P. A. Steerenberg

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

SummaryTo 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 α (TNFα), and interferon γ (IFNγ) 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, TNFα was only detected after repeated BCG instillations. Generally the highest TNF levels were found after BCG instillation 5. The presence of IFNγ 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-1α. In conclusion, activation of BCG-specific T cells was indicated by the detection of IL-2. The presence of IL-1, IL-6, and TNFα 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.

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

SummaryTo 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 (t0) and 24 h (t24) and 48 h (t48) 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 att0,t24, andt48 after repeated BCG instillations. The lymphocyte population consisted mainly of T cells (86% CD3+,t0). Most of the T cells were CD4+ (helper/inducer) and were significantly decreased at 48 h (62±9% att0 vs 49±6% att48). Lymphocytes partly expressed HLA-DR antigens (44%,t0). 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% att0 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.

Leukocytes in the urine after intravesical BCG treatment for superficial bladder cancer

SummaryCellular immunologic reactions occurring in the bladder after intravesical treatment with bacillus Calmette-Guérin (BCG) were investigated by flow cytofluorometric analysis of leukocytes present in the urine. Urine specimens from 11 superficial bladder cancer patients were collected before and 5, 24, 48 and 72 h after repeated BCG instillations. Monoclonal antibodies specific for granulocytes, monocytes/macrophages, and T-and B-lymphocytes were used to characterize and quantify leukocyte subpopulations. The total number of cells in urine was found to be 2- to 485-fold increased 24 h after BCG administration. The predominant cell type present was the polymorphonuclear granulocyte, probably representing a defense mechanism against mycobacteria. The main mononuclear leukocytes in urine specimens were monocytes/macrophages and T-lymphocytes, indicating an ongoing immune response in the bladder wall. Although percentages of lymphocytes were low, T- and B-cells could be identified using a selective cell measurement procedure. In conclusion, a clear increase in the numbers of granulocytes, monocytes/macrophages and T-lymphocytes in urine after intravesical BCG administration was demonstrated, indicating local activation of the immune system.

Induction of an antibody response in mice against human papillomavirus (HPV) type 16 after immunization with HPV recombinant Salmonella strains

Abstract Human papillomaviruses (HPV) are present in approximately 95% of all cervical carcinomas and the HPV E6 and E7 genes are continuously expressed in these lesions. There is also circumstantial evidence that often natural immunity against HPV is generated and that this is of influence on HPV-induced lesions. Stimulation of the immune system by proper presentation of relevant HPV antigens might, therefore, lead to a prophylactic or therapeutic immunological intervention for HPV-induced lesions. For this purpose we have expressed the E6 and E7 protein of HPV 16 in an attenuated strain of Salmonella typhimurium (SL3261, aroA mutation), which has been used extensively as a live vector. Live recombinant Salmonella vaccines have the ability to elicit humoral, secretory and cell-mediated immune responses, including cytotoxic T cells, against the heterologous antigens they express. This report describes the construction of recombinant Salmonella strains expressing the HPV 16 E6 and E7 proteins, and the induction of an HPV-16-specific immune response in mice after immunization with these live vectors.

Local interleukin-2 therapy in bovine ocular squamous cell carcinoma. A pilot study.

SummaryFive cows bearing bovine ocular squamous cell carcinoma (BOSCC) were treated with low doses of recombinant human interleukin-2 (rhIL-2). A dose of 2500 U rhIL-2 was injected intralesionally and another 2500 U were injected into the subparotid regional lymph node once a day during a period of 5 consecutive days. This cycle of 5 days was repeated after an interval of 2 days. Total regression of the tumor was observed in three out of five animals. One cow showed tumor regression (> 80%) accompanied by metastases to the regional lymph node that were observed from the fifth week after the beginning of the treatment. Growth of the tumor of the fifth animal was retarded after treatment. In vitro proliferation of peripheral blood lymphocytes was investigated in two animals and tumor-infiltrating lymphocytes in one animal during incubation in various rhIL-2 concentrations. Cytotoxic activity of both cell populations against P815, Yac-1 and BOSCC-derived cell lines increased during incubation with rhIL-2. Cultured BOSCC-infiltrating lymphocytes showed predominant killing of the BOSCC-derived autologous cell line after 4 weeks of culture. Preliminary phenotype analysis did not give conclusive results with respect to the types of cells responsible for killing.

Liposomes as drug carrier system for cis-diamminedichloroplatinum (II)

SummaryIn this study we have investigated the use of liposomes as a drug carrier system for cis-diamminedichloroplatinum (II) (cis-DDP) in order to reduce the nephrotoxicity with preservation of antitumor activity. Liposomes (PC/PS/Chol 10:1:4) were prepared using hydration media containing no or a relatively low concentration of NaCl. It was found that cis-DDP containing liposomes (lip cis-DDP) injected i.v. to IgM immunocytoma-bearing LOU/M rats at a dose of 1 mg cis-DDP/kg (cumulative dose 7 mg cis-DDP/kg) showed less antitumor activity than the free drug. The optimal cumulative dose of free cis-DDP for induction of antitumor activity in this tumor system is 7 mg/kg (7x1 mg/kg). At a dose of 2 mg lip cis-DDP/kg (cumulative dose 14 mg cis-DDP/kg) the antitumor activity was almost equal to that of free cis-DDP. The antitumor activity could not be increased by choosing another phospholipid composition of the liposomes [DPPC/DPPG/Chol (10:1:10)] cis-DPP incorporated in DPPC/ DPPG/Chol liposomes showed a similar antitumor activity to cis-DDP incorporated in PC/PS/Chol liposomes. After an i.v. dose of 2mg lip cis-DDP/kg (PC/PS/Chol) kidney damage was less compared to the treatment with free cis-DDP (1 mg/kg). However, after a single dose of 2 mg cis-DDP/kg or a cumulative dose of 8 or 16 mg cis-DDP/kg, kedneys of rats treated with lip cis-DDP contained twice as much Pt as after treatment with free cis-DDP. Moreover, after treatment with lip cis-DDP, a twofold increase of the amount of Pt in tumor tissue was measured. In vitro studies with Pt recovered from spleens obtained from rats treated with lip cis-DDP i.v. showed that based on the equal amounts of Pt recovered the antitumor activity of the recovered Pt was reduced, indicating inactivation of cis-DDP in vivo. As during treatment with free cis-DDP, recurrence of the tumor was observed during the continued treatment with lip cis-DDP. It was found that these recurrent tumors were resistant to further therapy with cis-DDP. In conclusion, cis-DDP encapsulation into liposomes decreased the nephrotoxicity. The antitumor activity of cis-DDP is preserved by liposome encapsulation when it was used at a dose of 2 mg/kg, but it was reduced in terms of earlier onset of regrowth.

Impaired Immune Response by Isoniazid Treatment During Intravesical BCG Administration in the Guinea Pig

At present, isoniazid (INH) is being used prophylactically to reduce the side effects of intravesical BCG therapy for superficial bladder cancer, although it is not clear whether or not this reduces the antitumor efficacy of BCG. In this study the impact of INH treatment on the immune response after repeated intravesical BCG administration was investigated in guinea pigs. INH was given during 3 days starting on the day prior to the BCG administration. It was found that the administration of INH severely impaired the immunological effects of BCG. The induction of mononuclear cell infiltration in the bladder wall was reduced. Enlargement of the regional lymph nodes (weight and number of cells), and increase of Major Histocompatibility Complex (MHC) class II expression on the lymph node cells, normally observed after intravesical BCG administration, were inhibited by INH. Systemic immunity, measured by the DTH reaction in the skin to PPD, was also diminished due to the combined treatment of BCG with INH. A five-fold increase of the dose of BCG did not overcome the effect of INH. INH probably did not exert a direct suppression of the immune system of the guinea pig as the DNCB skin reactivity was not influenced. Although INH concentrations in the urine were high at the onset of the instillation, in vitro experiments indicated that the effect of INH was probably not caused by killing of the BCG organisms shortly after application in the bladder. In conclusion, our data in guinea pigs suggest that the use of INH may impair the immune response to intravesical BCG. As this response may be important for the antitumor effect of BCG, urologists should be cautious with the prophylactic use of INH. The influence on the antitumor efficacy is now investigated in man.

A comparative study on the antitumor effect, cardiotoxicity and nephrotoxicity of doxorubicin given as a bolus, continuous infusion or entrapped in liposomes in the Lou/M Wsl rat

SummaryLiposome encapsulation of doxorubicin (DXR) has been shown to increase the therapeutic index of the drug in several animal systems. The prevention of peak plasma concentrations of free drug might be a major factor contributing to the beneficial effects resulting from liposome encapsulation. If so, the administration of DXR as a continuous infusion should also lead to an improved therapeutic index. In the present paper, the administration of liposome-encapsulated DXR is compared with the infusion of DXR with regard to their potential to preserve antitumor activity, enhance survival and reduce cardiomyo- and nephropathy in IgM immunocytoma-bearing Lou/M Wsl rats. Plasma concentrations of DXR were determined to correlate the biological results with pharmacokinetic parameters. Liposomes containing phosphatidylcholine, phosphatidylserine and cholesterol (extrusion-multilamellar vesicles) were used. Bolus injections of free DXR (free DXR) and DXR liposomes (lip-DXR) in a multiple-dose regimen were compared with 24-h infusions of the same cumulative doses of DXR (inf-DXR). The antitumor activity of inf-DXR equalled that of free DXR as well as that of lip-DXR at doses of >0.25 mg/kg. The overall survival of tumor-bearing animals treated with 2.0 mg/kg lip-DXR was significantly prolonged (P<0.01) in comparison with that of animals treated with 2.0 mg/kg free DXR; however, treatment with 2.0 mg/kg inf-DXR did not induce a significant prolongation of survival. At a cumulative dose of 12 mg/kg, inf-DXR appeared to be as effective as lip-DXR in reducing the severity of cardiomyopathy induced by free DXR. However, for the reduction of nephropathy, only therapy with lip-DXR was effective Inf-DXR induced high nephropathy scores comparable with those obtained with free DXR. For the first 24 h after an injection of 2.0 mg/kg or after the start of a continuous infusion of 2.0 mg/kg given over 24 h, similar areas under the plasma concentration-time curves (AUC) were calculated for free DXR and inf-DXR. However, for lip-DXR a much higher value was calculated. The higher plasma levels of lip-DXR did not result in higher cardiac levels. After five daily doses of 2.0 mg/kg, a much lower DXR concentration was found in cardiac tissue after the administration of lip-DXR than after the administration of free DXR or inf-DXR. This suggests that an important parameter to be determined and correlated with biological results is the free (i.e. not bound to liposomes) circulating fraction of DXR in lip-DXR-injected animals. In conclusion, in the IgM immunocytoma system the administration of DXR as a continuous infusion was as effective as DXR encapsulated in liposomes in reducing cardiotoxicity while preserving the antitumor effect; this indicates that the avoidance of peak plasma levels is an important component of the mode of action of DXR-containing liposomes. However, if both antitumor efficacy and nephrotoxicity are taken into account, lip-DXR could be considered to be superior to inf-DXR.

Morphological aspects of the interaction of Bacillus Calmette-Guérin with urothelial bladder cells in vivo and in vitro: relevance for antitumor activity?

SummaryIntravesical administration of Bacillus Calmette-Guérin (BCG) has been shown to be effective in the treatment of patients with superficial bladder cancer. For a better understanding of the mechanism of this antitumor activity, scanning and transmission electron microscope (SEM, TEM) studies were carried out to investigate morphological aspects of the interaction of BCG with the bladder wall in vivo and in vitro. Adherence of BCG to the bladder wall in vivo was studied 1 and 24 h after single or multiple (6×) BCG instillations in intact and in electrocauterized guinea pig bladders. Despite extensive search with SEM for its presence, virtually no BCG was found on the intact urothelium, and BCG was only occasionally observed in the coagulation lesions. SEM and TEM studies revealed adherence and phagocytosis of BCG by the T24 human bladder carcinoma cell line in vitro. Time sequence studies on the phagocytosis and fate of BCG showed that T24 cells are capable of progressively degrading the mycobacteria in phagolysosomes. However, BCG did not alter MHC class II antigen expression on T24 cells in vitro. In contrast, 54 urine sediments and bladder washings of 11 bladder cancer patients, taken prior to or after several intravesical BCG instillations, failed to demonstrate urothelial (tumor) cells showing evidence of BCG phagocytosis (682 cells screened by TEM), while BCG was phagocytized avidly by leukocytes. These data suggest that a direct interaction of BCG with urothelial bladder cells in vivo can be called in question.

Presence of interleukin-2 in urine of superficial bladder cancer patients after intravesical treatment with bacillus Calmette-Guérin

SummaryUrine samples were obtained from patients with superficial bladder cancer after immunotherapy with bacillus Calmette-Guérin (BCG). The patients were repeatedly (once a week for 6 consecutive weeks) treated with intravesical administration of approximately 5 × 108 culturable particles of BCG. Some patients received more than six BCG instillations. The urine samples were investigated for the presence of interleukin-2 (IL-2) in an in vitro bioassay using a murine cytotoxic T cell line (CTTL-16) that shows IL-2-dependent growth. Preliminary experiments indicated the presence of inhibitory factors in the urine. This inhibitory activity was abolished after 24 h dialysis. In a neutralization assay with both polyvalent and monoclonal anti-(human IL-2) antibody it was demonstrated that there was indeed IL-2 in the urine samples. In 8 of 11 patients the presence of IL-2 in the urine was demonstrated. The IL-2 production was directly related to the BCG administration as samples obtained just before the BCG instillation were always negative. In IL-2-positive samples a maximum level of IL-2 was observed between 2 h and 6 h after the BCG instillation. In urine samples obtained 24 h after the BCG IL-2 was not detected. In most patients the urine became positive after the third or fourth BCG instillation