The urinary bladder is especially subject to infection by virtue of its direct connection to the external urethral opening, and it is natural to anticipate the presence of a well-developed immunological mechanism to respond to this potential threat. The present study describes small, very highly vascular lymph nodes located in the wall of the rabbit bladder, which may be involved in a local response to foreign antigens. The vasculature and structure of these lymph nodes was described using a combination of vascular corrosion casting, ink injection, and light and electron microscopy. The distal abdominal aorta was cannulated, and after clearing the bladder vasculature with buffered saline, one of the following procedures was used: 1) the bladder was perfuse-fixed in preparation for light and electron microscopy; 2) the bladder vasculature was filled with India ink for vessel tracing; or 3) vascular corrosion casts of the vasculature were prepared by infusing resin comprised of a mixture of Mercox, methyl methacrylate monomer, and catalyst. The resulting casts were cleaned with KOH, formic acid, and water in preparation for scanning electron microscopy. Vascular casts and India ink injections revealed the presence of a number of isolated capillary tufts consisting of clusters of one to five 'glomeruli,' closely associated with the major vesicular vessels along the lateral walls of the bladder, and supplied by tertiary branches of these vessels. Light and electron microscopy showed that the capillary tufts represented the blood supply to small, ovoid lymph nodes located near the serosal surface of the bladder wall and usually restricted to the basal half of the bladder. These nodes were encapsulated and exhibited subcapsular sinuses, numerous small blood vessels, a limited number of high endothelial cells, and, occasionally, nerves and a follicular substructure. The nodes contained abundant lymphocytes, stellate stromal cells, macrophages, and eosinophils, but lacked the obvious cortical and medullary organization and germinal centers often seen in larger lymph nodes. Vascular corrosion casts, vascular ink injections, and microscopic examination confirmed the presence of small, highly vascular lymph nodes closely associated with the main vesicular vessels along the lateral walls of the rabbit bladder. A follicular substructure of the nodes appears to correspond with the 'glomerular' capillary arrangement within the nodes as seen with corrosion casts. The rich blood supply may be indicative of the high metabolic demand of lymphatic tissue, and may be altered in response to the level of activity of the node. The close association between the lymphatic tissue and the rich blood supply to the nodes may allow a rapid mobilization of lymphocytes during a local immune response to foreign agents.

Background: The urinary bladder requires a rich blood supply to maintain its functions, the storage and release of urine. Specialized properties of the bladder vasculature might be anticipated to ensure the integrity of this blood supply, because it is known that blood flow is reduced by distension during bladder filling. However, the bladder vasculature has been described in detail only at the gross level. A comprehensive, threedimensional view of the blood supply to the bladder wall is presented here. Methods: The microvasculature of the bladder of male New Zealand white rabbits was described using the combination of vascular corrosion casting, alkali digestion, light microscopy, and scanning and transmission electron microscopy. Following administration of an anticoagulant and an overdose of anesthetic, the abdominal aorta was cannulated just above the inferior mesenteric artery to permit flushing of the distal vasculature. The bladder vasculature was cleared of blood with buffered saline and then either perfuse‐fixed with buffered 2% glutaraldehyde and sectioned, or filled with “Mercox” resin to prepare vascular corrosion casts. Casts were cleaned with NaOH, formic acid, and water. In some cases fixed bladders were partially digested with NaOH to expose the mucosal capillary plexus. Results: The bladder is supplied with blood by single, left and right vesicular branches of the internal or external iliac arteries. The serpentine vesicular arteries extend along the lateral borders of the bladder from base to apex just deep to the serosal surface and send dorsal and ventral branches to supply the dorsal and ventral bladder walls. Veins accompany the arteries and exhibit numerous valves. A very dense complex of vessels at the apex of the bladder apparently serves to accommodate bladder distension. The muscularis and submucosa contains few vessels, but the mucosa is well vascularized. An especially dense capillary plexus is present in the lamina propria at its junction with the transitional epithelium. In the relaxed bladder these capillaries lie in grooves formed by the basal layers of the epithelium. The endothelial cells of these capillaries display few cytoplasmic vesicles and are continuous or fenestrated. These capillaries are often invested with pericytes. The mucosal capillary plexus may be associated with an epithelial transport function or may be necessary for urothelial metabolism or maintenance of the barrier function of the urothelium. Unusual capillary tufts, possibly associated with vascular lymphatic tissue, are found associated with the main vessels on the lateral walls in the basal half of the bladder. Conclusions: These methods present a clear, comprehensive, three‐dimensional view of the microvasculature of the bladder wall. They also identify several unique features of this vasculature and provide a basis for studies of the response of this vasculature to pathologic states and experimental manipulation.

These experiments were done to determine the effect of glibenclamide and diazoxide on the growth of human bladder carcinoma (HTB-9) cells in vitro. Cell growth was assayed by cell counts, protein accumulation, and 3H-thymidine uptake. Glibenclamide added at 75 and 150 μM for 48 hr reduced cell proliferation. Dose-inhibition curves showed that glibenclamide added for 48 hr reduced cell growth at concentrations as low as 1 μM (IC50 = 73 μM) when growth was assayed in the absence of added serum. This μM-effect on cell growth was in agreement with the dose range in which glibenclamide decreased open probability of membrane K(ATP) channels. Addition of glibenclamide for 48 hr also altered the distribution of cells within stages of the cell cycle as determined by flow cytometry using 10-5 M bromodeoxyuridine. Glibenclamide (100 μM) increased the percentage of cells in G0/G1 from 33.6% (vehicle control) to 38.3% (P < 0.05), and it reduced the percentage of cells in S phase from 38.3% to 30.6%. On the other hand, diazoxide, which opens membrane K(ATP) channels in HTB-9 cells, stimulated growth measured by protein accumulation, but it did not increase the cell number. We conclude that the sulfonylurea receptor and the corresponding membrane K(ATP) channel are involved in mechanisms controlling HTB-9 cell growth. However, K(ATP) is not rate-limiting among the signaling mechanisms or molecular switches that regulate the cell cycle.

Bladder cancer, as one of the most common cancer types and with high recurrence risk, is considered a candidate for novel immunotherapy strategies. An important aspect of the research for immunotherapy drug development for bladder cancer is to study the tumor microenvironment (TME) and it’s immune contexture. Besides tumor-infiltrating lymphocytes (TILs) as the main drivers of anti-tumor response, recent studies revealed the importance of tumor-associated tertiary lymphoid structures (TLSs) and high endothelial venules (HEVs) in the TME. Structures similar to these were found to spontaneously form in the orthotopic MB49 model used for bladder cancer research in our group. The aim of this study was to perform a deeper characterization of the TME in this model, by using immunofluorescent staining and microscopy. Specifically, the co-localization of tumor infiltrating lymphocytes (CD8+ and CD4+ T cells, CD19+ B cells), CD11c+ dendritic cells and HEVs along with CCL21 signaling were analyzed within orthotopic MB49 tumors, with and without immune stimulation. The quantification of cells expressing CD8, CD19 and CD11c immune markers, CCL21 levels, vascular density and numbers of HEVs, showed higher densities within the immune-stimulated tumors, indicating a rapid effect of immune stimulation on increasing immune cell infiltration and vascular density after only 24 hours post CpG therapy. Also, the highest frequency of TILs, CCL21 chemokine and vascular density was located in regions of the tumor border indicating that these regions should be studied further in depth as a potential target for entry of cells to the tumor with immunotherapy or as a model of the tumor microenvironment since tumor cell density is maintained high in these locations.

Real-time multi-circle detection has been a challenging problem in the field of biomedical image processing, due to the variable sizes and non-ideal shapes of cells in microscopic images. In this study, two new multi-circle detection algorithms are developed to facilitate an automatic bladder cancer diagnosis system: one is a modified circular Hough Transform algorithm integrated with edge gradient information; and the other one is a stochastic search approach based on real valued artificial immune systems. Computer simulation results show both algorithms outperform traditional methods such as the Hough Transform and the geometric feature based method, in terms of both precision and speed.

Interstitial Cystitis/Bladder Pain Syndrome (IC/BPS) is a disabling chronic disease of still unknown origin and complex pathophysiology. The disease affects mainly female patients, with a female to male ratio of about 9 to 1. Prevalence ranges from 52 to 500/100,000 in females and 8 to 41/100,000 in males. The diagnosis of IC/BPS is mainly hampered by the lack of appropriate biomarkers and, therefore, extensive clinical examinations are required to exclude “confusable” diseases [1]. In consequence, most patients experience several years of ineffective treatments of various urinary tract symptoms often associated with, but by themselves not characteristic of, IC/BPS. Unequivocal diagnosis of IC/BPS is the prerequisite to find more effective therapeutic approaches. Therefore, more specific biomarkers are needed to facilitate IC/BPS diagnosis and to stratify patients for treatment at earlier stages of the disease. In this Special Issue, we gathered reviews and original work elucidating the current developments in IC/BPS biomarker research.

In pediatric pelvic health, sensory processing is not often considered as a significant factor. The current gold standard for addressing bowel and bladder dysfunction is urotherapy and medication. Urotherapy encompasses education regarding the anatomy and function, behavior modifications including fluid intake, removing or managing bowel and bladder irritants, diet changes, timed or scheduled voids, toilet postures and avoidance of holding maneuvers, manual therapy, and biofeedback. These forms of treatment have shown a roughly 50% success rate six months to several years after treatment is concluded (Pijpers et al., 2010 and Noordhoff et al., 2018). While the previously mentioned treatment methods are often necessary and appropriate, they fail to consider the central role that sensory processing, sensory integration, and emotional regulation play in basic biological functions and homeostasis. This doctoral project involved the creation and piloting of the Sensory Modulation – Bowel and Bladder Questionnaire (SM-BBQ) questionnaire to help diagnose children who have bowel or bladder dysfunction due to sensory under-responsivity. Results demonstrated a strong positive correlation between children who leaked both urine and stool and demonstrated hypo-responsiveness on the Sensory Processing Measure -2 (SPM-2) and their SM-BBQ scores. Significant findings were also noted between the SPM-2 scale scores and scores on both the SM-BBQ. In contemplating how these findings fit with Ayres Sensory Integration theory, perception and threshold may be more relevant than responsivity, as most participants were noted to be hypo- and hyper- responsive to input. A larger scale follow-up study will need to be conducted to ensure that the SM-BBQ is a reliable and valid measure for identifying children with toileting dysfunction related to sensory perception and helping those families to find appropriate services. / 2025-05-08T00:00:00Z

Bladder dysfunction is seen as a public health problem with a strongly increasing incidence in older men. Despite this, the man's experience of bladder dysfunction is a topic that has not been researched in the empirical field as much as women’s experiences. Not highlighting the man's experience leads to suffering and constitutes an obstacle to equal care. The aim of this study was to illustrate the man's experience of living with bladder dysfunction. This study is intended to illustrate experiences, therefore it was well suited to make an integrative compilation of qualitative research – inspired by meta-synthesis. Nine qualitative articles that described the man's experiences were analysed schematically and resulted in three themes and six sub-themes. Experiences that were noticed in the man were the impact of the environment in bladder dysfunction which included environmental and health care responses. Emotional impact in bladder dysfunction, which highlighted the experiences that arose in connection with bladder dysfunction. The life adaptations in bladder dysfunction was an experience which described the changes the man made to achieve a normality in everyday life as well as changes to hide the condition. The man used a range of strategies to maintain normality in life and to keep his condition a secret from those around him. This is seen because of the stigmatization that emerged through this study. Healthcare failed to care for the man with bladder dysfunction and was seen as partially responsible for the stigmatization the man experienced.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第24191号 / 医博第4885号 / 新制||医||1060(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 中山 健夫, 教授 松村 由美, 教授 万代 昌紀 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Background: Bladder cancer (BCa) is the most frequent malignancy in the urinary tract. Despite great progress in our understanding of BCa in the past decades, we still lack significant improvement in the development of new BCa chemotherapeutics, which is largely attributed to the fact that 2D tumor models are used as the predominant platform for cell-based assays. The conventional 2D tumor models, although simple and convenient, fail to recreate an in vivo-like tumor microenvironment (TME). 3D tumor models more faithfully recapitulate the complexity of TME than 2D-based models. 3D-organoid models reproduce many biological characteristics of the real solid tumor, including biochemical gradients, different proliferating states, complex cell-cell interaction, ECM deposition, and chemo-resistance, thus providing a promising tool in tumor biology research. There have been many studies about the patient-derived BCa organoids. However, we still lack a research about the development and characterization of multiple bladder organoid models that mimicked both normal bladder tissue and bladder tumors representing the entire range of malignant grades. Aims: The study aimed to investigate the formation and characteristics of hetero-typed bladder organoids derived from the three major cell types in the human bladder, including either non-cancerous urothelial cells or different bladder cancer cell lines. Hypotheses: 1. Bladder-like organoids can formed by self-organization from mixed bladder cell suspension 2. The degree of histological organization depends on the urothelial cells used 3. The different cell types can be identified in the bladder organoids by immunohistochemistry Materials and Methods: We used RT4, RT112, T24, and CAL29 cells (transitional cancer cell lines, histological grade: G1-G4), non-malignant HBLAK (bladder epithelium progenitors), primary human bladder fibroblasts (hBF), and human bladder smooth muscle cells (hBSMC) in this study. The following figure displays the construction process of hetero-typed bladder organoids by ultra-low attachment (ULA) 96-well microplate method. At day 4 post seeding, bladder organoids were harvested, fixed, processed, and sectioned. Then, we characterized the bladder organoids by histology and immunohistochemistry (IHC) staining. Bladder organoids were stained for panCK, Vimentin, α-SMCA, CK7, CK13, CK20, Ki67, Claudin4, ZO-1, and fibronectin and the immunoreactivity (IR) of specific antigen was analyzed using ImageJ plus IHC profiler plugin. We also did H&E and Crossmon staining to investigate the histology and ECM deposition of the bladder organoids. Results: Mixed cell suspensions self-organized into compact organoids in the ULA plate within 24 hours after seeding. At day 4 post seeding, the organoids were grown to 650-1,000 μm in diameter, with BCa organoids significantly bigger than HBLAK organoids. The morphology of bladder organoids greatly varied depending on the urothelial cells used. Besides, urothelial cells were mainly located in the periphery and supportive cells (hBF and hBSMC) were in the core of the organoids. High-grade RT112, T24 and CAL29 organoids showed significantly thicker urothelial cell layers and more urothelial cells in the periphery than low-grade RT4 organoids and non-malignant HBLAK organoids. The CK7, CK13 and CK20-IR greatly varied between the organoid-cultured urothelial cells. HBLAK cells showed significant lower panCK, CK7, CK13 and CK20-IR than BCa cells. CK-IR was higher in RT4 and RT112 cells than in T24 cells. CAL29 organoids showed a stratified layering: CK7-IR in superficial cells, CK13-IR in intermediate cells, and CK20 in all cells. The urothelial cells in organoid culture consisted of not only proliferating cells but also a large portion of quiescent cells. High-grade RT112, T24 and CAL29 cells (30-50%) showed a significantly higher proliferation index than the low-grade RT4 (2%) and non-malignant HBLAK (6.6%) cells. All urothelial cells in organoid culture expressed Claudin4(CLDN4) positively. In addition, high-grade T24 and CAL29 cells showed higher Claudin4-IR than low-grade RT4 and RT112 cells. In contrast, all bladder organoids showed very low ZO-1-IR. Fibronectin and Crossmon staining indicated fibronectin, collagen, and reticular fibers deposition in the bladder organoids. Besides, high-grade T24 and CAL29 cells showed remarkably higher fibronectin-IR than low-grade RT4 and RT112 cells. Discussion: All urothelial cells were able to form compact and reproducibly sized organoids with hBSMC and hBF by spontaneous cell aggregation in ULA 96-well plate. The compactness of bladder organoids might reflect the adhesion between the cells and the expression of underlying adhesion molecules. The size of organoids and number of urothelial cells in the periphery could be influenced by multiply factors. The organoids formed a bladder-like structure with outside urothelial cells and a core of supportive cells, indicating they could be useful tools in the testing of anti-cancer drugs. The cytokeratin expression profiles reflect the differentiation status of tumors. All urothelial cells retained the CK7/CK13/CK20 expression patterns of their original tissues in organoid culture, supporting that the bladder organoids mimicked both the normal bladder urothelium and bladder tumors of different grades. In addition, RT112 and CAL29 organoids formed a stratified urothelium. Urothelial cells in organoid culture were at different proliferation stages, better mimicking the in vivo bladder tumors in terms of proliferation and cell heterogeneity than 2D-based BCa models. Besides, the organoid-cultured urothelial cells retained the proliferation characteristics of their original tissues. Bladder organoids showed abundant fibronectin deposition, which could affect their response to drug treatments. Besides, the fibronectin expression level in BCa cells was correlated with their primary origins, supporting the view that the BCa cells in organoid culture retained the invasive and metastatic feature of their parental bladder tumors. The fibronectin, collagen, and reticular fibers deposition indicated bladder organoids mimicked the in-situ situation of bladder tumors in respect to ECM deposition. The expression of CLDN4 in urothelial cells indicated the formation of para-cellular barriers in the bladder organoids, which could limit the penetration and diffusion of anticancer drugs into bladder organoids. Especially, the RT112 and CAL29 organoids showed high expression of CLDN4 on the apical membrane, indicating that they could be helpful in the investigation of drug penetration into tumor tissues. The ULA microplate method is easy, fast, and suitable for massive production of reproducibly sized organoids. The imageJ plus IHC profiler plugin method was able to perform fast and automatic quantitative analysis for DAB-stained IHC images and comparisons of the expression of specific antigens in formalin-fixed tissues Conclusion: The bladder organoids represented a bladder-like architecture by self-organization, with a peripheral urothelium surrounding a supportive core of hBF and SMCs. These organoids exhibit characteristics of the in situ normal urothelium and bladder tumors of different grades in cell composition, proliferation, stratified urothelium, epithelial diferentiation, and ECM deposition. Thus, they can be useful tools in cancer biology research and anti-cancer drug development.:1. Title page 2. Table of contents 3. List of Abbreviations 4. Introduction 4.1 Bladder cancer 4.2 Currently available bladder cancer models 4.3 Advantages of 3D cell culture over 2D cell culture 4.4 3D tumor spheroid/organoid models 4.5 Spheroids/organoids construction methods 4.6 Application of 3D organoid culture in bladder cancer research 4.7 Limitations of previous research of bladder cancer organoids 5. Aims 5.1 Hypothesis 5.2 Tasks 6. Materials and Methods 6.1 Cell lines used in the study 6.2 Monolayer culture of the cell lines 6.3 Bladder organoids construction by ULA microplate method 6.4 Fixation, processing, sectioning, and size measurement 6.5 Histology and immunohistochemistry 6.6 Image acquisition 6.7 Image analysis with ImageJ plus IHC profiler plugin 6.8 Statistical analysis 7. Results 7.1 Spontaneous formation of packed bladder organoids in the ULA plate 7.2 Bladder-like self-organization of the organoids 7.3 Immunohistochemical characterization of the organoids 7.4 Expression of urothelial cell differentiation makers 7.5 Proliferation of urothelial cells in 3D organoid culture 7.6 Tight junction protein expression in the organoids 7.7 ECM deposition in bladder organoids 8. Discussion 8.1 Organoid models in cancer research 8.2 Morphology and Size 8.3 Bladder-like internal structure 8.4 Formation of a stratified urothelium in bladder organoids 8.5 Proliferation characteristics of the urothelial cells in organoid culture 8.6 ECM deposition in bladder organoids 8.7 TJ protein expression in bladder organoids 8.8 Possible application of bladder organoids 8.9 Review of methods used in this project 8.10 Limitations of the research 9. Summary of the work 10. References 11. Appendix 12. Declaration of independence 13. Curriculum vitae 14. Acknowledgment