Sin Mun Tham

Lactobacillus rhamnosus GG Activation of Dendritic Cells and Neutrophils Depends on the Dose and Time of Exposure

This study evaluates the ability of Lactobacillus rhamnosus GG (LGG) to activate DC and neutrophils and modulate T cell activation and the impact of bacterial dose on these responses. Murine bone marrow derived DC or neutrophils were stimulated with LGG at ratios of 5 : 1, 10 : 1, and 100 : 1 (LGG : cells) and DC maturation (CD40, CD80, CD86, CD83, and MHC class II) and cytokine production (IL-10, TNF-α, and IL-12p70) were examined after 2 h and 18 h coculture and compared to the ability of BCG (the present immunotherapeutic agent for bladder cancer) to stimulate these cells. A 2 h exposure to 100 : 1 (high dose) or an 18 h exposure to 5 : 1 or 10 : 1 (low dose), LGG : cells, induced the highest production of IL-12 and upregulation of CD40, CD80, CD86, and MHC II on DC. In DCs stimulated with LGG activated neutrophils IL-12 production decreased with increasing dose. LGG induced 10-fold greater IL-12 production than BCG. T cell IFNγ and IL-2 production was significantly greater when stimulated with DC activated with low dose LGG. In conclusion, DC or DC activated with neutrophils exposed to low dose LGG induced greater Th1 polarization in T cells and this could potentially exert stronger antitumor effects. Thus the dose of LGG used for immunotherapy could determine treatment efficacy.

Tumor and Microenvironment Modification during Progression of Murine Orthotopic Bladder Cancer

The aim of this study was to monitor changes in the expression of immune-related genes in the bladder after tumor implantation. Mice were orthotopically implanted with MB49-PSA cells (C57BL/6 mice) on day 1 and terminated on days 7, 14, 21, and 28. Another mouse model (MBT-2/C3H mice) was examined at day 7. Gene expression analysis was performed using a TaqMan Low Density Mouse Immune Panel (Applied Biosystems, USA) on RNA extracted from the bladders. Selected genes were reconfirmed by real-time PCR analysis and RT-PCR on the mRNA from other animals. Immune suppressive (IL13, IL1β, PTGS2, NOS2, IL10, CTLA4, and CCL22) and immune stimulatory genes (CSF2, GZMB, IFNγ, CXCL10, TNFα, CD80, IL12a, and IL6) and AGTR2 were increased by day 7. By day 28, IL10, CCL2, CCL5, CXCL11, CTLA4, GZMB, IFNγ, CSF2, and IL6 were significantly increased. Therapeutic strategies involving TH1 induction and TH2 dampening may improve responses to immunotherapy.

MP65-05 IFNα MODULATES THE RESPONSE TO BCG IMMUNOTHERAPY IN BLADDER CANCER PATIENTS WITH SPECIFIC CTLA4 SINGLE NUCLEOTIDE POLYMORPHISMS

respect to the GSTT2B genotypes. The impact of BCG instillation (numbers) on recurrence was analyzed in a subset of patients for whom complete 10y follow-up data was available. Analysis was performed using SPSS 23.0 and p<0.05 was taken to be significant. RESULTS: GSTT2 was silenced in MGH cells (GSTT2B homozygous full length (GSTT2B)) and overexpressed in UMUC3 and U937 cells (GSTT2B homozygous deleted (GSTT2B)). A 2h exposure to BCG resulted in decreased ROS in GSTT2 silenced cells (p<0.05) and increased ROS in GSTT2 overexpressing cells (p<0.05). There was no difference in cellular cytotoxicity to BCG with respect to GSTT2 expression. However, intracellular BCG survival increased at 2 hours when GSTT2 was silenced (p<0.05) and decreased when GSTT2 was overexpressed (p<0.05). There was no significant difference between these groups at 24h. The majority of patients with complete 10y follow-up data, completed a 6+3 BCG schedule (n1⁄463) and n1⁄422 had less than 8 instillations. Patients with GSTT2B genotype (n1⁄46) who received 8 or less BCG instillations, were recurrence free (Likelihood ratio 1⁄4 0.040, p1⁄40.054). In the group that received at least 9 instillations of BCG, the GSTT2B was associated with earlier recurrence. CONCLUSIONS: GSTT2 expression decreases cellular ROS and BCG survival. GSTT2B was associated with lower likelihood of recurrence for patients who received 8 or less BCG instillations. In contrast patients with GSTT2B who received 9 or more instillations of BCG had earlier recurrences. Hence GSTT2B could be used as a marker for patients who will do well with less BCG therapy.

A Murine Orthotopic Bladder Tumor Model and Tumor Detection System.

This protocol describes the generation of bladder tumors in female C57BL/6J mice using the murine bladder cancer cell line MB49, which has been modified to secrete human Prostate Specific Antigen (PSA), and the procedure for the confirmation of tumor implantation. In brief, mice are anesthetized using injectable drugs and are made to lay in the dorsal position. Urine is vacated from the bladder and 50 µL of poly-L-lysine (PLL) is slowly instilled at a rate of 10 µL/20 s using a 24 G IV catheter. It is left in the bladder for 20 min by stoppering the catheter. The catheter is removed and PLL is vacated by gentle pressure on the bladder. This is followed by instillation of the murine bladder cancer cell line (1 x 105 cells/50 µL) at a rate of 10 µL/20 s. The catheter is stoppered to prevent premature evacuation. After 1 h, the mice are revived with a reversal drug, and the bladder is vacated. The slow instillation rate is important, as it reduces vesico-ureteral reflux, which can cause tumors to occur in the upper urinary tract and in the kidneys. The cell line should be well re-suspended to reduce clumping of cells, as this can lead to uneven tumor sizes after implantation. This technique induces tumors with high efficiency. Tumor growth is monitored by urinary PSA secretion. PSA marker monitoring is more reliable than ultrasound or fluorescence imaging for the detection of the presence of tumors in the bladder. Tumors in mice generally reach a maximum size that negatively impacts health by about 3 - 4 weeks if left untreated. By monitoring tumor growth, it is possible to differentiate mice that were cured from those that were not successfully implanted with tumors. With only end-point analysis, the latter may be mistakenly assumed to have been cured by therapy.

Gene Therapy in Urology

Bladder cancer is the 7th most common cancer worldwide. It has a natural history of superficial recurrences and local progression. It is estimated that within 18 months of first diagnosis approximately 50% of patients will have a recurrence (Anderson & Naish 2008). Thus there is a need for frequent monitoring of these patients. In the US the estimated lifetime cost of therapy for bladder cancer patients with non-muscle invasive disease was US

MP56-19 LONG TERM RESULTS OF A DOUBLE-BLIND RANDOMIZED CONTROLLED TRIAL OF INTERFERON ALPHA-2B AND LOW DOSE BCG IN PATIENTS WITH HIGH RISK NON-MUSCLE-INVASIVE BLADDER CANCER

21.03 million based on a Medicare database (Cooksley et al. 2008). The majority of this is spent on surveillance and the treatment of recurrences. Tumors occur on the luminal surface of the bladder and the architecture of the bladder permits topical intravesical therapies. The bladder is isolated from other organs and tissues and intravesical therapy permits contact with the entire internal surface of the bladder with minimal systemic side-effects. The present gold standard therapy for superficial bladder cancers is immunotherapy with Mycobacterium bovis, Bacillus Calmette Guerin (BCG) following local transurethral resection of the bladder tumor (TURBT). BCG induces a mononuclear and neutrophilic infiltrate in the bladder wall which results in an inflammatory response as measured by cytokine production that causes sloughing of both tumor and normal cells (Herr and Morales 2008). The presence of IL-2, IL-8 and IL-18 in the urine of patients has been reported to correlate with response to therapy (Thalmann et al. 1997; Thalmann et al. 2000; Saint et al. 2003). Unfortunately, BCG has several shortcomings: it is a live vaccine and commonly causes side effects and occasionally septicemia. In addition some patients (2042%) do not respond to therapy (Kamat and Lamm 2000). In place of BCG, recombinant cytokines such as IFN-γ, TNF-α, and IL-2 have been used in a number of clinical trials with encouraging results (Glazier et al. 1995; Den Otter et al. 1998; Stavropoulos et al. 2002). However, recombinant cytokines are costly, unstable in urine and have poor permeability