Complex relationships between shallow muddy benthic assemblages, sediment chemistry and toxicity in estuaries in southern New South Wales, Australia.

Synoptic sediment quality triad (contaminants, benthic assemblages, toxicity testing) data were collected for sites in Sydney estuary, adjacent Cooks River and five less-modified southern estuaries. Matching data tested relationships between contaminants and benthic assemblages, correlations with specific contaminants, and the ability of sediment quality guidelines to predict the risk of adverse effects. Significant but weak relationships occurred in complex patterns between assemblages, contaminant concentrations and environmental variables. Maximum benthos abundance occurred where sediment contamination was high and was dominated by polychaetes. Spionidae (polychaete) and Galeommatidae (mollusc) abundances were strongly correlated with site environmental characteristics and with varying mixtures of metals and organic contaminants. The risk of adverse effects on benthic assemblage structure increased with increasing sediment toxicity except for areas of very high contamination and for non-bioavailable anthropogenic chemicals. The overall weight-of-evidence scores differentiated the highly modified sites from less-contaminated southern estuaries, where toxicity scores were higher than predicted.

Anti-interleukin-10R1 monoclonal antibody in combination with bacillus Calmette--Guérin is protective against bladder cancer metastasis in a murine orthotopic tumour model and demonstrates systemic specific anti-tumour immunity.

Effective treatment of bladder cancer with bacillus Calmette-Guérin (BCG) depends on the induction of a T helper type (Th) 1 immune response. Interleukin (IL)-10 down-regulates the Th1 response and is associated with BCG failure. In this study, we investigated whether blocking IL-10 signalling could enhance the BCG-induced Th1 response and anti-tumour immunity in a murine orthotopic tumour model. Treatment with BCG and anti-IL-10 receptor 1 monoclonal antibody (anti-IL-10R1 mAb) increased the interferon (IFN)-γ to IL-10 ratio in both splenocyte cultures and urine. Mice bearing luciferase-expressing MB49 (MB49-Luc) tumours were treated and followed for tumour growth by bioluminescent imaging, bladder weight and histology. Mice treated with phosphate-buffered saline (PBS) (group 1), BCG plus control immunoglobulin (Ig)G1 (group 2) or BCG plus anti-IL-10R1 mAb (group 3) showed 0, 6 and 22% tumour regression, respectively. The mean bladder weight of group 3 mice was substantially lower than those of groups 1 and 2 mice. Remarkably, 36% of group 1 and 53% of group 2 mice but no group 3 mice developed lung metastasis (P = 0·02). To investigate the mechanisms underlying the effect of combination therapy, splenocytes were stimulated with S12 peptide (serine mutation at codon 12 of the K-ras oncogene) known to be expressed in MB49-Luc cells. Induction of ras mutation-specific IFN-γ and cytotoxicity was observed in mice treated with combination therapy. These observations indicate that BCG, in combination with anti-IL-10R1 mAb, induces enhanced anti-tumour immunity that is protective against lung metastasis. Anti-IL-10R1 mAb demonstrates systemic effects and may prove useful in clinical practice for treating bladder cancer in high-risk patients.

The API2-MALT1 fusion exploits TNFR pathway-associated RIP1 ubiquitination to promote oncogenic NF-κB signaling.

The API2-MALT1 fusion oncoprotein is created by the recurrent t(11;18)(q21;q21) chromosomal translocation in mucosa-associated lymphoid tissue (MALT) lymphoma. We identified receptor interacting protein-1 (RIP1) as a novel API2-MALT1-associated protein, and demonstrate that RIP1 is required for API2-MALT1 to stimulate canonical nuclear factor kappa B (NF-κB). API2-MALT1 promotes ubiquitination of RIP1 at lysine (K) 377, which is necessary for full NF-κB activation. Furthermore, we found that TNF receptor-associated factor 2 (TRAF2) recruitment is required for API2-MALT1 to induce RIP1 ubiquitination, NF-κB activation and cellular transformation. Although both TRAF2 and RIP1 interact with the API2 moiety of API2-MALT1, this moiety alone is insufficient to induce RIP1 ubiquitination or activate NF-κB, indicating that API2-MALT1-dependent RIP1 ubiquitination represents a gain of function requiring the concerted actions of both the API2 and MALT1 moieties of the fusion. Intriguingly, constitutive RIP1 ubiquitination was recently demonstrated in several solid tumors, and now our study implicates RIP1 ubiquitination as a critical component of API2-MALT1-dependent lymphomagenesis.

NEMO/IKKgamma regulates an early NF-kappaB-independent cell-death checkpoint during TNF signaling.

TNF receptor 1 (TNFR1) ligation can result in cell survival or cell death. What determines which of the two opposing responses is triggered is not fully understood. The current model suggests that it is the activation of the NF-kappaB pathway and its induction of prosurvival genes, or the lack thereof, which determines the outcome. NF-kappaB essential modifier (NEMO)/IkappaB kinase-gamma (IKKgamma)-deficient cells are highly sensitive to apoptosis, and as NEMO is essential for NF-kappaB activation, it has been assumed that this is due to the lack of NF-kappaB. This study demonstrates that this assumption was incorrect and that NEMO has another antiapoptotic function that is independent of its role in the NF-kappaB pathway. NEMO prevents receptor interacting protein-1 (RIP1) from engaging CASPASE-8 before NF-kappaB-mediated induction of antiapoptotic genes. Without NEMO, RIP1 associates with CASPASE-8 resulting in rapid tumor necrosis factor (TNF)-induced apoptosis. These results suggest that there are two cell-death checkpoints following TNF stimulation: an early transcription-independent checkpoint whereby NEMO restrains RIP1 from activating the caspase cascade, followed by a later checkpoint dependent on NF-kappaB-mediated transcription of prosurvival genes.

Dose-dependent synergy of Th1-stimulating cytokines on bacille Calmette-Guérin-induced interferon-gamma production by human mononuclear cells.

Successful bacille Calmette-Guérin (BCG) immunotherapy of bladder cancer depends on the proper induction of a T helper-type 1 (Th1) immune response. In this study we investigated the possible involvement of Th1-stimulating cytokines in BCG-induced interferon (IFN)-gamma production as well as their potential roles in enhancing BCG-induced IFN-gamma from human peripheral blood mononuclear cells (PBMCs). BCG efficiently induced IFN-gamma production by PBMCs in a dose-dependent manner. Neutralization of endogenous cytokines interleukin (IL)-2, IL-12 and IFN-alpha reduced BCG-induced IFN-gamma by 38%, 67% and 49%, respectively. Although single recombinant (r) IL-2, rIL-12 and rIFN-alpha induced no or a marginal amount of IFN-gamma, a combination of any two or three cytokines increased IFN-gamma production. When BCG (a subsaturated dose) was combined with mono, dual or triple cytokines, a synergy on IFN-gamma production was observed. Such a synergy was readily achievable even when minimal or low doses of cytokines were used. No saturation of IFN-gamma production was observed even when a subsaturated BCG dose was combined with very high doses of cytokines. A robust IFN-gamma production was also observed when a minimal BCG dose was combined with minimal doses of triple cytokines. In addition, we demonstrated that IL-2- and IFN-alpha-expressing rBCGs were superior to wild-type BCG for PBMC IFN-gamma induction and that combination of both rBCGs showed a synergy in IFN-gamma production. Taken together, these results suggest that combination of BCG with certain exogenous or endogenous (expressed by rBCGs) Th1-stimulating cytokines is a rational candidate for further study in bladder cancer treatment.

Mycobacterium bovis bacillus Calmette-Guérin (BCG) induces human CC- and CXC-chemokines in vitro and in vivo.

Both CC- and CXC-chemokines are known to be potent leucocyte activators and chemoattractants and play important roles in inflammatory responses. However, chemokine response to bacillus Calmette-Guérin (BCG) infection remains incompletely defined. In this study, we investigated human CC- [macrophage-derived chemokine (MDC), monocyte chemoattractant protein (MCP)-1, macrophage inflammatory protein (MIP)-1alpha and eosinophil chemoattractant activity (eotaxin)] and CXC-interferon-inducible protein (IP)-10 chemokine production in response to BCG stimulation. BCG efficiently induced all chemokines tested in the urine of four bladder cancer patients undergoing intravesical BCG immunotherapy. The peak urinary chemokine responses occurred generally between the fourth and sixth weekly treatment, except eotaxin, which was less predictable. To evaluate the effect of BCG on induction of chemokines in vitro, urothelial cell lines and peripheral blood mononuclear cells (PBMCs) were used. Although BCG induced no or marginal chemokines from urothelial SV-HUC-1, RT4 and T24 cells, BCG-derived cytokines [interleukin (IL)-1beta, interferon (IFN)-gamma and tumour necrosis factor (TNF)-alpha] induced all chemokines tested except eotaxin from these cell lines. BCG also efficiently induced all chemokines tested except eotaxin from PBMCs of both BCG-naive and BCG-vaccinated subjects. MCP-1 and MIP-1alpha emerged at 4-5 h post-BCG exposure (early chemokines); IP-10 elevated at day 1 and peaked at day 2 (intermediate chemokine); and MDC elevated at day 1 and peaked at day 7 (late chemokine). This kinetic pattern was paralleled with that of BCG-induced cytokines [early: TNF-alpha; intermediate: IL-6 and IL-10; and late: IFN-gamma and granulocyte-macrophage colony-stimulating factor (GM-CSF)]. Taken together, these results indicate that BCG directly or indirectly induces human CC- and CXC-chemokine production, which may represent one of the mechanisms by which BCG exerts its anti-tumour activity.

Recombinant Mycobacterium bovis bacillus Calmette-Guérin (BCG) expressing mouse IL-18 augments Th1 immunity and macrophage cytotoxicity.

Interleukin-18 (IL-18) has been demonstrated to synergize with BCG for induction of a T-helper-type 1 (Th1) immune response. Since successful treatment of superficial bladder cancer with BCG requires proper induction of Th1 immunity, we have developed a recombinant (r) BCG strain that functionally secretes mouse (m) IL-18. This rBCG-mIL-18 strain significantly increased production of the major Th1 cytokine IFN-gamma in splenocyte cultures, at levels comparable to that elicited by control BCG plus exogenous rIL-18. IFN-gamma production by splenocytes was eliminated by addition of neutralizing anti-IL-18 antibody. Endogenous IL-12 played a favourable role whereas IL-10 played an adverse role in rBCG-mIL-18-induced IFN-gamma production. Enhanced host antimycobacterial immunity was observed in mice infected with rBCG-mIL-18 which showed less splenic enlargement and reduced bacterial load compared to control mice infected with BCG. Further, splenocytes from rBCG-mIL-18-infected mice, in response to BCG antigen, displayed increased production of IFN-gamma and GMCSF, decreased production of IL-10, elevated cellular proliferation and higher differentiation of IFN-gamma-secreting cells. rBCG-mIL-18 also enhanced BCG-induced macrophage cytotoxicity against bladder cancer MBT-2 cells in a dose-dependent manner. Neutralizing all endogenous macrophage-derived cytokines tested (IL-12, IL-18 and TNF-alpha) as well as IFN-gamma severely diminished the rBCG-mIL-18-induced macrophage cytolytic activity, indicating a critical role for these cytokines in this process. Cytokine analysis for supernatants of macrophage-BCG mixture cultures manifested higher levels of IFN-gamma and TNF-alpha in rBCG-mIL-18 cultures than in control BCG cultures. Taken together, this rBCG-mIL-18 strain augments BCG's immunostimulatory property and may serve as a better agent for bladder cancer immunotherapy and antimycobacterial immunization.

Interleukin 10 induced augmentation of delayed-type hypersensitivity (DTH) enhances Mycobacterium bovis bacillus Calmette-Guérin (BCG) mediated antitumour activity.

Intravesical BCG therapy is effective in the treatment of superficial bladder cancer. Both clinical and experimental results suggest a role for cytokines and delayed-type hypersensitivity (DTH) in BCG-induced antitumour immunity. We characterized the modulatory effects of BCG on bladder cytokine expression and determined the relationship between DTH and BCG antitumour activity. The bladders of mice were instilled with BCG through a catheter. Bladder tissue RNA and urine were collected for evaluation of cytokine expression using reverse transcriptase-polymerase chain reaction (RT-PCR) and/or ELISA. IFN-gamma and TNF-alpha, the two major cytokines associated with DTH, were efficiently induced by BCG. IL10, an important down-regulator of DTH, was also induced by BCG. Constitutive levels of IL4 and IL5 were observed, but neither IL4 nor IL5 were modulated by BCG. Similar results were observed in the kinetic analysis of urinary cytokines in patients after intravesical BCG therapy. Production of Th1 (T helper type 1) cytokines (IFN-gamma, IL2 and IL12) preceded that of the Th2 (T helper type 2) cytokine IL10. A tendency toward higher ratios of IFN-gamma versus IL10 for BCG responders also was observed. In animal studies the absence of IL10 abrogated either by antibody inhibition or the use of genetically modified, IL10 deficient (IL10-/-) mice resulted in enhanced DTH responses. Under conditions of enhanced DTH, a significant enhancement in antitumour activity was observed. These data demonstrate that DTH and its associated mononuclear infiltration and cytokine production are important to the antitumour activity of intravesical BCG therapy, and suggest that effects to diminish IL10 production may have therapeutic value.

Salvage intravesical therapy with interferon-alpha 2b plus low dose bacillus Calmette-Guerin is effective in patients with superficial bladder cancer in whom bacillus Calmette-Guerin alone previously failed.

PURPOSE: We determined whether combining low dose bacillus Calmette-Guerin (BCG) interferon-alpha 2B would be effective for patients in whom previous BCG failed. MATERIALS AND METHODS: A total of 40 patients in whom 1 (19) or more (21) previous induction courses of BCG failed received 6 to 8 weekly treatments of 1/3 dose (27 mg.) BCG plus 50 million units interferon-alpha 2B. Additional 3 week miniseries of further decreased BCG (1/10, 1/30 or 1/100) titrated to symptoms without changing the interferon-alpha 2B dose were given at 5, 11 and 17 months. In 12 patients a second induction course was given with 1/10 BCG plus 100 million units interferon-alpha 2B. There was multifocal disease in 39 patients, previous BCG had failed within 6 months in 34, disease was aggressive (stage T1, grade 3 or carcinoma in situ in 31, there had been 2 or more previous recurrences in 25 and disease history was greater than 4 years in 13. RESULTS: At a median followup of 30 months 63% and 53% of patients were disease-free at 12 and 24 months, respectively. Patients in whom 2 or more previous BCG courses had failed fared as well as those with 1 failure. Of the 18 failures 14 occurred at the initial cystoscopy evaluation. Of 22 patients initially counseled to undergo cystectomy 12 (55%) are disease-free with a functioning bladder. Combination therapy was well tolerated. CONCLUSIONS: While longer followup and larger multicenter studies are required to validate these encouraging findings, intravesical low dose BCG plus interferon-alpha 2B appears to be effective in many cases of high risk disease previously deemed BCG refractory. However, early failure while on this regimen should be aggressively pursued with more radical treatment options.

Recombinant bacille Calmette-Guérin (BCG) expressing human interferon-alpha 2B demonstrates enhanced immunogenicity.

To increase its immunostimulatory properties, BCG was genetically engineered to secrete recombinant human interferon-alpha 2B (rhIFN-alpha) under control of the mycobacterial heat shock protein (hsp)60 promoter and the alpha antigen signal sequence. Expression of rhIFN-alpha was readily detectable by ELISA and on Western blotting. When compared with control BCG, rhIFN-alpha BCG was substantially more active in inducing the production of IFN-gamma 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-alpha. Among 10 patients tested, rhIFN-alpha BCG enhanced IFN-gamma 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-alpha BCG decreased IL-10 production in all patients by 1.2-4.8-fold. The onset of IFN-gamma production induced by rhIFN-alpha BCG was also more rapid, occurring within 4 h after stimulation versus > 24 h with wild-type BCG. The observation that the maximum IFN-gamma induction depends on the simultaneous presence of both IFN-alpha and BCG highlights the advantages of rhIFN-alpha BCG. Taken together, these immunostimulatory properties of rhIFN-alpha BCG suggest that it may be a superior agent for immunotherapeutic protocols involving live BCG in humans.

BCG-induced urinary cytokines inhibit microvascular endothelial cell proliferation.

PURPOSE: Angiogenesis is thought to depend on a net balance of molecules that inhibit or stimulate microvascular endothelial cells. A variety of molecules that affect angiogenesis are induced locally by the administration of intravesical bacille Calmette-Guerin (BCG) for superficial bladder cancer. We sought to determine whether BCG-induced urinary cytokines alter the effects of patient urine on assays of angiogenic activity. MATERIALS AND METHODS: Patients undergoing BCG treatment provided urine samples before and at peak cytokine production times after BCG instillation. Fifty-four urine samples from 8 patients were analyzed by ELISA for a panel of molecules known to affect angiogenesis, and tested for angiogenic activity in human dermal microvascular endothelial cell (HDMEC) proliferation and migration assays. To assess the role of specific BCG-induced cytokines, urinary HDMEC proliferation assays were repeated in the presence of neutralizing antibodies to tumor necrosis factor-alpha (TNF-alpha), interferon-inducible protein-10 (IP-10), and/or interferon-gamma (IFN-gamma). RESULTS: Urinary IFN-gamma, IP-10, TNF-alpha, and vascular endothelial growth factor (VEGF) were induced to nanogram/ml amounts by BCG treatment. While pre-BCG treatment urine samples minimally stimulated microvascular endothelial cell proliferation (+ 9%), post-BCG treatment urine became progressively inhibitory to endothelial cells (to -85%, p = 0.005) during weekly treatment courses. Neutralizing antibodies to TNF-alpha or to IP-10, either alone or in combination, greatly reduced this inhibitory effect. CONCLUSIONS: Intravesical BCG induces a cytokine-rich urinary microenvironment that is inhibitory to human endothelial cells. Urinary cytokine profiles and assays of angiogenic inhibition may provide prognostically important information regarding BCG treatment outcomes.

Interleukin-12. Opportunities for the treatment of bladder cancer.

Despite incomplete understanding of the human immune system, the rapid progress in tumor immunology provides a framework for more effective and safe interventions in the near future. Early approaches in patients with cancer that have focused on the nonspecific and broad stimulation of the immune system by interferons and IL-12 will be replaced by the highly specific stimulation of immune reactions targeting precisely defined tumor antigens. IL-12 has several biologic properties that seem useful in immune therapy for bladder cancer. The striking antitumor responses with IL-12 in preclinical animal models of bladder cancer provide optimism that future clinical trials involving this agent may impact on the risk and mortality associated with this disease.

Clinical and pathological characteristics of micropapillary transitional cell carcinoma: a highly aggressive variant.

PURPOSE: We present preliminary clinical, histochemical and molecular findings for 5 patients with micropapillary transitional cell carcinoma of the bladder, a rare histological variant not widely recognized in the urological literature. MATERIALS AND METHODS: The 5 patients were prospectively identified. In 3 cases immunohistochemical staining for expression of CD31, p53, E-cadherin, and alpha, beta and gamma-catenin was performed on paraffin embedded tissue. Sequencing was used to identify point mutations in exons 5 to 9 of p53, and exons 1 and 2 of H-ras. RESULTS: Of the patients 2 died within 1 year of presentation to our institution with rapid local extension along the bladder serosal surface and ureteral sheaths. Another patient had progression to invasive disease within 22 months. In the 3 cases with immunohistochemical staining p53 was negative, despite positive staining of nonmicropapillary transitional cell carcinoma within the same specimen. Stains for the angiotrophic marker CD31 were negative. In all 3 cases normal membrane associated alpha, beta and gamma-catenin expression was present. Examination of p53 sequences revealed a single point mutation in exon 8 of 1 case. In 2 cases different mutations in exon 1 of H-ras were noted. CONCLUSIONS: Micropapillary transitional cell carcinoma is a rare and highly aggressive variant. Paradoxically, our study demonstrated no significant p53 abnormalities. The lacunar histological pattern did not appear to represent invasion of vascular spaces. Rather, these tumors seemed to have the ability to disrupt and replace the normal stromal matrix to achieve rapid nonendothelial extension. Thus, micropapillary histology may predict a lesser likelihood of surgical cure.

Co-expression of interleukin-2 and green fluorescent protein reporter in mycobacteria: in vivo application for monitoring antimycobacterial immunity.

Recombinant Mycobacterium bovis bacillus Calmette-Guérin expressing green fluorescent protein (rBCG-GFP), driven by the mycobacterial heat shock protein 70 (HSP 70) promoter from an autonomously replicating plasmid, was genetically engineered to co-express mouse interleukin-2 (IL-2) by introduction of an independent HSP 60 promoter. To monitor host antimycobacterial immunity, C57BL/6 mice were intravenously infected with IL-2 expressing and non-expressing GFP rBCGs. Both rBCGs were clearly imaged and easily quantified with ultraviolet microscopy of tissue sections and whole organ suspensions. Enhanced mycobacterial clearance from the spleens of mice infected with the rBCG-IL-2/GFP strain was apparent by both diminished bacterial counts and spleen weights during the first 6 weeks post-infection relative to rBCG-GFP. T helper type 1 (TH1) cytokine production and proliferative response to BCG restimulation was also elevated from in vitro splenocyte cultures taken from the rBCG-IL-2/GFP-infected group. Taken together, these results suggest that IL-2 expression from rBCG augmented host protective immunity to mycobacterial infection via an enhanced TH1 immune response. Mycobacterial expression vectors that allow simultaneous but independent production of reporter proteins and bioactive substances provide an ideal means for monitoring the in vivo fate of recombinant mycobacteria.

Role of IL-12 in the induction and potentiation of IFN-gamma in response to bacillus Calmette-Guérin.

Although Mycobacterium bovis bacillus Calmette-Guérin (BCG) has been accepted as the most effective agent in clinical use against superficial bladder cancer, its mechanism of action remains incompletely understood. A kinetic analysis in assessing the potential role of cytokines from BCG-stimulated murine splenocytes showed that IL-12 expression preceded that of other cytokines. Experiments subtracting endogenous BCG-driven IL-12 using neutralizing Ab or augmenting its activity with supplemental rIL-12 revealed not only that IL-12 plays a dominant role in IFN-gamma induction but also that it is normally dose limiting. A striking increase in IFN-gamma production could be generated in both mouse and human immunocompetent cell culture by the addition of even a small amount of rIL-12. Moreover, this same synergistic effect could be replicated during in vivo administration of BCG plus rIL-12 into the mouse bladder and was observed in a patient receiving intravesical combination therapy. In costimulation cultures, this synergy appeared to partially rely on IL-18 and IL-2 and could be down-regulated by IL-10. This suggests that a dynamic interplay between Th1 and Th2 cytokines is responsible for net IFN-gamma production. The ability of supplemental exogenous IL-12 to strongly shift this balance toward Th1 provides an immunological basis for using it in conjunction with intravesical BCG for bladder cancer immunotherapy.

Mutated ras p21 as a target for cancer therapy in mouse transitional cell carcinoma.

PURPOSE: To establish an experimental mouse model for bladder cancer immunotherapy using mutated ras as a target. MATERIALS AND METHODS: A tumorigenic mouse bladder transitional cell carcinoma (TCC) line MB49 (C57BL/6 origin) was analyzed for its c-ras gene status by DNA cloning and sequencing. Aberrant expression of the ras gene was measured with Western blotting. 13-mer peptides corresponding to residues 5 to 17 of the ras protein were synthesized and tested for immunogenicity in syngeneic C57BL/6 mice. Induction of specific immune responses was evaluated by analyzing splenocyte activity in vitro and tumor suppression in vivo. RESULTS: MB49 cells were found to contain a single amino acid substitution of serine for glycine at codon 12 in K-ras loci and an abundant amount of cellular mutated ras p21 protein. C57BL/6 mice immunized with the 13-mer serine-containing ras peptide exhibited mutation-specific immune responses in splenocyte proliferation, cytokine production and cytotoxicity. Specific antitumor immunity in the form of tumor growth delay in vivo was observed in mice immunized with the same mutant peptide followed by subcutaneous MB49 tumor challenge and was enhanced by the addition of low dose interleukin-12. CONCLUSIONS: The mouse bladder TCC line MB49 contains a serine mutation at codon 12 of its K-ras gene that is sufficient to induce mutation-specific immune responses in vitro and specific protective immunity to MB49 tumor in vivo. Mutated oncoproteins may be ideal targets for the development of specific immunotherapy regimens for bladder cancer immunotherapy.

An in vivo comparison of bacillus Calmette-Guérin (BCG) and cytokine-secreting BCG vaccines.

A recombinant bacillus Calmette-Guérin (BCG) vaccine has been developed, which constitutively secretes interleukin (IL)-2. Groups of deer were immunized with either normal BCG (Pasteur 1173 P2 strain) or recombinant BCG (rBCG/IL-2) and their immune responses were monitored over 3 months. Animals gained weight over this period and showed no signs of adverse reactions to either vaccine. Lymphocyte transformation responses did not differ significantly between the two groups. No antibody that was specific for BCG was detected in any animal. Intradermal skin-test responses to BCG antigens showed that the rBCG/IL-2 induced a smaller delayed-type hypersensitivity response than the normal BCG. Cytokine transcription was determined by reverse transcription-polymerase chain reaction (RT-PCR). While IL-2 and interferon-gamma (IFN-gamma) levels did not differ significantly between the two groups, the level of IL-4 was found to be lower in the group given rBCG/IL-2. This resulted in a strong interferon-gamma:IL-4 ratio, suggesting a skewing of the immune response towards a Type 1 response. The rate at which the vaccine was eliminated from the host was the same regardless of whether BCG or rBCG was used. At autopsy (3 months after vaccination) 99.99% of the organisms had been eliminated. The small number of organisms isolated from the draining lymph node of animals given rBCG/IL-2 were grown in antibiotic-containing media. They were shown to still contain the shuttle plasmid and to secrete biologically active IL-2, indicating that the plasmid was stably maintained despite the host's immune response and in the absence of antibiotic selection.