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京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第17946号 / 医博第3830号 / 新制||医||1000(附属図書館) / 30776 / 京都大学大学院医学研究科医学専攻 / (主査)教授 稲垣 暢也, 教授 小西 郁生, 教授 安達 泰治 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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Bladder pain syndrome (BPS)/interstitial cystitis (IC) is a chronic pain disorder characterized by at least six weeks of lower urinary tract symptoms and unpleasant sensations (pain, pressure and discomfort) thought to be related to the urinary bladder and not meeting exclusion criteria. While the etiology is not known, BPS/IC may involve a "vicious circle" of uroepithelial dysfunction, inflammation and peripheral and central sensitization. We propose that the urinary bladder inflammatory insult partly mediates voiding dysfunction and visceral neurogenic pain characteristic of BPS/IC. Several studies from our laboratory have already demonstrated the role(s) of cytokines and their downstream targets in the functional alterations in micturition reflex pathways following chemically (cyclophosphamide, CYP)-induced cystitis. More recently, the pleiotropic protein, TGF-β, has been implicated in the pathogenesis of CYP-induced cystitis. TGF-β is activated locally at the initial site of injury by protease-dependent or protease-independent mechanisms to initiate a proinflammatory milieu. Depending on its contextual cues, TGF-β may then aid in resolving the primary immune response and support tissue repair. Though TGF-β is necessary to maintain normal immunological function, its aberrant expression and activation may have detrimental effects on responding tissues and cell types. A sustained increase in peripheral TGF-β reactivity, such as what may be observed in chronic inflammatory bladder conditions, may influence bladder afferent excitability to amplify nociceptive transmission and CNS input. The subsequent sensitization of peripheral afferent nociceptors at the level of the DRG or urothelium may promote spinal cord "wind-up" and cascade into visceral hyperalgesia and allodynia. In the first aim of this dissertation we investigated the functional profile of TGF-β isoforms and receptor (TβR) variants in the normal and inflamed (CYP-induced cystitis) urinary bladder with qRT-PCR, ELISA, IHC and in vivo cystometry. Our studies determined (i) the involvement of TGF-β in lower urinary tract neuroplasticity following urinary bladder inflammation, (ii) a functional role for TGF-β signaling in the afferent limb of the micturition reflex and (iii) urinary bladder TβR-1 as a viable target to reduce voiding frequency with cystitis. In the second aim of this dissertation we investigated the sensory components of the urinary bladder that may underlie the pathophysiology of aberrant TGF-β activation with bladder-pelvic nerve electrophysiology and luciferin-luciferase assays for ATP measurement. Our studies determined that TGF-β1 increased bladder afferent nerve excitability by stimulating ATP release from the urothelium via vesicular exocytosis mechanisms with minimal contribution from pannexin-1 channels. Furthermore, blocking aberrant TGF-β signaling in CYP-induced cystitis with TβR-1 inhibition decreased afferent nerve excitability with an equivalent decrease in ATP release. Taken together, these results establish a causal link between an inflammatory mediator, TGF-β, and intrinsic signaling mechanisms of the urothelium that may contribute to the altered sensory processing of bladder filling to facilitate increased voiding frequency. The distinct interactions of multiple mediators underscore the challenges for single target therapies and support the development of combinatory therapeutics for bladder dysfunction. Ultimately, these studies have increased our understanding of functional disorders and visceral pain and have the potential to improve the health of those suffering from inflammation-associated bladder syndromes.

Overactive bladder (OAB), involuntary contractions during bladder filling, is a common condition affecting 17% of the adult population worldwide, and in the U.S. ranks ahead of diabetes in a list of the 10 most common chronic disorders (Mullins 2009). Mechanical mechanisms contributing to OAB are not completely understood and because of the unique function and broad volume range of the bladder, there may be mechanical characteristics that distinguish detrusor smooth muscle (DSM) in bladder from other smooth muscles. Recent studies have shown that the length-passive tension curve in DSM exhibits adjustable passive stiffness (APS) characterized by a passive curve that can be shifted along the length axis as a function of strain history and activation history; however, the mechanical mechanisms responsible for APS remain to be determined. Also, whether DSM exhibits a dynamic length-active tension relationship, as has been identified in airway and vascular smooth muscles, has not been investigated. This dissertation focused on both the passive and active length-tension relationships in DSM and the mechanical mechanisms responsible for these relationships. The first objective was to study the impact of APS on the length-total tension relationship and identify the mechanical mechanisms responsible for generating APS. The second objective was to determine whether the length-active tension relationship is adaptive and identify specific mechanical mechanisms contributing to any adaptive behavior. The results showed that a shift in the length-passive tension curve due to APS corresponded with a shift in the length-total tension curve in DSM, and that APS was 27.0±8.4% of active tension at the optimum length for active tension generation. Most importantly, low-grade rhythmic contraction (RC), which can occur spontaneously in rabbit and human bladders, regenerated APS. Results also showed that the length-active tension curve shifted due to stretch to and then activation at long lengths, as well as either multiple KCl-induced maximal contractions or RC. Thus, DSM exhibits length adaptation, and RC may contribute to both APS and length adaptation. Because increased RC has been correlated with OAB, understanding RC, APS and length-adaptation in bladder may enable the identification of specific targets for new treatments for OAB.

Introdução: Ocupação foi identificada como o segundo fator de risco mais importante para o câncer de bexiga depois de fumar sendo responsável por até 20% de todos os cânceres de bexiga em países industrializados. Apesar dos esforços consideráveis para investigar ocupações em relação ao risco de câncer de bexiga, muitas não foram encontrados de forma consistente. Material e Métodos: Foram incluídos 200 pacientes com diagnóstico de câncer de bexiga entre os anos de 2009 e 2013. Foi aplicado um questionário para obter informações sobre a profissão, tempo de exposição e hábitos diários, sintomas, e também dados de doenças incluindo estágio, grau e número e tamanho de lesões. Os pacientes do Grupo 1 foram aqueles sem emprego previamente associados com o risco de câncer de bexiga. Grupo 2 representado pacientes em risco devido a profissões. Resultados: Os pacientes do Grupo 2 apresentaram uma proporção significativamente maior de pT2 CaB (P = 0,037), enquanto que os pacientes do grupo 1 apresentaram significativamente mais pTa (p = 0,002) da doença. Analisando preditores de pT2, a presença de ocupação aumento de alto risco por 2,80 vezes a chance de desenvolver uma doença invasiva. Ao analisar o grau do tumor descobriram que um tempo de exposição de 10 anos ou mais aumenta o risco de tumores de alto grau em 4,28 vezes (p = 0,001). Conclusão: Pacientes com história de exposição a agentes cancerígenos devido à sua atividade profissional podem estar em maior risco de desenvolvimento de tumores invasivos e aqueles que estão expostos a estes agentes para mais de 10 anos podem desenvolver doença de alto grau com mais freqüência. População em risco pode, portanto, beneficiar de rastreamento para o câncer de bexiga / Occupation was identified as the second most important risk factor for bladder cancer after smoking accounting for up to 20% of all bladder cancers in industrialized countries. Despite considerable efforts to investigate occupation against the risk of bladder cancer, many have not been found consistently. We included 200 patients diagnosed with bladder cancer between 2009 and 2013. A questionnaire was applied to obtain information about the profession, exposure time and daily habits, symptoms, and also diseases of data including stage, grade and number and lesion size. Patients in Group 1 were those without jobs previously associated with the risk of bladder cancer. Group 2 represented patients at risk because of professions. Group 2 patients had a significantly higher proportion of pT2 CaB (P = 0.037), whereas patients in group 1 had significantly more pTa (p = 0.002) of the disease. Analyzing predictors of pT2, the presence of high-risk occupation increases by 2.80 times the chance of developing invasive disease. In considering the degree of tumor found that a time of 10 or more years of exposure increases the risk of high-grade tumors in 4.28 times (p = 0.001). Patients with a history of exposure to carcinogens because of their duties may be at greater risk of developing invasive tumors and those who are exposed to these agents for more than 10 years can develop high-grade disease more often. Population at risk can therefore benefit from screening for bladder cancer

Introdução: Um dos principais obstáculos para compreensão dos eventos biológicos envolvidos no câncer é a falta de modelos adequados para o estudo in vitro em especial em relação ao câncer de próstata (CaP) e ao câncer de bexiga (CaB). Há um número limitado de linhagens celulares de CaP e de CaB sendo a maioria proveniente de tumores invasivos e metastáticos. Sabe-se ainda, que existem diferenças étnicas entre as populações quanto ao comportamento de neoplasias. Desta forma, a pesquisa baseada em linhagens de uma população homogênea seria fonte de resultados limitados, não contemplando a diversidade que sabidamente ocorre entre os diferentes grupos. Além desse aspecto, as linhagens celulares comerciais são na sua maioria adquiridas na Coleção Americana de Culturas de Tecido (ATCC, do inglês American Tissue Cell Culture) que apesar de serem bem padronizadas, requerem processos de importação com aumento do custo e demandas burocráticas que dificultam a pesquisa. Portanto, consideramos vital para a compreensão dos fenômenos relacionados à carcinogênese, assim como estudos de resistência a drogas, quimioprevenção e novas estratégias terapêuticas, o desenvolvimento de linhagens tumorais derivadas de tumores primários que acometem a nossa população, peculiarmente miscigenada. No presente trabalho, fragmentos de carcinoma urotelial da bexiga e de adenocarcinoma da próstata foram obtidos durante cirurgia para remoção de tumores primários de pacientes tratados e acompanhados na Divisão de Urologia da Faculdade de Medicina da Universidade de São Paulo (FMUSP) e no Hospital Sírio Libanês. As linhagens estabelecidas a partir destes fragmentos foram caracterizadas através da análise da cinética de crescimento, análises imunocitoquímicas e anormalidades cromossômicas incluindo cariótipo e hibridização in situ por fluorescência (FISH). Além disso, as linhagens obtidas foram submetidas a estudos de quimiossensibilidade com o uso dos compostos curcumin e Prima-1. Avaliamos ainda, a tumorigenicidade de nossas linhagens em camundongos atímicos. Os resultados deste trabalho demonstram o desenvolvimento de três linhagens de CaB e três linhagens de CaP sendo as mesmas não tumorigênicas em camundongos atímicos. Além disso, demonstramos que o curcumin na concentração de 50 M induziu morte celular em todas as linhagens estudadas, sendo seu efeito mais evidente nas linhagens de CaP. Por fim, Prima-1 reduziu a viabilidade celular independente do status de p53 nas linhagens de CaB / Introduction: One of the main obstacles for understanding biological events involved in cancer is the lack of appropriated models for in vitro studies especially for prostate cancer (PC) and bladder cancer (BC). There are a limited number of PC and BC cell lines being the majority originated from metastatic and invasive tumors. Also it is well known that there are ethnic differences between populations concerning the behavior of tumors. In such a way, the research based on cell lines derived from a homogenous population should be source of limited results, not contemplating the diversity known to occur among different groups. In addition the commercial cell lines are generally acquired at American Tissue Cell Culture (ATCC) that although wellestablished requires importation processes with cost increase and bureaucratic demands that difficult the research. Therefore we consider vital to the comprehension of the carcinogenesis phenomena, as well as drug resistance studies, chemoprevention and new therapeutic strategies, the development of tumor lineages derived from primary tumors that assail our miscigenated population. At the present work, fragments of bladder urothelial carcinoma and prostate adenocarcinoma were obtained by surgical resection of primary tumors from patients treated and followed in the Division of Urology of the Clinical Hospital of the São Paulo University (FMUSP) and Syrian Lebanese Hospital. The cell lines established from these fragments were characterized through growth kinetic, immunocytochemistry and chromosome abnormalities including karyotyping and Fluorescence in situ hybridization (FISH). Moreover, the cell lines were submitted to chemosensitivity studies using curcumin and Prima-1 and analyzed regarding their tumorigenicity in athymic mice. The results of this work show the development of three BC and three PC cell lines that were not tumorigenic in athymic mice. Curcumin at 50 M concentration induced cell death in all studied lineages, being more effective in PC cell lines. Finally, PRIMA-1 reduced the cellular viability independent of the p53 status in BC cell lines

Conclusion. Impairment in bladder and brain functions was identified in children with severe NE. Post-treatment studies indicated that brain function normalized in parallel with amelioration of bladder dysfunction. Interaction between brain and bladder dysfunction is likely to have an important implication in the pathophysiology and resolution of NE. / Objective. To (1) investigate sleep pattern and cortical arousals in enuretic children; (2) assess brain and bladder function in enuretic children; (3) evaluate post-treatment brain and bladder functional changes in enuretic children and correlate these with the treatment outcomes. / Part II. Fifty-two patients with severe PNE and 15 normal controls were recruited. Bladder and brain functions (sleep arousal threshold, P300 ERPs latency and PPI of startle amplitude) in enuretic children were evaluated, and brain function was compared with normal controls. / Part II. Markedly reduced nocturnal FBC and impaired brain function were found in enuretic patients. Higher sleep arousal threshold was negatively correlated to lower FBC. Prolonged P300 ERPs and higher PPI of startle amplitude were positively correlated to a higher sleep arousal threshold. / Part III. NE episodes and bladder function were re-evaluated in 52 severely enuretic children (Part II) at 3 and 6 months on treatment. Brain function was re-evaluated in 41/52 enuretic children at 6 months on treatment. / Part III. Post-treatment FBC significantly increased, and sleep arousal threshold, number of awakenings, P300 ERPs latency and PPI of startle amplitude normalized in treatment responders. NE episodes reduction was significantly correlated to the improvement in FBC and brain function. Greater decrease in sleep arousal threshold was positively correlated to higher FBC increase. Higher P300 ERPs latency and PPI of startle amplitude reduction were positively correlated to greater decrease in sleep arousal threshold. / Patients and methods. Part I. Thirty-five children with refractory PNE and 21 normal controls were recruited. Overactive bladder contractions, NE episodes and volume in enuretic children, sleep stages and cortical arousals in all children were recorded. / Results. Part I. Underlying bladder dysfunction, abnormal sleep architecture and increased cortical arousal index were found in enuretic children. Cortical arousal index was positively correlated to the frequency of overactive bladder contractions. / Diao Mei. / "May 2005." / Adviser: Chung-Kwong Yeung. / Source: Dissertation Abstracts International, Volume: 67-07, Section: B, page: 3693. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (p. 135-159). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract in English and Chinese. / School code: 1307.

Guang Fu Chen. / "May 2002." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (p. 194-221). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

Background and purpose. Recent studies have shown that the children with severe nocturnal enuresis, or bedwetting, often have underlying bladder dysfunction as well as various types of brainstem disorders, including arousal inability, a deficient response to startle sounds, or prepulse inhibition. Since the pontine micturition centre is anatomically very close, even overlapping with the nuclei responsible for sleep arousal, one may speculate that there may be close inter-relationships between abnormal bladder function, brainstem dysfunction and sleep-arousal disturbance. We hypothesize that the brainstem function would be changed in response to alteration of bladder function. Using conventional-fill cystometric study, functional magnetic resonance imaging (fMRI) scanning and immunohistochemistry approaches, we propose to characterize the functional changes in the brainstem in response to altered bladder function (i.e. surgically reduced bladder capacity). / Conclusions. Our results showed that bladder dysfunction elicited by surgical reduction in bladder capacity can induce functional changes in the central nervous system. In response to surgical reduction in bladder capacity, deactivation in the vlPAG was detected suggesting that the vlPAG plays a role in the biofeedback of bladder dysfunction. / Data are expressed as the mean +/-1SD unless otherwise specified. Appropriate statistical tests were used for parametric and non-parametric testing between the groups by using the SPSS computer program. In all comparisons a statistical significance level of 95% (p<0.05) was chosen. / Immunohistochemistry study showed a significant decrease in the reaction of dopaminergic neuron in the correspondent regions, suggesting a dopaminergic dependent change in the vlPAG in response to the bladder dysfunction. / In addition, we will explore the use of electroacupuncture (EA), a traditional Chinese therapy that has been broadly used for treatment of bladder functional disorders, to modulate functional changes in the central nervous system. We hypothesize that functional change in various nuclei in the central nervous system that are responsible for micturition control can also be affected by acupuncture treatment. A further aim of this study was to identify brain areas involved in EA to acupoint Chiliao (BL 32), a special acupoint broadly used for the treatment of bladder disorders. / Materials and methods. The study was divided into four parts. Seventy-five male New Zealand white rabbits (14-16 weeks, mean body weight: 3.0-3.5 kg) were used. / Moreover, in this study, we also found a notable activation in the vlPAG and dlPONS in response to the acupuncture stimulations to acupoint Chiliao (BL 32). The changes were identical to that induced by the bladder distension, suggesting a neuromodulation in central nervous system in response to acupuncture therapy. / Results. Study I: Bladder dysfunction elicited by surgical reduction in bladder capacity. Compared to sham animals, the maximum cystometric capacity in animals with RBC operation was markedly decreased at week 4 (35.3+/-8.2 ml vs. 71.6+/-12.9 ml, p<0.05), and week 8 (46.2+/-12.1ml vs. 82.7+/-20.1 ml, p<0.05) groups respectively; however, the maximum voiding detrusor pressure was significantly increased at week 4 (24.4+/-7.0 vs.16.5+/-7.2 cm water, p<0.05) and week 8 (27.7+/-8.p vs. 16.8+/-7.5 cm water, p<0.05) groups respectively, and their corresponding vesical pressure was also enhanced. Other parameters including maximum flow rate, and bladder emptying efficiency did not change significantly in between the sham and RBC subgroups. / Study I: Establishment of the dysfunctional bladder animal model with small bladder capacity. Forty rabbits underwent either sham operation (n=20) or operation for reduced bladder capacity (RBC) (n=20). The sham-operated and the RBC animals were further divided into two groups, i.e. four, and eight weeks after operation (n=10 in each sham and RBC subgroup). A conventional-fill cystometric study was performed on these animals whilst awake in order to evaluate the functional changes (if any) in response to surgical bladder capacity reduction, compared to sham subgroup. / Study II: Detection of functional changes in the brainstem in response to bladder dysfunction. FMRI scanning was performed at the brainstem region in sham-operated and RBC rabbits (12 in each group) at four weeks postoperatively. Bladder stimulation was provided by warm saline (37°C) infusion through a urethral catheter until bladder distended to 70% of the maximum capacity. Area(s) of brainstem activation were assessed by comparing the fMRI scans performed before and after warm saline infusion. / Study II: Functional changes in brainstem in response to bladder dysfunction. FMRI scanning results demonstrated that for the sham animals, there were two activated regions in the brainstem in response to bladder distention, one in the ventrolateral region of periaqueductal gray (vlPAG, 83.3%, 10/12), and the other in the dorsolateral region of pons (dlPONS, 91.7%, 11/12). In animals with RBC operation, only 25% (3/12) showed vlPAG activation compared to 83.3% (10/12) in sham group (p<0.05); however, 83.3% (10/12) of animals showed similar dlPONS activation compared to 91.7% in sham group (p>0.05). / Study III: Catecholaminergic neurotransmitters changes in the brainstem affected areas in response to bladder dysfunction. Brainstem immunohistochemistry results showed that a large number of dopaminergic neuron scattered throughout the whole vlPAG, rarely appeared in dlPAG (dorsolateral region of periaqueductal gray) (lambda +6.0 to +3.0 mm); and an abundant noradrenergic neurons were also accumulated in a restricted region of dlPONS (lambda +3.0 to 0 mm). Compared with the sham group, the density of TH-positive neurons in the vlPAG was significantly decreased in RBC group (38.38+/-4.71 vs.51.57+/-8.38/field, p<0.05); for the another region of dlPONS, although the density of TH-positive neurons decreased slightly in RBC group compared to sham group, the results showed no statistical difference between groups (106.89 +/- 21.61 vs.120.61 +/- 17.03/field, p>0.05). / Study III: Investigation of the catecholaminergic neurotransmitters changes in brainstem affected areas in response to bladder dysfunction. After fMRI examination, all animals were euthanized, and their brainstems were collected for immunohistochemistry study with tyrosine hydroxylase, dopamine-beta-hydroxylase assays to investigate the changes of catecholaminergic neurotransmitters including dopaminergic, noradrenergic and adrenergic in response to bladder dysfunction elicited by surgical reduction in bladder capacity. / Study IV: Electroacupuncture modulation via acupoint Chiliao (BL 32) on bladder and the brainstem activated sites. FMRI study showed that the two brainstem micturition centers of the vlPAG (72.7%, 8/11) and dlPONS (82.8%, 9/11) can be activated by EA on BL 32, and there were no significant difference compared with stimulation of bladder distention (72.7% vs. 83.3% in vlPAG and 82.8% vs. 91.7% in dlPONS respectively, p>0.05). Urodynamic results showed that, bladder contraction obviously evoked in response to EA on BL 32 (ON-EA state) compared to before EA state (OFF-EA state), displaying a significantly increased detrusor pressure (14.04+/-3.17 vs. 8.19+/-0.69 cm water, p<0.05) and vesical pressure (13.48+/-1.61vs. 7.90+/-0.81 cm water, p<0.05). In addition, dissection of BL 32 showed that the stem of S1 and S2 pass through the region 0.5 cm around the acupuncuture needle. / Study IV: Investigation of electroacupuncture modulation via acupoint Chiliao (BL 32) on bladder and the brainstem activated sites. FMRI scanning and urodynamic evaluation were performed respectively during ON/OFF EA on acupoint Chiliao (BL 32) on sham-operated animals (n=12) at four weeks post-operation. At last, dissection of acupoint BL 32 was performed on seven sham animals. / We also found that surgically induced bladder dysfunction, mainly displaying as reduced bladder capacity and maximum voiding detrusor pressure enhanced, was elicited at four to eight weeks after the surgical reduction in bladder capacity in rabbit. / Xiang Bo. / "August 2006." / Adviser: Chung Kwong Yeung. / Source: Dissertation Abstracts International, Volume: 68-03, Section: B, page: 1551. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (p. 161-196). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / School code: 1307.