Halting targeted and collateral damage to red blood cells by the complement system.

The complement system is an important defense mechanism against pathogens; however, in certain pathologies, the system also attacks human cells, such as red blood cells (RBCs). In paroxysmal nocturnal hemoglobinuria (PNH), RBCs lack certain complement regulators which sensitize them to complement-mediated lysis, while in autoimmune hemolytic anemia (AIHA), antibodies against RBCs may initiate complement-mediated hemolysis. In recent years, complement inhibition has improved treatment prospects for these patients, with eculizumab now the standard of care for PNH patients. Current complement inhibitors are however not sufficient for all patients, and they come with high costs, patient burden, and increased infection risk. This review gives an overview of the underlying pathophysiology of complement-mediated hemolysis in PNH and AIHA, the role of therapeutic complement inhibition nowadays, and the high number of complement inhibitors currently under investigation, as for almost every complement protein, an inhibitor is being developed. The focus lies with novel therapeutics that inhibit complement activity specifically in the pathway that causes pathology or those that reduce costs or patient burden through novel administration routes.

Immunostimulation in the urinary bladder by local application of Nocardia rubra cell-wall skeletons (Rubratin) and bacillus Calmette-Guérin as therapy for superficial bladder cancer: a comparative study.

Twelve patients with superficial bladder cancer were treated with intravesical instillations of Rubratin (ASTA Pharma AG, Frankfurt, Germany), a cell-wall preparation of Nocardia rubra. The objective was to compare the immunostimulating effect of Rubratin with that of bacillus Calmette-Guérin (BCG). Local immunostimulation was determined by cytokine induction in serially collected urine samples during the first 24 h after each instillation, leukocyte influx into the urine, and phenotypic analysis of the lymphocyte fraction. Levels of Rubratin-induced interleukin (IL)-1 beta, IL-6, and tumor necrosis factor-alpha were significantly elevated compared with pretherapy levels. Rubratin induced leukocyte influx into the urine. T-cell activation (IL-2 receptor and human leukocyte antigen-DR expression) can be induced, and CD4:CD8 cell ratios can be increased. All parameters indicated that Rubratin-induced immunostimulation was less than that associated with BCG. In conclusion, although local Rubratin-induced immunostimulation occurs in a limited number of patients, the amount of immunocompetent cells attracted to the bladder seems to be less than that associated with BCG therapy, thus resulting in lower levels of cytokine production (which may reflect less clinical efficacy).

Urinary cytokines reflecting the immunological response in the urinary bladder to biological response modifiers: their practical use.

BACKGROUND: Intravesical bacillus Calmette-Guérin (BCG) immunotherapy is currently the most effective treatment for superficial transitional cell carcinoma (TCC) of the urinary bladder. In recent years, the substantial number of patients not responding to BCG or experiencing considerable toxicities has stimulated studies addressing either the development of improved BCG treatment schedules or the exploration of the therapeutic value of a series of (novel) biological response modifiers, like interferons (IFNs), interleukin (IL) 2 and keyhole limpet hemocyanin. Although the actual mechanism by which BCG exerts its antitumor effect still needs detailed unraveling, current available knowledge suggests the induction of a T helper 1 (Th1) or Th1-like cytokine profile, represented by IL-2, IL-12 and IFN-gamma, as essential in the development of a cell-mediated antitumor activity. CONCLUSIONS: In this review, it is argued that incorporation of urinary cytokine determinations, like IL-2 and possibly IL-12 and IFN-gamma, may represent a valuable approach in the optimization and individualization of the BCG therapy and an early, initial evaluation of the potential efficacy of novel immunomodulating agents in the treatment of superficial TCC.

Retrovirus type C in the mouse bladder carcinoma cell line MBT-2.

PURPOSE: The presence of replicating type C retrovirus in MBT-2 mouse bladder carcinoma cells is reported. This MBT-2 tumor cell line is nowadays globally distributed. The cells have been and are still used to study various aspects of bladder cancer. While studying the phagocytic capacity of MBT-2 cells for BCG organisms by electron microscopic methods, the presence of this retrovirus was noticed. MATERIALS AND METHODS: MBT-2 cells that were cultured in vitro as well as cells from intravesically and intradermally grown MBT-2 tumors from syngeneic mice were investigated using transmission electron microscopy (TEM) and scanning electron microscopy (SEM) techniques. RESULTS: All samples including the earliest generation MBT-2 cells that could be traced from stocks of other research groups contained the C type retrovirus, suggesting a contamination in all available generations of the MBT-2 cell line. CONCLUSIONS: As this tumor cell line is widely used in immunologic studies of the response to bladder cancer, it is important to consider the possible presence of type C viruses and associated antigens, since they could contribute to or interfere with the responses being measured. Studies should be initiated to determine whether viral antigen expression is involved in the immune rejection of MBT-2 bladder cancer. As a consequence, clinical implementation of immunological treatment strategies should not be based on results obtained with the MBT-2 model alone, but preferably should be confirmed with other (bladder) carcinoma models.

Polymerase chain reaction based method for the detection of BCG retention after intravesical instillation in guinea pig bladders.

OBJECTIVE: In intravesical Bacille bilié de Calmette-Guérin (BCG) immunotherapy of superficial bladder cancer, a T cell mediated immunological reaction is associated with the antitumor activity. To gain insight into the approximate number of BCG bacteria retained in the normal, noninjured, urinary bladder after intravesical application responsible for induction of the immune reaction, the utility of a sensitive polymerase chain reaction (PCR) based assay was investigated in a guinea pig model. METHODS: After one single or six subsequent weekly instillations with 1x10(7) CFU of BCG, the bladders were resected and processed for BCG determination with PCR. The bladders were resected 24 h after instillation, aiming at (semi)quantifying the number of BCG organisms able to resist the natural voiding washout of the bladder. The PCR was based on amplification of a 249 base pair fragment of the insertion element IS6110 and is specific for bacteria belonging to the Mycobacterium tuberculosis complex which includes Mycobacterium bovis BCG. RESULTS: After one single instillation no detectable BCG retention was found. However, after six weekly instillations, BCG bacteria could be demonstrated in 2 out of 5 guinea pig bladders, indicating that the number of adhering BCG organisms was around the detection limit of the assay (600-1,000 BCG bacteria per bladder). CONCLUSIONS: The data suggest that after six instillations, the retention of BCG in the guinea pig bladder is enhanced as compared with one single instillation. This finding is suggestive of a role of the inflammatory process that is, besides immune system mediated reactions, associated with intravesical BCG instillations. The nature of the molecules involved in enhanced BCG retention after repeated instillations remains to be investigated.

Urinary interleukin-2 monitoring during prolonged bacillus Calmette-Guerin treatment: can it predict the optimal number of instillations?

PURPOSE: In patients with superficial bladder cancer treated with a first 6-week instillation course of bacillus Calmette-Guerin (BCG) the induction pattern of urinary interleukin (IL)-2 has been described, and the levels of urinary IL-2 were associated with the clinical response to BCG treatment. We evaluated urinary IL-2 kinetics in patients with recurrent superficial bladder tumor receiving a second or third 6-week BCG instillation course. To our knowledge there have been no studies of prolonged BCG treatment and urinary cytokine responses. MATERIALS AND METHODS: Urinary IL-2 was determined in 12 patients with superficial transitional cell carcinoma of the bladder receiving a complete (6-week) second or third BCG instillation course and in 3 patients receiving 3 BCG instillations during a maintenance schedule at month 3. Urinary IL-2 was determined with an enzyme-linked immunosorbent assay using an oligoclonal system. RESULTS: Of 12 patients 10 had a urinary IL-2 positive response during the subsequent BCG course and at week 1 urinary IL-2 was already increased. Comparing the urinary IL-2 kinetics observed during a second or third with a first course, urinary IL-2 tended to be higher during the first and lower during the last weeks. If the interval between subsequent courses was short (12 months or less) significantly higher urinary IL-2 levels at weeks 1 and 2, and a lower level at week 6 were observed. CONCLUSIONS: During a repeat BCG instillation course urinary IL-2 reached a maximum at an earlier week, especially if the interval between the subsequent courses was short. Since an association between urinary IL-2 levels and response to BCG treatment during an induction course has been observed, these immunological data argue in favor of a limited number of instillations during prolonged BCG therapy which could reduce side effects as well as costs.

The Bard BTA test: its mode of action, sensitivity and specificity, compared to cytology of voided urine, in the diagnosis of superficial bladder cancer.

OBJECTIVES: Application of immunocytology directed against antigens of urothelial tumor cells or tumor-associated breakdown products intends to improve the sensitivity of the diagnosis of superficial transitional cell carcinoma (TCC) of the urinary bladder. In this study, the mode of action, sensitivity and specificity of the Bard(R) BTA test, detecting a tumor-associated release of a basement membrane complex, was addressed and compared with voided urine cytology (VUC). RESULTS: Contrary to grade, a significant (p = 0. 003) relationship between tumor stage and BTA test sensitivity was observed, being 23.8, 33.3 to 100% for Ta (n = 42), T1 (n = 6) to >/=T2 (n = 5), respectively. These data suggest an association between an increase of the BTA test sensitivity with an increase of basement membrane degradation or interruption. With regard to this mechanism, the BTA test may be of special importance for monitoring tumor progression or increase in tumor invasiveness. Detection of low-stage, low-grade tumors by noninvasive techniques remains a challenge. The sensitivity of the BTA test for the presence of TCC was 32.3%, while that of VUC was 17.7%, but in this study the difference was not statistically significant. Furthermore, the BTA test was not more effective in identifying the various tumor grades, stages or stage/grade groupings. However, dividing the patients in two groups of low risk (TaG1/TaG2) and high risk (TaG3 to >/=T2) leads to a significant (p = 0.008) increased sensitivity of the BTA test (27.3%) in detecting patients with low-risk tumors compared to VUC (3.0%). The specificity of the BTA test in patients with a history of TCC was 81.6%, while that of VUC was 98.9%. CONCLUSION: The sensitivity of the BTA test is at least equivalent to VUC and may be suited to monitor increase in stage in patients suffering from bladder carcinoma, but cannot replace cystoscopy in patients suspected for a bladder tumor.

BCG-induced interleukin-6 upregulation and BCG internalization in well and poorly differentiated human bladder cancer cell lines.

Intravesical bacillus Calmette-Guerin (BCG) is a successful therapy for superficial bladder cancer. However, the working mechanism of BCG after intravesical instillation is not completely understood. A functional role of urothelial (tumor) cells in the initiation of the BCG-induced immune reaction should be considered. Here, the possibility of a causal relationship between BCG-induced interleukin-6 (IL-6) synthesis and BCG internalization by urothelial tumor cells was examined in a series of human transitional bladder cancer (TCC) cell lines with different degrees of differentiation. The results showed that the well differentiated TCC cell lines, RT4, SBC-2, and SBC-7, did not possess the capacity to internalize BCG, which was associated with an inability to upregulate IL-6 synthesis when stimulated with BCG. Moreover, these cell lines expressed a low level of constitutive IL-6 synthesis. In contrast, the poorly differentiated TCC cells, T-24, TCC-SUP and J-82, were able to internalize BCG. In T24 and J82, but not in TCC-SUP cells, BCG internalization appeared to result in an upregulation of IL-6 synthesis. Constitutive IL-6 synthesis of the high grade cell lines was found to be cell line-dependent: both TCC-SUP and J82 cells exhibited a high level of constitutive IL-6 synthesis, whereas T24 cells exhibited a low level. The possible relationship between BCG internalization and IL-6 upregulation was studied in detail with the T24 cell line, which exhibited a low constitutive and high BCG-inducible IL-6 synthesis, using anti-BCG antibodies (alphaBCG) and Cytochalasin B as internalization inhibitors. Upregulation of IL-6 synthesis was significantly inhibited by alphaBCG or Cytochalasin B, indicating that internalization is a prerequisite for BCG-induced upregulation of IL-6 synthesis. In conclusion, upregulation of IL-6 production due to BCG internalization by poorly differentiated bladder carcinoma cells may be part of the mode of action of intravesical BCG therapy.

[Changes in the cytokine concentration during immunotherapy of superficial bladder tumors]. / Vizelet cytokinek koncentrációjának változása felületes hólyagdaganatok immunterápiája során.

A growing number of data support the importance of urinary cytokines in the BCG immunotherapy of superficial bladder tumours. To investigate kinetics and stability, urinary levels of IL-8, IL-2 and IL-6 cytokines after BCG treatment, were determined. Significant elevation in the level of IL-8 was established immediately following the first BCG instillation and it reached its highest value 4-6 hours after the treatment. During the first three weeks of the treatment and follow-up period IL-8 peaked significantly earlier than IL-2 or IL-6. Its early appearance associated with high stability makes IL-8 a good candidate for being considered as an effective agent with high predictive value in BCG immunotherapy.

Effects of isoniazid on the proliferation and cytokine production of bladder cancer cells in vitro induced by bacille Calmette-Guérin.

OBJECTIVE: To determine the effects of isoniazid (isonicotinic acid hydrazide), used to reduce the serious side-effects of immunotherapy of superficial bladder cancer with bacille Calmette-Guérin (BCG), on the proliferation and constitutive BCG-induced synthesis of interleukins 6 (IL6) and 8 (IL8) in human bladder cancer cells cultured in vitro. MATERIALS AND METHODS: Three poorly differentiated human cell lines, T24, TCC-SUP and BT-B, were used to study the effect of isoniazid on cell proliferation. Cells were inoculated in tissue culture plates and various concentrations of isoniazid added to the medium. Cell density was then monitored for up to 6 days using a colorimetric assay. To determine the effects of isoniazid on constitutive and BCG-induced cytokine synthesis, cells were cultured in medium containing no additions, BCG, isoniazid or BCG with isoniazid, at various concentrations. Samples of medium were collected regularly for 6 h and the cytokine content (IL6 and IL8) determined using enzyme-linked immunosorbent assays. RESULTS: Continuous incubation of proliferating T24, TCC-SUP and BT-B cells with isoniazid at concentrations of 0-100 micrograms/mL did not affect the rate of proliferation. Unlike TCC-SUP and BT-B cells, T24 cells released more IL6 and IL8 during incubation with BCG. At 6 h after the addition of BCG, the cumulative mean (SD) IL6 and IL8 production of T24 cells was 2.6 (0.1) and 2.3 (0.4) ng per 3 x 10(5) cells, compared with a constitutive level of 0.1 (0.0) and 1.3 (0.2) ng, respectively. There was no significant effect of isoniazid (1-100 micrograms/mL) on either the constitutive or BCG-induced synthesis of IL6 and IL8 in T24 cells. CONCLUSION: Assuming an essential role of (tumour) epithelial cells in the local immune response induced by BCG, these in vitro results suggest that the administration of isoniazid does not interfere with this part of the mechanisms by which BCG operates.

Role of interleukin-8 in onset of the immune response in intravesical BCG therapy for superficial bladder cancer.

In intravesical therapy for superficial bladder carcinoma urothelial cells may, through the production of cytokines, contribute to the bacillus Calmette-Guerin (BCG)-induced local immunological reaction and associated antitumor efficacy. The aim of this study was to investigate such a role for the neutrophil-attracting cytokine interleukin-8 (IL-8). The appearance of IL-8 in patients urine after BCG therapy was compared with BCG-induced IL-6 and IL-2 and the stability of IL-8 in urine was tested. Compared to IL-6 and IL-2, a rapid induction of IL-8 was observed, occurring after the first BCG instillation. Urinary IL-8 was highly stable, even after 24 h incubation at 37 degrees C. The IL-8 concentration after the first instillation seemed to be associated with subsequent development of an immune response. Consequently, IL-8 seems an attractive candidate for investigation of its prognostic value for a clinical response to BCG therapy.

Immunostimulation in the urinary bladder by local application of Nocardia rubra cell wall skeleton preparation (Rubratin) for superficial bladder cancer immunotherapy--a phase I/II study.

OBJECTIVES: Twelve patients with superficial papillary transitional cell carcinoma of the bladder (pTa, pT1) were treated with six consecutive weekly intravesical instillations of Rubratin (in a dose of 1.5, 3.0, or 4.5 mg), a cell wall skeleton preparation of Nocardia rubra (NCW). The main objective of this study was to look for local immunomodulating effects of NCW and in the first four patients the effect on a marker lesion was also investigated. METHODS: Local immunostimulation in all 12 patients was determined by (1) measurement of cytokine induction [interleukin 1 beta (IL1 beta), IL2, IL6, and tumor necrosis factor alpha (TNF alpha)], (2) leukocyte influx into the urine, and (3) phenotypic analysis of the lymphocyte fraction of these leukocytes. RESULTS: Significantly elevated levels of Rubratin-induced IL1 beta (P < 0.001), IL2 (P < 0.001), IL6 (P < 0.01), and TNF alpha (P < 0.001) were found compared to control pretherapy levels. Rubratin also induced leukocyte influx into the urine. Fluorescence-activated cell sorter (FACS) analysis of the urinary leukocytes indicated T-cell activation (IL2 receptor and HLA-DR expression), while in two out of five patients the CD4/CD8 ratios were increased. Urinary cytokine induction by Rubratin was comparable with cytokine induction observed in nonresponding bacillus Calmette-Guérin (BCG) patients (recurrent tumor within 6 months), but less compared with responding BCG patients (no recurrent tumor within 6 months). Clinical results showed no response on the marker lesion and in five out of eight patients early recurrence was found after complete transurethral resection (TUR) of the bladder tumors. This biological response modifier caused no local or systemic side effects at the doses used. CONCLUSION: Although local immunostimulation by intravesical Rubratin administration can be induced, the amount of immunocompetent cells attracted to the bladder is not as high as observed in BCG-responding patients, resulting in lower amounts of cytokines produced. This could also explain the lack of clinical efficacy.

Double fluorescent flow cytometric assessment of bacterial internalization and binding by epithelial cells.

This study describes a new flow cytometric method for assessment of phagocytosis of specific bacteria (bacillus Calmette-Guérin (BCG) and Escherichia coli) by bladder epithelial cells. The internalization assay consisted of labeling bacteria chemically with fluorescein isothiocyanate (FITC). Subsequent to incubation of fluoresceinated bacteria with internalizing cells, adherent nonphagocytosed bacteria were marked by two-step labeling using specific antibodies and phycoerythrin (PE)-conjugated antibodies. Double fluorescent FACS analysis differentiated between bacterial phagocytosis and adherence. The validity of the method was shown by inhibition of BCG phagocytosis at 4 degrees C by cytochalasin B, by removal of excess free bacteria, and by anti-BCG antibodies. BCG-phagocytizing and -nonphagocytizing cell lines were discriminated by applying this technique to a series of bladder carcinoma cell lines. There seemed to be a relationship between phagocytic capacity and grade of differentiation in these cell lines, which may have implications for topical BCG immunotherapy in superficial bladder cancer. In conclusion, a new, reliable, rapid, and relatively simple double fluorescent method is described for quantification of specific bacterial internalization by large numbers of (bladder) epithelial cells. This method should be generally applicable to the study of in vitro interaction between bacteria and different types of host cells.

Urinary cytokines during intravesical bacillus Calmette-Guerin therapy for superficial bladder cancer: processing, stability and prognostic value.

PURPOSE: An accurate prognostic indicator to identify nonresponding patients with superficial transitional cell carcinoma of the bladder at an early stage of intravesical bacillus Calmette-Guerin (BCG) therapy is urgently needed. MATERIALS AND METHODS: The processing conditions and stability of several BCG induced urinary cytokines were analyzed, as was the possible correlation between these cytokines (indicating immune responsiveness to BCG) and bladder tumor recurrence. We studied 23 patients with superficial transitional cell carcinoma of the bladder. Monitoring was performed by serial collection of urine during the first 24 hours after each of the 6 consecutive weekly intravesical BCG instillations. Baseline pre-therapy cytokine levels were 3.9 +/- 4.7 pg./mumol creatinine for interleukin-6 and 0.1 +/- 0.2 pg./mumol creatinine for tumor necrosis factor-alpha (all measured by enzyme-linked immunosorbent assay). To investigate the correlation between interleukin-2 and bladder tumor recurrence, patients were stratified into 2 groups based on an early (6 months or less) or late (greater than 6 months) recurrent tumor. For each patient the highest cytokine value measured during the 6-week BCG treatment course was evaluated. RESULTS: The results were positive if the level in urine exceeded 0.34 units interleukin-2 per mumol. creatinine. A significant correlation between urinary interleukin-2 and tumor recurrence was found (p = 0.003, 23 patients). Of the studied cytokines obtained from BCG treated patients, interleukin-1 beta, 2 and 6 but not tumor necrosis factor-alpha were stable in urine at 4C and 20C. At 37C all cytokines were unstable. Interferon-gamma could only be detected in immediately dialyzed urine and its occurrence correlated most with that of interleukin-2. Processing of urine by centrifugation to remove leukocytes immediately after collection was not required for reliable measurements of interleukins-2 and 6. Based on these results interleukins-2 and 6 were preferred for extensive monitoring of the BCG induced immune reaction. CONCLUSIONS: Our study provides significant evidence for a correlation between urinary cytokine induction and clinical response following intravesical BCG therapy. Particularly, monitoring of interleukin-2 may have the potential for prognostic value provided that strict precautions regarding urine collection, such as maximal 2-hour sampling and immediate cooling, are taken.

Effects of sequential intravesical administration of mitomycin C and bacillus Calmette-Guérin on the immune response in the guinea pig bladder.

It has been suggested that intravesical treatment with mitomycin C (MMC) before instillation of bacillus Calmette-Guérin (BCG) improves the antitumor activity of BCG in human bladder cancer. Therefore, we studied the immunological effects of sequential intravesical treatment with MMC and BCG in the guinea pig. Four weekly intravesical instillations with MMC preceded six weekly intravesical BCG instillations. The delayed-type hypersensitivity (DTH) skin reaction evoked by tuberculin purified protein derivative (PPD) in guinea pigs receiving BCG intravesically appeared slightly earlier in animals pretreated intravesically with MMC than in phosphate-buffered saline (PBS)-pretreated animals. However, after completing BCG instillations no differences in DTH reaction were observed between these treatment groups. The extent of the local inflammatory reaction in the bladder wall, as well as the parameters measured in the draining iliacal lymph nodes (i.e., the weight, the number of leukocytes, and the composition of leukocyte subpopulations), did not differ in animals treated with BCG alone or in combination with MMC. A slight increase in the MHC class II expression on the bladder urothelium was shown if MMC and BCG treatment was combined. The adherence of mycobacteria to the bladder wall, measured using 3H-labeled mycobacteria, dit not differ between MMC/BCG- and BCG-treated animals. We conclude that MMC does not enhance the immune response against mycobacteria. Therefore, we hypothesize that a possible increased antitumor activity by the combination of MMC and BCG might be due to separate, rather than synergistic, effects of the drugs, namely a cytostatic effect of MMC on tumor cells and a local immune response in the bladder evoked by BCG.

Interaction between bacteria and the lumenal bladder surface: modulation by pentosan polysulfate, an experimental and theoretical approach with clinical implication.

An attempt was made to interpret bacterial adsorption to the lumenal surface of the urinary bladder wall under normal and pathological conditions according to the DLVO theory of lyophobic colloid stability, which describes the interaction between a bacterium and the bladder-wall surfaces as a balance of attraction and repulsion forces. Computer modeling suggested that a decrease in the surface potential of the bladder wall may well explain an increased bacterial adsorption, possibly associated with bacterial cystitis. With the intent of preventing bacterial adsorption, treatment of bacterial cystitis by intravesical instillation of pentosan polysulfate (PPS) was evaluated. PPS is a polysaccharide with high affinity (4 +/- 2 mg/bladder) to the bladder. The attachment of PPS strongly depends on the intrinsic properties of the bacterial surface. Theoretical considerations indicate that either complete coverage of the bacterium with PPS or an absence of PPS affinity is a prerequisite for obtaining steric interaction or prevention of bacterial sorption. Experimentally, an absence of PPS affinity (0-0.7 microgram/mg bacteria) was found for bacteria commonly found during cystitis. Immunological treatment of superficial bladder cancer by bacillus Calmette-Guérin (BCG) depends on the interaction of BCG with the bladder wall. Improvement of the treatment may be obtained by increasing BCG adsorption. In this respect, the phenomenon of bridging in which PPS binds simultaneously to both BCG and the bladder-wall surface was investigated. Theoretical considerations and experimental results appeared to be in good agreement. It was found that BCG binds a considerable amount of PPS (3.4 +/- 0.3 microgram/mg BCG). In a guinea pig model the theoretical considerations, indicating the occurrence of bridging at a low and narrow range of PPS concentrations, seemed to be confirmed. In contrast to a high PPS concentration of 10 mg/ml, at a low (0.1 mg/ml) PPS concentration a significant stimulation of the BCG-associated immune reaction(s) was observed. The results suggest that to obtain PPS-induced bridging between BCG and the bladder wall and to prevent steric interaction, PPS should be instilled prior to BCG, separated by extensive washout of free PPS.

Cytokine production by the human bladder carcinoma cell line T24 in the presence of bacillus Calmette-Guerin (BCG).

The study was initiated as an in vitro approach to the situation existing during intravesical bacillus Calmette-Guerin (BCG) instillation in patients with superficial bladder cancer. Cytokine secretion of a human bladder carcinoma cell line T24 treated with BCG was investigated. A 24-h treatment of T24 cells with BCG resulted in a tenfold higher secretion of interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF alpha) when compared with T24 cells treated with Escherichia coli, Streptococcus faecalis or a cell wall preparation of Nocardia rubra (N-CWS). No secretion of IL-1 beta and IL-2 was detected. Pre-exposing T24 cells to BCG for various periods of time indicated that a minimum exposure time of 0.5-1 h was required to upregulate IL-6 and TNF alpha production. Extending the BCG pre-exposure time to 2 and 3 h further increased the rate of cytokine production. No significant difference was found, however, between the rate of secretion initiated after a 2-h or 3-h pre-exposure period. The amounts of these cytokines secreted in the presence of BCG-conditioned medium did not differ significantly from the constitutively secreted amounts, excluding an effect of products possibly secreted by BCG on the upregulation of IL-6 and TNF alpha. In addition, upregulation of cytokine production appeared to be dependent on the concentration of BCG. The results suggest that cytokines may be produced by urothelial tumor cells after intravesical instillation in patients with superficial bladder cancer, which may play a role in the mode of action of BCG.