rate of 67%. This value was significantly higher than that obtained by administering divided doses. The response to intravesical instillation therapy with rIFN- varies with treatment protocol. Frequent and longer exposure to rIFN- may induce better regression of superficial bladder cancer. Six incidences of side-effects were found in five cases (9.8%): pollakiuria in one, pain on micturition in two, fever in two, and eruption in one case. All of these side-effects were slight and reversible after drug withdrawal. Laboratory tests showed only a few changes with low severity. Thus, rIFN- is potentially a new drug for instillation therapy of superficial bladder cancer, in view of the absence of adverse effects.