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Keywords:

  • BCG;
  • interferon-alpha;
  • Th1 response;
  • bladder cancer

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

To increase its immunostimulatory properties, BCG was genetically engineered to secrete recombinant human interferon-alpha 2B (rhIFN-α) under control of the mycobacterial heat shock protein (hsp)60 promoter and the α antigen signal sequence. Expression of rhIFN-α was readily detectable by ELISA and on Western blotting. When compared with control BCG, rhIFN-α BCG was substantially more active in inducing the production of IFN-γ and IFN-inducible protein 10 (IP-10) from human peripheral blood mononuclear cells, while IL-10 production was correspondingly decreased. These effects were reversible upon antibody neutralization of rhIFN-α. Among 10 patients tested, rhIFN-α BCG enhanced IFN-γ production in all patients ranging from 1·4- to 23·7-fold with a general trend toward greatest enhancement among those with weakest baseline responses to control BCG. Correspondingly, rhIFN-α BCG decreased IL-10 production in all patients by 1·2–4·8-fold. The onset of IFN-γ production induced by rhIFN-α BCG was also more rapid, occurring within 4 h after stimulation versus > 24 h with wild-type BCG. The observation that the maximum IFN-γ induction depends on the simultaneous presence of both IFN-α and BCG highlights the advantages of rhIFN-α BCG. Taken together, these immunostimulatory properties of rhIFN-α BCG suggest that it may be a superior agent for immunotherapeutic protocols involving live BCG in humans.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Intravesical administration of BCG is currently accepted as the most effective therapy for superficial bladder cancer and carcinoma in situ (CIS) of the bladder [1,2]. However, 25–40% of patients do not respond to BCG therapy and long-term remission (> 5 years) is only achieved in 50% of patients [3]. Furthermore, side-effects of BCG therapy are common with occurrence of severe adverse effects in 5% of patients and life-threatening symptoms in 0·5% of patients [4]. These disadvantages limit its use in clinical practice.

Other than BCG, interferon-alpha (IFN-α) has also been used as an intravesical agent for treating superficial bladder cancer. Although its 40% response rate is clearly inferior to that of BCG therapy [5–7], a proportion of BCG non-responders have been shown to benefit from IFN-α monotherapy [6,8]. In addition, intravesical IFN-α produces only minimal local and systemic toxicity [5,6]. However, it is very costly and requires repeated applications.

In an attempt to obtain an improved remedy for bladder cancer immunotherapy, an alternative schedule with concomitant administration of low-dose BCG plus IFN-α has been proposed. A number of pilot clinical trials have shown that combination therapy is well tolerated and can yield a high complete response rate [6,9,10]. In murine models, combination therapy is also very encouraging. BCG plus IFN-α or BCG plus the interferon inducer bropirimine were observed to be superior to either agent alone [11,12]. These limited clinical trials and animal studies have shown that adding IFN-α to BCG bladder cancer immunotherapy could lower BCG toxicity due to the reduced BCG dose while at the same time preserving or enhancing BCG activity against tumours.

Although the exact mechanism through which IFN-α enhances BCG-mediated anti-tumour immunity remains unclear, recent studies including ours have shown that adding IFN-α could enhance BCG immune activities in several aspects. These include synergistically augmenting IFN-γ and IL-12 production from immune cells [13], significantly increasing αβ+ T cell populations [11], and enhancing the direct effects on bladder cancer cells such as anti-proliferation and cytokine induction (IL-6, IL-8, granulocyte-macrophage colony-stimulating factor (GM-CSF) and tumour necrosis factor-alpha (TNF-α)) [14,15]. These favourable synergies between BCG and IFN-α are believed to contribute to the improved efficacy observed in anti-bladder cancer immunotherapy.

Our laboratory has pioneered methods to genetically engineer BCG, which allows it to express biologically active molecules of interest [16–18]. Based on the current understanding of the combination therapy in bladder cancer, in this study we intended to make a new strain of BCG that constitutively expresses rhIFN-α. Using our established cytokine induction bioassays [13], we evaluated the newly constructed rhIFN-α BCG. We demonstrated that rhIFN-α BCG possesses enhanced immunogenicity. Compared with wild-type BCG, it not only augments but also accelerates the overall T-helper type-1 (Th1) immune response.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Construction of human IFN-α 2B expression plasmid and the recombinant BCG strain

The previously described rat IL-2 expression vector pMAO-4 [16] was engineered to express human IFN-α 2B by replacing the rat IL-2 coding sequence with human IFN-α 2B coding sequence at the BamHI-EcoRI site. The insert sequence was derived from polymerase chain reaction (PCR) amplification of human IFN-α-containing plasmid (ATCC, Rockville, MD) using a pair of primers for the mature form of the cytokine. The sequence of the sense primer was CAAGggatccTGTGATCTGCCTCAAACCCACAG (BamHI site in lower case) and that of the antisense primer was GCCGgaattcTCATTCCTTACTTCTTAAACTTTCTTG (EcoRI site in lower case). The newly constructed plasmid phIFN-α is illustrated in Fig. 1a. Escherichia coli competent cells (XL1-Blue MR) obtained from Stratagene (La Jolla, CA) were transformed with the E. coli–BCG shuttle plasmid according to the manufacturer's instructions and selected on kanamycin (30 μg/ml) Luria-Bertani agar plates. The E. coli-derived plasmid with correct structures verified by restriction analysis and sequencing were then transformed into BCG Pasteur strain by electroporation as described previously [18]. The transformed BCG cells were plated on Middlebrook 7H10 Bacto agar (Difco, Detroit, MI) supplemented with 10% ADC (5% bovine serum albumin fraction V, 2% dextrose and 0·85% NaCl) and 30 μg of kanamycin per ml. Individual colonies containing phIFN-α verified by PCR were picked and grown in Middlebrook 7H9 Bacto broth (Difco) supplemented with 10% ADC, 0·05% Tween 80 (Sigma, St Louis, MO), and 30 μg of kanamycin per ml.

image

Figure 1. (a) Schematic illustration of the human IFN-α 2B-containing Escherichia coli–BCG shuttle plasmid. The IFN-α 2B coding sequence is placed downstream of BCG α-antigen signal sequence (SS) and influenza virus haemagglutinin epitope tag sequence (T). Expression of the cytokine is driven by the BCG heat shock protein (hsp)60 promoter (black box). The arrow indicates the direction of transcription. The kanamycin resistance cassette (open box) and a mycobacterial as well as an E. coli origin of replication are indicated. The illustration is not to scale. (b) Expression of IFN-α 2B in Western blot of BCG culture supernatant (S) and pellet lysate (P) from human IFN-α 2B BCG recombinant. Purified recombinant human IFN-α 2B is used as a positive control shown on the right. The blot is probed with a specific mouse anti-human IFN-α MoAb. Standard molecular weights are indicated.

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Western blot analysis

Cellular lysates were prepared from log-phase BCG cultures at 1 OD600 density (one unit of optical density at 600 nm was calculated as 2·5 × 107 colony-forming units (CFU)) using a lysis buffer consisting of 20 m m HEPES pH 7·5, 150 m m NaCl, 1% NP40, 1 m m EDTA, 1 m m DTT, and four types of protease inhibitor (2 μg/ml for each of aprotinin, leupeptin and pepstatin A, and 100 μg/ml for 4-(2-aminoethyl)-benzenesulphonylfluoride ; all purchased from Sigma). Culture supernatants were concentrated using a protein G immunoprecipitation kit (Roche Molecular Biochemicals, Indianapolis, IN, USA) according to the manufacturer's instructions. Briefly, 1·5 ml of culture supernatants collected from 1 OD600 density of log-phase BCG was precleared by incubation with 50 μl of protein G agarose at 4°C for 3 h on a rocking platform. After centrifugation, supernatants were collected and incubated with 20 μg of sheep anti-human IFN-α polyclonal antibody (Endogen, Boston, MA) at 4°C for 1 h followed by further incubation with 100 μl of protein G agarose at 4°C overnight. The bound IFN-α was released from protein G agarose after three cycle washes. Protein concentrations were measured using the coomassie plus assay (Pierce, Rockford, IL). Following electrophoresis in a 15% SDS–polyacrylamide gel (BioRad, Richmond, CA) and transfer to nitrocellulose, the filters were blocked in 5% non-fat powdered milk and then probed with mouse anti-human IFN-α MoAb (Sertotec, Raleigh, NC, USA) . The blots were subsequently probed with a horseradish peroxidase-labelled goat anti-mouse IgG antibody (Pierce) and a chemiluminescent substrate (Amersham, Arlington Heights, VA) to detect membrane-bound IFN-α 2B protein.

Peripheral blood mononuclear cell culture

Human peripheral blood mononuclear cells (PBMC) were prepared from buffy coat leucocytes purified on Ficoll–Paque (Pharmacia, Uppsala, Sweden). Viability by trypan blue exclusion usually exceeded 95%. PBMC were suspended in RPMI 1640 medium containing 10% fetal calf serum (FCS) and 30 μg per ml of kanamycin, and incubated at 37°C in a humidified 5% CO2 incubator at a density of 8 × 105 cells/200 μl per well in 96-well tissue culture plates in the presence or absence of designated doses of BCG, human IFN-α or both. To define the functional specificity of rhIFN-α secreted by rBCG, neutralizing rabbit anti-human IFN-α (PeproTech, Rocky Hill, NJ, USA) was used. The plates were incubated for the designated time and then frozen at −70°C until cytokine ELISA assays were performed.

ELISA assays and reagents

ELISA reagents including recombinant human cytokines and paired monoclonal capture and detecting antibodies for the cytokines were obtained from Endogen for IFN-α and -γ and from PharMingen (San Diego, CA) for IL-10 and IFN-inducible protein 10 (IP-10). Samples of conditioned PBMC cultures were assayed by ELISA using a sandwich format according to the manufacturer's instructions. Cytokine concentrations were calculated in standard mass/volume format using standard curves derived from purified recombinant cytokine standards. For all of the above measured cytokines, 1 IU is equal to approximately 200 pg of purified cytokine.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Construction of rhIFN-α BCG

The PCR-derived human IFN-α coding sequence was inserted into the immediate downstream site of the BCG a-antigen signal sequence (SS) and an influenza virus haemagglutinin epitope tag sequence (T) under control of the constitutively active heat shock protein (hsp)60 promoter ( Fig. 1a). From BCG transformed with this construct, rhIFN-α was detected in both BCG lysates and culture supernatants on Western blot ( Fig. 1b). Due to partial cleavage of SS, two bands are visible for the supernatant sample of rhIFN-α BCG, the lower band resulting from cleavage of the SS. MV261 BCG, a control BCG strain carrying the vector alone, showed no specific band on the same blots. The concentration of rhIFN-α in culture was quantified by ELISA and calculated to be approximately 30 IU/ml (data not shown).

Enhanced production of IFN-γ from human PBMC by this rhIFN-α BCG was readily demonstrable ( Fig. 2). It is worthy of note that, although the kinetic pattern of IFN-γ production induced by both MV261 and rhIFN-α BCG was similar and peaked at day 3, a burst of IFN-γ induction by rhIFN-α BCG (10-fold higher than that induced by MV261 BCG) occurred already by day 1 ( Fig. 2a). Furthermore, rhIFN-α BCG continued to induce higher IFN-γ responses to a dose up to 0·1 OD, a point at which wild-type BCG began to lose Th1 inductive capacity ( Fig. 2b). This ability of rhIFN-α BCG to retain its biological activity at higher doses is potentially favourable from a clinical perspective, since the standard BCG dose for intravesical treatment of bladder cancer is typically 2–10 × 108 CFU of BCG in 50 ml of physiologic saline per instillation.

image

Figure 2. (a) Time course of IFN-γ production in human peripheral blood mononuclear cell (PBMC) cultures. PBMC were incubated with medium, MV261 BCG (0·01 OD600/ml), or rhIFN-α BCG (rBCG, 0·01 OD600/ml) for up to 7 days. ○, None; ●, MV261 BCG; ▪, rBCG. (b) Dose–response of IFN-γ production in human PBMC cultures. PBMC were incubated with medium or various doses of control MV261 BCG or rhIFN-α BCG (rBCG) for 3 days. IFN-γ in the cultures was quantified and the values were expressed as means ± s.d. from triplicate-well incubations. □, None; hatched, MV261 BCG; ▪, rBCG.

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RhIFN-α BCG exerts a stronger Th1 immune stimulation

In our previous work, we demonstrated that adding exogenous IFN-α into the mixture cultures of human PBMC and BCG synergistically increased IFN-γ production and meanwhile significantly decreased IL-10 production [13]. In this study we made use of the same parameters to evaluate rhIFN-α BCG. As expected, rhIFN-α BCG was observed to increase IFN-γ production 30-fold while decreasing IL-10 by 40% when compared with MV261 BCG in a biological activity assay ( Fig. 3). These effects are comparable to those obtained from the combined stimulation using the same dose of MV261 BCG plus exogenous IFN-α (50 IU/ml). Expression of IP-10, a chemokine known to respond to IFN-α stimulation [19] (our unpublished observations), was also up-regulated by rhIFN-α BCG (98-fold increase compared with MV261 BCG). The inductive effect of rhIFN-α BCG on these cytokines is dependent on the specific expression of IFN-α, since neutralizing antibody to IFN-α could efficiently reduce or abolish such effects.

image

Figure 3. Specificity of IFN-α expressed by rhIFN-α BCG in regulating human peripheral blood mononuclear cell (PBMC) IFN-γ, IL-10 and IFN-inducible protein 10 (IP-10) production. PBMC were incubated with rhIFN-α BCG (rBCG, 0·01 OD600/ml) or rBCG (0·01 OD600/ml) plus neutralizing anti-IFN-α antibody (3 μg/ml) for 3 days. As controls, PBMC were incubated with medium, IFN-α (50 IU/ml), MV261 BCG (0·01 OD600/ml), or MV261 BCG (0·01 OD600/ml) plus IFN-α (50 IU/ml). IFN-γ, IL-10 and IP-10 in the cultures were quantified and the values were expressed as means ± s.d. from triplicate-well incubations.

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The favourable effects of rhIFN-α BCG on induction of both IFN-γ and IL-10 were further demonstrated in a group of 10 different individuals (seven control patients and three bladder cancer patients) as shown in Fig. 4. Compared with MV261 BCG, rhIFN-α BCG increased IFN-γ production in all patients ranging from 1·4- to 23·7-fold, and meanwhile decreased IL-10 production in all patients ranging from 1·2- to 4·8-fold. There was no obvious difference between the control group and the bladder cancer group in the ratio of changes in both IFN-γ and IL-10 induction. These results were consistent with the observations in our previous study that PBMC from 10 other patients in that study all showed an increased production of IFN-γ and a decreased production of IL-10 in response to MV261 BCG plus exogenous IFN-α[13].

image

Figure 4. Effect of rhIFN-α BCG on human peripheral blood mononuclear cell (PBMC) IFN-γ and IL-10 production. PBMC from 10 patients (seven control patients: MO, RE, JA, DC, SS, YZ and HC; three bladder cancer patients: MF, SKS and MR) were incubated with medium, MV261 BCG (0·01 OD600/ml), or rhIFN-α BCG (rBCG, 0·01 OD600/ml) for 3 days. IFN-γ and IL-10 in the cultures were quantified and the values were expressed as means ± s.d. from triplicate-well incubations. The ratio of rBCG to MV261 BCG responses for IFN-γ and IL-10 is given for each patient. □, None; hatched, MV261 BCG; ▪, rBCG.

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RhIFN-α BCG accelerates IFN-γ induction

As shown in Fig. 2a, a burst of IFN-γ production from human PBMC occurs at 24 h after rhIFN-α BCG stimulation, at which time the level of IFN-γ induced by wild-type BCG is usually low. To investigate the early role of IFN-α in BCG-induced IFN-γ production, PBMC were cultured with either MV261 BCG (0·01 OD600/ml), MV261 BCG (0·01 OD600/ml) plus exogenous IFN-α (100 IU/ml), or rhIFN-α BCG (0·01 OD600/ml) for a period of 4 h (data not shown). MV261 BCG did not induce IFN-γ production during the 4-h incubation. However, IFN-γ was fairly measurable (2100 pg/ml) at 4 h in the presence of rhIFN-α BCG. This early IFN-γ induction could be reproduced by combined stimulation with MV261 BCG plus exogenous IFN-α (1800 pg/ml of IFN-γ). Thus, it is clear that IFN-α has a role in early triggering of IFN-γ production in response to BCG.

Maximum IFN-γ induction depends on simultaneous presence of both BCG and IFN-α

To dissect further the early role of IFN-α in BCG-induced IFN-γ production, PBMC were incubated with either MV261 BCG or IFN-α for up to 4 h. At the different time points, either IFN-α (for MV261 BCG prior cultured group) or MV261 BCG (for IFN-α prior cultured group) was added and the incubation continued for 3 days. Compared with IFN-γ production in the control group, where both biologicals (BCG and IFN-α) were simultaneously present from the beginning of incubation, a time-dependent decrease in IFN-γ production occurred as the interval between BCG and IFN-α recombination was lengthened ( Fig. 5). This was especially evident when BCG was added first. After 3 h or longer prior incubation with BCG, nearly all of the augmentation effect of IFN-α was abolished. The observation that maximum IFN-γ induction in the combination system requires the simultaneous presence of both BCG and IFN-α highlights the advantages of rhIFN-α BCG and provides a rationale for its use in clinical practice.

image

Figure 5. Maximum human peripheral blood mononuclear cell (PBMC) IFN-γ induction requires simultaneous presence of both BCG and IFN-α. PBMC were incubated with either MV261 BCG (0·01 OD600/ml) or IFN-α (100 IU/ml) for up to 4 h. At the indicated time points, the same doses of either IFN-α (for MV261 BCG prior cultured group) or MV261 BCG (for IFN-α prior cultured group) were added and the incubation continued for 3 days. IFN-γ in the cultures was quantified and the values were expressed as means ± s.d. from triplicate-well incubations.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Based on the observations that combination therapy with BCG plus IFN-α favours bladder cancer treatment in clinic and animal models, in this study we intended to create a new therapeutic agent by combining these two biologicals in one using modern BCG cloning techniques. We demonstrated that the newly constructed IFN-α-expressing BCG strain possesses superior immunostimulatory properties to wild-type BCG, as shown in the induction of Th1 and Th2 cytokines from human PBMC.

The advantages of supplementing IFN-α to BCG for anti-bladder cancer therapy have been documented in both in vivo[9–11] and in vitro studies [11,13–15]. However, IFN-α therapy has several shortcomings, such as high cost and repeated administration. In clinical practice, a standard intravesical IFN-α administration (either monotherapy or combined with BCG) is carried out with a dose of 50–100 million units of IFN-α in 50 ml of physiologic saline. After intravesical instillation, IFN-α is maintained in the bladder for 2 h before elimination by urination. Although such administration guarantees a high concentration of IFN-α inside the bladder, it consumes a large quantity of IFN-α that may be far in excess from the amount needed for inducing a favourable localized anti-tumour immunity. Furthermore, while BCG physically binds to the urothelium [20], even such a high pulse dose of IFN-α may not be sufficient to induce optimal immunity due to its short retention time inside the bladder. The rhIFN-α BCG constructed in this study could potentially cope with these shortcomings. Upon intravesical instillation, BCG attaches to bladder epithelial cells via fibronectin binding [20]; it then is either retained on epithelial cells or enters into macrophages and epithelial cells as an intracellular pathogen [20]. As it may remain viable inside the bladder for several days to months [21], rBCG could durably express any designated cytokines that have been engineered into it. Although the amount of IFN-α expressed by rhIFN-α BCG is fairly low compared with the large doses used in clinical intravesical therapy, such low but consistently expressed IFN-α is sufficient in enhancing BCG-induced immune responses. In our previous studies, a plateau level of IFN-γ induced from human PBMC in response to BCG plus IFN-α was well maintained within a wide range of IFN-α concentrations and even when the dose of IFN-α went as low as 10 IU/ml [13].

Although the mechanism by which IFN-α enhances BCG anti-tumour effect needs to be further explored, our previous studies showed that adding IFN-α could polarize BCG-mediated immune responses toward the Th1 pathway, as demonstrated by an increased induction of IFN-γ and IL-12 and a decreased induction of IL-10 [13]. In the present study, we demonstrated that rhIFN-α BCG could exert the same biological activities as the two single biologicals do mixed together. Compared with control BCG, rhIFN-α BCG not only enhanced the IFN-γ response but also kinetically accelerated the response. Such early polarization to the Th1 immune pathway is extremely important in anti-bladder cancer immunotherapy, since bladder tumour destruction is believed to be largely dependent on cellular immunity [20,22–24].

The observation that maximum IFN-γ induction in costimulation requires the simultaneous presence of BCG and IFN-α favours the newly constructed rhIFN-α BCG. Although IFN-α by itself only induces negligible amount of IFN-γ, timely addition of IFN-α could synergistically enhance BCG-induced IFN-γ. The reasons why optimal IFN-γ induction could not be achieved if IFN-α is added either before or after BCG stimulation are not clear. IFN-α has been reported to have paradoxical effects on regulating immune responses. It positively influences Th1 development through activating Th1 cells for IFN-γ production and meanwhile suppressing Th2 cells for B cell antibody production [25,26]. It also inhibits bladder cancer cell proliferation [14,27], inhibits tumour vascularization [28], up-regulates MHC class I expression on target tumour cells [29], and activates natural killer (NK) cells, lymphokine-activated killer (LAK) cells and T lymphocytes [30,31]. In contrast to these, IFN-α also exhibits immunosuppressive activities through down-modulating MHC class II and intercellular adhesion molecule-1 expression on antigen-presenting cells and interfering with various anti-inflammatory cytokine syntheses in immune cells [32]. The mechanism by which IFN-α acts on BCG to trigger favourably immune cell interactions and cytokine network cascades needs further investigation. Nevertheless, rhIFN-α BCG ideally meets the requirement for the maximum Th1 immune induction, as it combines the two biologicals into one.

In conclusion, this novel rhIFN-α BCG strain possesses enhanced immunogenicity for inducing Th1 immune responses compared with wild-type BCG. Its biological effects are equivalent to, if not any better than, those of exogenously combined BCG plus IFN-α. Its potential clinical use for bladder cancer immunotherapy is worthy of evaluation.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

This work was supported by National Institutes of Health Grants R29CA64230 and RR01032, and a grant from Schering Corporation.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
  • 1
    Alexandroff AB, Jackson AM, O'Donnell MA, James K. BCG immunotherapy of bladder cancer: 20 years on. Lancet 1999; 353:1689 94.DOI: 10.1016/s0140-6736(98)07422-4
  • 2
    O'Donnell MA & DeWolf WC. Bacillus Calmette–Guerin immunotherapy for superficial bladder cancer: new prospects for an old warhorse. Surg Oncol Clin North Am 1995; 4:189 202.
  • 3
    Lamm DL. Long-term results of intravesical therapy for superficial bladder cancer. Urol Clin North Am 1992; 19:573 80.
  • 4
    Steg A, Adjiman S, Debre B. BCG therapy in superficial bladder tumours—complications and precautions. Eur Urol 1992; 21 (Suppl. 2):35 40.
  • 5
    Williams RD. Intravesical interferon alfa in the treatment of superficial bladder cancer. Semin Oncol 1988; 15:10 13.
  • 6
    Belldegrun AS, Franklin JR, O'Donnell MA et al. Superficial bladder cancer: the role of interferon-alpha. J Urol 1998; 159:1793 801.
  • 7
    Naitoh J, Franklin J, O'Donnell MA, Belldegrun AS. Interferon alpha for the treatment of superficial bladder cancer. Adv Exp Med Biol 1999; 462:371 86.
  • 8
    Sargent ER & Williams RD. Immunotherapeutic alternatives in superficial bladder cancer: interferon, interleukin-2 and keyhole limpet hemocyanin. Urol Clin North Am 1992; 19:581 9.
  • 9
    Bercovich E, Deriu M, Manferrari F, Irianni G. BCG vs. BCG plus recombinant alpha-interferon 2b in superficial tumors of the bladder. Arch Ital Urol Androl 1995; 67:257 60.
  • 10
    Stricker P, Pryor K, Nicholson T et al. Bacillus Calmette–Guerin plus intravesical interferon alpha-2b in patients with superficial bladder cancer. Urology 1996; 48:957 61.
  • 11
    Gan YH, Zhang Y, Khoo HE, Esuvaranathan K. Antitumor immunity of bacillus Calmette–Guerin and interferon alpha in murine bladder cancer. Eur J Cancer 1999; 35:1123 9.DOI: 10.1016/s0959-8049(99)00057-x
  • 12
    Sarosdy MF & Kierum CA. Combination immunotherapy of murine transitional cell cancer using BCG and an interferon-inducing pyrimidinone. J Urol 1989; 142:1376 9.
  • 13
    Luo Y, Chen X, Downs TM, DeWolf WC, O'Donnell MA. IFN-α 2B enhances Th1 cytokine responses in bladder cancer patients receiving Mycobacterium bovis bacillus Calmette–Guerin immunotherapy . J Immunol 1999; 162:2399 405.
  • 14
    Pryor K, Stricker P, Russell P, Golovsky D, Penny R. Antiproliferative effects of bacillus Calmette–Guerin and interferon-α 2b on human bladder cancer cells in vitro. Cancer Immunol Immunother 1995; 41:309 16.DOI: 10.1007/s002620050233
  • 15
    Zhang Y, Khoo HE, Esuvaranathan K. Effects of bacillus Calmette–Guerin and interferon alpha-2B on cytokine production in human bladder cancer cell lines. J Urol 1999; 161:977 83.
  • 16
    O'Donnell MA, Aldovini A, Duda RB, Yang H, Szilvasi A, Young RA, DeWolf WC. Recombinant Mycobacterium bovis BCG secreting functional interleukin-2 enhances gamma interferon production by splenocytes. Infect Immun 1994; 62:2508 14.
  • 17
    O'Donnell MA. The genetic reconstruction of BCG as a new immunotherapeutic tool. Trends Biotechnol 1997; 15:512 7.DOI: 10.1016/s0167-7799(97)01134-7
  • 18
    Luo Y, Szilvasi A, Chen X, DeWolf WC, O'Donnell MA. A novel method for monitoring Mycobacterium bovis BCG trafficking using recombinant BCG expressing green fluorescent protein. Clin Diag Lab Immunol 1996; 3:761 8.
  • 19
    Farber JM. Mig and IP-10: CXC chemokines that target lymphocytes. J Leuk Biol 1997; 61:246 57.
  • 20
    Ratliff TL. Role of the immune response in BCG for bladder cancer. Eur Urol 1992; 21 (Suppl. 2):17 21.
  • 21
    Bowyer L, Hall RR, Reading J, March MM. The persistence of bacille Calmette–Guerin in the bladder after intravesical treatment of bladder cancer. Br J Urol 1995; 75:199 202.
  • 22
    Ratliff TL, Gillen DP, Catalona WJ. Requirement of a thymus dependent immune response for BCG-mediated antitumor activity. J Urol 1987; 137:155 8.
  • 23
    Ratliff TL, Ritchey JK, Yuan JJ, Andriole GL, Catalona WJ. T-cell subsets required for intravesical BCG immunotherapy for bladder cancer. J Urol 1993; 150:1018 23.
  • 24
    Bohle A, Gerdes J, Ulmer AJ, Hofstetter AG, Flad HD. Effects of local bacillus Calmette–Guerin therapy in patients with bladder carcinoma on immunocompetent cells of the bladder wall. J Urol 1990; 144:53 58.
  • 25
    Brinkmann V, Geiger T, Alkan S, Heusser CH. Interferon α increases the frequency of interferon γ-producing human CD4+ T cells. J Exp Med 1993; 178:1655 63.
  • 26
    Brinkmann V, Heusser CH, Baer J, Kilchherr E, Erard F. Interferon α suppresses the capacity of T cells to help antibody production by human B cells. J Interferon Res 1992; 12:267 74.
  • 27
    Borden EC, Groveman DS, Nasu T, Renzikoff C, Bryan GT. Antiproliferative activities of interferons against human bladder carcinoma cell lines in vitro. J Urol 1984; 132:800 3.
  • 28
    Slaton JW, Perrotte P, Inoue K, Dinney CP, Fidler IJ. Interferon-alpha-mediated down-regulation of angiogenesis-related genes and therapy of bladder cancer are dependent on optimization of biological dose and schedule. Clin Cancer Res 1999; 5:2726 34.
  • 29
    Tzai TS & Lin SN. Interferon-α can alter the lytic susceptibility of murine bladder transitional cell carcinoma (MBT-2) by their original poor specific cytotoxic tumor infiltrating lymphocytes. J Urol 1992; 147:523 7.
  • 30
    Alvarez-Mon M, Molto LM, Manzano L, Olivier C, Carballido JA. Immunomodulatory effect of interferon-alpha 2b on natural killer cells and T lymphocytes from patients with transitional cell carcinoma of the bladder. Anticancer Drugs 1992; 3 (Suppl. 1):5 8.
  • 31
    Wang Z, Cheng Y, Zheng R, Qin D, Liu G. Effect of TNF-alpha and IFN-alpha on the proliferation and cytotoxicity of lymphokine-activated killer cells in patients with bladder cancer. Chin Med J 1997; 110:180 3.
  • 32
    Tilg H & Peschel C. Interferon alpha and its effects on the cytokine cascade: a pro- and anti-inflammatory cytokine. Leuk Lymphoma 1996; 23:55 60.