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1148 THE ROLE OF MACROPHAGES IN CHRONIC BLADDER INFLAMMATION-ASSOCIATED GRANULOMA FORMATION, FIBROSIS, AND SEPSIS
Vol. 189, No. 4S, Supplement, Monday, May 6, 2013
tract has been implicated in UCPPS. We used culture-independent molecular methods to identify the microbiota of the lower urinary tract in men and women with UCPPS. METHODS: Uniformly collected urine specimens were obtained in UCPPS and age matched healthy controls (including positive controls with CFS, FM, IBS). Specimens were tested by Ibis T-5000 Universal Biosensor using BAC plate assay. Mass spectometric technology measures unique molecular weights of multiple DNA amplicons generated by PCR to detect the presence and identify the species of all bacteria. Differences in species composition for cases versus controls were assessed using “distance measure” methods from microbial ecology. PERMANOVA and Cochran-Mantel-Haenszel methods were used to test differences by cohort type in the presence and/or richness of species. Testing, adjusted for sex, was completed for differences in overall composition at the species, genus, and gram-stain level. RESULTS: Baseline urine specimens were obtained from 257 cases (161 female; 96 male) and 261 controls (164 female; 97 male). A total of 136 species (57 genera) were detected in VB1; VB2 contained 109 species (52 genera). Mean VB2 species count per person was 2.50 and 2.29 among female UCPPS patients and controls respectively; 1.33 and 1.08 for males respectively. Trends were similar for VB1. Overall species composition was not significantly associated with cohort type at any level (p⫽0.198, 0.612 species level, p⫽0.313, 0.085 genus level, p⫽0.279, 0.086 Gram-stain level in VB1 and VB2, respectively). At the species level in VB1, Lactobacillus gasseri and Streptococcus pneumoniae were more prevalent among cases than controls, while Staphylococcus capitis/caprae was underrepresented among cases (OR⫽3.74, p⫽0.007 L. gasseri, OR⫽0.263, p⫽0.011 S. capitis/caprae, OR⫽3.45,p⫽0.007 S. Pneumoniae). VB1 samples among cases were also more likely to have gram-negative organisms than controls (OR⫽1.88, p⫽0.008), For VB2 the only statistical difference observed was at the genus level for Corynebacterium (lower prevalence among UCPPS patients compared to controls OR⫽0.34, p⫽0.011). CONCLUSIONS: Despite observing some provocative trends, we did not find significant differences in the microbiome detected in lower urinary tract urine specimens from men and women with UCPPS compared to controls using state-of-the-art detection methods.
THE JOURNAL OF UROLOGY姞
e469
and fewer infiltrating leukocytes (Figure 1C). None of the control vehicle-treated mice receiving eggs died, whereas 60% of macrophage-depleted mice receiving high doses of eggs died by day 11 post-egg injection, indicating an unexpected yet crucial role for macrophages in prevention of bladder granuloma-associated sepsis (Figure 2). CONCLUSIONS: Our results confirm that macrophages are critical to bladder granuloma pathogenesis, even in the setting of a single exposure to an inciting agent. This suggests that macrophages may be a suitable therapeutic target for bladder granuloma-associated diseases.
Source of Funding: NIH/NIDDK
1148 THE ROLE OF MACROPHAGES IN CHRONIC BLADDER INFLAMMATION-ASSOCIATED GRANULOMA FORMATION, FIBROSIS, AND SEPSIS
Source of Funding: NIH T32AI007290 (CF), the Society of Pediatric Urology (MH), NIH K08-DK087895 (MH), Lucile Packard Foundation for Children’s Health (LPFCH) [MH], the Stanford NIH CTSA (UL1 RR025744) [MH], the Stanford Consortium for Innovation, Design, Evaluation and Action (C-IDEA) [NIH RC4TW008781] [MH], a Stinehart/Reed Award from the Institute for Stem Cell Biology and Regenerative Medicine at Stanford(MH), the UBS Optimus Foundation [MH], and NIH/NCRR UCSF-CTSI Grant Number UL1 RR024131
Chi-Ling Fu, Stanford, CA; Kim Thai, Portland, OR; Justin Odegaard, Michael Hsieh*, Stanford, CA INTRODUCTION AND OBJECTIVES: Chronic bladder inflammation leading to granuloma formation and fibrosis can be caused by various stimuli, including suture material, BCG, and urogenital schistosomiasis (infection by Schistosoma haematobium worms). Although macrophages are the defining feature of the granuloma and associated fibrosis, the importance of these cells in bladder granuloma formation and fibrosis is poorly understood. We used the first tractable animal (mouse) model of urogenital schistosomiasis (published by us in PLOS Pathogens) as a starting point for defining the role of macrophages in bladder granuloma formation and fibrosis. METHODS: Mice underwent bladder wall injection with S. haematobium eggs or vehicle controls. Next, mice were administered systemic followed by intravesical liposomal clodronate (which selectively depletes macrophages) or control vehicle liposomes. Mice were sacrificed at various time points and their bladders harvested and subjected to either flow cytometry or biochemical assays for fibrosis. RESULTS: Serial microultrasonography revealed zones of decreased echogenicity in the periphery of egg granulomas in macrophage-depleted versus -replete mice, suggestive of relative hypocellularity. This was confirmed by histology, biochemical assays for fibrosis, and flow cytometry, which revealed hypocellular peripheral zones in macrophage-depleted granulomas, less fibrosis (Figure 1A-B),
1149 BRILLIANT BLUE G, A P2X7 ANTAGONIST, ATTENUATES RENAL INFLAMMATION AND FIBROSIS IN UNILATERAL URETERAL OBSTRUCTION IN RATS José Monteiro Sad Pereira*, André Luis Barreira, Alberto Schanaider, Christina Maeda Takiya, Maurilo Leite Jr, Luis Carlos Miranda, Rio de Janeiro, Brazil INTRODUCTION AND OBJECTIVES: Purinergic receptors have been involved in inflammation and immunological response. These receptors expression have been demonstrated in granulocytes, monocytes/macrophages, dendritic cells and T and B lymphocytes. The present study investigated the effect of brilliant blue G (BBG), the most potent and selective irreversible antagonist, on rat kidneys after unilateral ureteral obstruction (UUO).
tract has been implicated in UCPPS. We used culture-independent molecular methods to identify the microbiota of the lower urinary tract in men and women with UCPPS. METHODS: Uniformly collected urine specimens were obtained in UCPPS and age matched healthy controls (including positive controls with CFS, FM, IBS). Specimens were tested by Ibis T-5000 Universal Biosensor using BAC plate assay. Mass spectometric technology measures unique molecular weights of multiple DNA amplicons generated by PCR to detect the presence and identify the species of all bacteria. Differences in species composition for cases versus controls were assessed using “distance measure” methods from microbial ecology. PERMANOVA and Cochran-Mantel-Haenszel methods were used to test differences by cohort type in the presence and/or richness of species. Testing, adjusted for sex, was completed for differences in overall composition at the species, genus, and gram-stain level. RESULTS: Baseline urine specimens were obtained from 257 cases (161 female; 96 male) and 261 controls (164 female; 97 male). A total of 136 species (57 genera) were detected in VB1; VB2 contained 109 species (52 genera). Mean VB2 species count per person was 2.50 and 2.29 among female UCPPS patients and controls respectively; 1.33 and 1.08 for males respectively. Trends were similar for VB1. Overall species composition was not significantly associated with cohort type at any level (p⫽0.198, 0.612 species level, p⫽0.313, 0.085 genus level, p⫽0.279, 0.086 Gram-stain level in VB1 and VB2, respectively). At the species level in VB1, Lactobacillus gasseri and Streptococcus pneumoniae were more prevalent among cases than controls, while Staphylococcus capitis/caprae was underrepresented among cases (OR⫽3.74, p⫽0.007 L. gasseri, OR⫽0.263, p⫽0.011 S. capitis/caprae, OR⫽3.45,p⫽0.007 S. Pneumoniae). VB1 samples among cases were also more likely to have gram-negative organisms than controls (OR⫽1.88, p⫽0.008), For VB2 the only statistical difference observed was at the genus level for Corynebacterium (lower prevalence among UCPPS patients compared to controls OR⫽0.34, p⫽0.011). CONCLUSIONS: Despite observing some provocative trends, we did not find significant differences in the microbiome detected in lower urinary tract urine specimens from men and women with UCPPS compared to controls using state-of-the-art detection methods.
THE JOURNAL OF UROLOGY姞
e469
and fewer infiltrating leukocytes (Figure 1C). None of the control vehicle-treated mice receiving eggs died, whereas 60% of macrophage-depleted mice receiving high doses of eggs died by day 11 post-egg injection, indicating an unexpected yet crucial role for macrophages in prevention of bladder granuloma-associated sepsis (Figure 2). CONCLUSIONS: Our results confirm that macrophages are critical to bladder granuloma pathogenesis, even in the setting of a single exposure to an inciting agent. This suggests that macrophages may be a suitable therapeutic target for bladder granuloma-associated diseases.
Source of Funding: NIH/NIDDK
1148 THE ROLE OF MACROPHAGES IN CHRONIC BLADDER INFLAMMATION-ASSOCIATED GRANULOMA FORMATION, FIBROSIS, AND SEPSIS
Source of Funding: NIH T32AI007290 (CF), the Society of Pediatric Urology (MH), NIH K08-DK087895 (MH), Lucile Packard Foundation for Children’s Health (LPFCH) [MH], the Stanford NIH CTSA (UL1 RR025744) [MH], the Stanford Consortium for Innovation, Design, Evaluation and Action (C-IDEA) [NIH RC4TW008781] [MH], a Stinehart/Reed Award from the Institute for Stem Cell Biology and Regenerative Medicine at Stanford(MH), the UBS Optimus Foundation [MH], and NIH/NCRR UCSF-CTSI Grant Number UL1 RR024131
Chi-Ling Fu, Stanford, CA; Kim Thai, Portland, OR; Justin Odegaard, Michael Hsieh*, Stanford, CA INTRODUCTION AND OBJECTIVES: Chronic bladder inflammation leading to granuloma formation and fibrosis can be caused by various stimuli, including suture material, BCG, and urogenital schistosomiasis (infection by Schistosoma haematobium worms). Although macrophages are the defining feature of the granuloma and associated fibrosis, the importance of these cells in bladder granuloma formation and fibrosis is poorly understood. We used the first tractable animal (mouse) model of urogenital schistosomiasis (published by us in PLOS Pathogens) as a starting point for defining the role of macrophages in bladder granuloma formation and fibrosis. METHODS: Mice underwent bladder wall injection with S. haematobium eggs or vehicle controls. Next, mice were administered systemic followed by intravesical liposomal clodronate (which selectively depletes macrophages) or control vehicle liposomes. Mice were sacrificed at various time points and their bladders harvested and subjected to either flow cytometry or biochemical assays for fibrosis. RESULTS: Serial microultrasonography revealed zones of decreased echogenicity in the periphery of egg granulomas in macrophage-depleted versus -replete mice, suggestive of relative hypocellularity. This was confirmed by histology, biochemical assays for fibrosis, and flow cytometry, which revealed hypocellular peripheral zones in macrophage-depleted granulomas, less fibrosis (Figure 1A-B),
1149 BRILLIANT BLUE G, A P2X7 ANTAGONIST, ATTENUATES RENAL INFLAMMATION AND FIBROSIS IN UNILATERAL URETERAL OBSTRUCTION IN RATS José Monteiro Sad Pereira*, André Luis Barreira, Alberto Schanaider, Christina Maeda Takiya, Maurilo Leite Jr, Luis Carlos Miranda, Rio de Janeiro, Brazil INTRODUCTION AND OBJECTIVES: Purinergic receptors have been involved in inflammation and immunological response. These receptors expression have been demonstrated in granulocytes, monocytes/macrophages, dendritic cells and T and B lymphocytes. The present study investigated the effect of brilliant blue G (BBG), the most potent and selective irreversible antagonist, on rat kidneys after unilateral ureteral obstruction (UUO).