Dissertations / Theses on the topic 'Urinary bladder infection – Rats'

The overall aim of this thesis was to evaluate methods for urinary catheter handling in patients undergoing hip surgery. The intention was to gain knowledge in order to provide optimal and cost- effective care regarding urinary catheterisation in this group of patients. In Study I , 45 of the 86 catheterised patients (52%) contracted nosocomial urinary tract infections (UTIs). Diabetes was a risk factor for developing UTI, and cloxacillin as a perioperative antibiotic prophylaxis seemed to offer a certain protection. Study II was a randomised controlled trial on the effect of clamping (n = 55) or not (n = 58) of the indwelling urinary catheter before removal. No significant differences were found between the groups with respect to time to normal bladder function, need for recatheterisation, or length of hospital stay. Study III was a randomised controlled trial among patients with hip fracture and hip arthroplasty, in which the patients were randomised to intermittent (n = 85) or indwelling (n = 85) urinary catheterisation. No significant differences in nosocomial UTIs (9% vs. 12%) or cost-effectiveness were shown. The patients in the intermittent group regained normal bladder function significantly sooner after surgery. Fourteen percent of the patients in the intermittent group did not need any catheterisation. In Study IV , 30 patients were interviewed about their experiences of bladder emptying and urinary catheterisation. The patients’ views were described through the main category ‘An issue but of varying impact’. Both bladder emptying through micturition and bladder emptying through catheterisation were described as convenient, but also as uncomfortable and an intrusion on dignity. The patients were aware of risks and complications of urinary catheterisation. In conclusion, this thesis indicates that UTI is common in hip surgery patients. Clamping of indwelling catheters seems not necessary. There is no preference for either intermittent or indwelling urinary catheterisation according to the results of this thesis, either for the development of nosocomial UTI or, for cost-effectiveness, or from the patient perspective. Nurses should be aware that catheterisation might make the patients feel exposed, and it is essential that their practice reflect the best available evidence.

L’infection des voies urinaires est l'une des infections bactériennes les plus courantes avec des coûts de soins de santé très élevés. On estime que 50% des femmes connaîtront une infection urinaire au cours de leur vie, ceci de manière récurrente chez la moitié d’entre elles. Le développement de thérapies efficaces a été limité par le manque de connaissance concernant la mise en place de la réponse immune adaptative lors de cette infection. Dans cette étude, nous avons démontré qu'une réponse adaptative est générée lors de l'infection urinaire, cependant celle-ci n’a pas d’action protectrice. Afin de comprendre les mécanismes aboutissant à ce phénomène, nous avons cherché à caractériser les cellules immunitaires présentes dans la vessie. Des tests d’absorption bactérienne ont montré que ces macrophages phagocytent la majorité des bactéries au début de l'infection. Pour évaluer l’influence de ces cellules sur la mise en place de la réponse immune adaptative, nous avons déplété les macrophages et évalué la clairance bactérienne lors d’une deuxième infection. En comparaison avec les animaux non traités, les souris déplétées présentaient une réduction de la charge bactérienne conséquente lors de la seconde infection, cette clairance dépendant de la réponse immune adaptative. Pour comprendre ce mécanisme d'inhibition par les macrophages, nous avons évalué le microenvironnement vésical et la phagocytose au début de l'infection chez les souris déplétées, et chez les souris non traitées. Bien que nous n’ayons pas observé de différences dans la production de cytokines, l'absorption bactérienne par les cellules dendritiques s’avère deux fois plus importante chez les animaux déplétés. Ces données suggèrent que l'absorption bactérienne par les macrophages tissulaires est néfaste pour la mise en place de la réponse adaptative, ouvrant de nouvelles options thérapeutiques. Nous avons également évalué le rôle des lymphocytes T dans ce processus en déplétant ces cellules au cours de l'infection primaire ou avant la deuxième infection. Ainsi, nous avons observé que les lymphocytes T sont nécessaires dans la réponse adaptative, mais ne sont cependant pas indispensables à la clairance bactérienne lors d'une réinfection. De plus, l'infection des souris Batf3-/-, déplétées en cellules dendritiques spécialisées dans la présentation croisée, a montré que ces souris contrôlent une seconde infection aussi bien que les souris contrôle. Ces résultats suggérent que la présence lymphocytes T CD8+ n’est pas nécessaire pour lutter contre l’infection urinaire. Notre étude révèle un mécanisme par lequel le système immunitaire est compromis lors de l'infection urinaire, offrant un point de départ intéressant pour une recherche plus approfondie sur le rôle du système immunitaire adaptatif dans ce contexte, élément fondamental dans le développement de nouvelles thérapies
Urinary tract infection (UTI) is one of the most common bacterial infections with exorbitant health care costs. It is estimated that 50% of women will experience a UTI during their lifetime and approximately half will suffer recurrent infections. Infected women are treated with antibiotics, however, antibiotic resistance is increasing, raising the need for new therapeutic options. Development of efficient therapies has been impeded by the lack of knowledge of events leading to adaptive immunity. In this study, we demonstrated that an adaptive immune response is generated during UTI, however this response does not confer protective immunity. To begin to understand why the response induced during UTI was not effective, we delineated the immune cell compartment of the bladder and identified macrophages as the most populous immune cell. We evaluated bacterial acquisition in the bladder observing that macrophages phagocytize the majority of the bacteria early in infection. To evaluate the impact of macrophages on the generation of adaptive immunity, we depleted bladder resident macrophages and evaluated bacterial clearance during a challenge infection. Interestingly, mice depleted of resident macrophages, prior to primary infection, exhibited a nearly 2-log reduction in bacterial burden following secondary challenge compared to untreated animals. This improvement in clearance was dependent on the adaptive immune system. To shed light on the mechanism of macrophage inhibition, we evaluated the bladder microenvironment and bacterial acquisition early in infection in macrophage-depleted and control-treated mice. While we did not observe differences in the cytokine microenvironment, bacterial uptake by dendritic cells was increased nearly 2-fold in macrophage-depleted animals. These data suggest that bacterial uptake by tissue macrophages negatively impacts the development of adaptive immunity, revealing a novel target for enhancing host responses to bacterial infection of the bladder. We also evaluated the role of T cells during UTI by depleting these cells during the course of the infection or just prior to challenge infection. We observed that T cells were necessary to mount an adaptive immune response to UTI, however, they were dispensable for bacterial killing during challenge infection. Additionally, infection of Batf3-/- mice, lacking cross-presenting dendritic cells, suggested that CD8+ T cells are dispensable for the response against UTI as these mice cleared a challenge infection as well as wildtype mice. Our study has revealed a mechanism by which the immune system is compromised during UTI, providing an interesting start point for further investigation of the role of the adaptive immune system during UTI, which will be fundamental for the development of new therapies to efficiently treat infection

Tyrimo tikslas: išsiaiškinti slaugytojų vaidmenį su šlapimo pūslės kateterizacija susijusių šlapimo takų infekcijų išsivystyme ir profilaktikoje. Tyrimo uždaviniai: 1. Įvertinti slaugytojų, dirbančių reanimacijos ir intensyvios terapijos skyriuose, teorines žinias apie šlapimo takų infekcijas, jų rizikos veiksnius bei profilaktiką; 2. Įvertinti ar slaugytojos, dirbančios reanimacijos ir intensyvios terapijos skyriuose, žino kaip taisyklingai atlikti šlapimo pūslės kateterizaciją laikantis slaugos procedūrų metodikos reikalavimų; 3. Nustatyti, kokias profilaktikos priemones bei metodus taiko reanimacijos ir intensyvios terapijos skyrių slaugytojos, kad būtų išvengta šlapimo takų infekcijų; 4. Pateikti praktines rekomendacijas. Tiriamoji grupė: tyrime dalyvavo 104 slaugytojos, dirbančios KMUK, Kauno 2-osios klinikinės ligoninės, Kauno apskrities ligoninės ir Kauno Raudonojo Kryžiaus klinikinės ligoninės reanimacijos ir intensyvios terapijos skyriuose. Tyrimo metodai: anketinė apklausa ir statistinė duomenų analizė. Tyrimas buvo atliekamas 2009 m. vasario – kovo mėnesiais. Apklausai naudota nestandartizuota autorinė anketa, kuri buvo sudaryta remiantis literatūros analize ir Lietuvos Respublikos sveikatos apsaugos ministro įsakymu patvirtinta higienos norma HN 47-1:2008 “Sveikatos priežiūros įstaigos. Higieninės ir epidemiologinės priežiūros reikalavimai.“, ir tyrėjos paruoštas veiklos testas. Tyrimo metu gauti duomenys apdoroti naudojant SPSS 13.0 versijos statistinį duomenų... [toliau žr. visą tekstą]
The goal of the research – to find out what is the role of the nurses in the development and prophylaxis of the catheter – associated urinary tract infections. Research tasks: 1. Evaluate the theoretical knowledge about the urinary tract infections and their risk’s factors and prophylaxis of the nurses working in the units of Intensive care; 2. Evaluate do the nurses working in the units of Intensive care know how to do the urinary bladder catheterization properly according to the requirements of the methodology of the care procedures; 3. Estimate what preventive measures and methods are used by the nurses of Intensive care units in order to avoid the urinary tract infections; 4. Give the practical recommendations. Investigative group: 104 nurses working in the units of intensive care in Kaunas University Hospital, the Second Kaunas Clinical Hospital, Kaunas Regional Hospital and Kaunas Red Cross Hospital took part in this research. The methods of the research: questionnaire and statistical data analyses. The research was made from January to March in the year 2009. The nonstandard authorized questionnaire which was made according to the analyses of the literature and certified hygiene norm HN 47-1:2008 „Health Care Institutions. Sanitary and epidemiological care requirements.“ affirmed by the Minister of Health of the Republic of Lithuania and the test prepared by the researcher. The data received during the research chiseled using the SPSS 13.0 version of the... [to full text]

Introdução: A bexiga é responsável em armazenar urina em volume adequado e de esvaziar seu conteúdo de forma plena. Suas propriedades miogênicas intrínsecas e viscoelásticas são as responsáveis por esta função. Disfunções vesicais podem ser decorrentes, dentre outras causas, de anormalidades intrínsecas da musculatura detrusora ou da composição de sua matriz extracelular (MEC). O colágeno corresponde a 50% do estroma vesical, possuindo importante papel na adaptação vesical a condições fisiopatológicas específicas. Os colágenos tipo I e III são os mais comuns, sendo o colágeno tipo III o primeiro a ser sintetizado em processos de reparação e fibrose. Diversas afecções como a obstrução infravesical (OIV) parcial crônica podem induzir estes processos através da remodelação da MEC e conseqüentemente alterar a função vesical. Acredita-se que a hipercolesterolemia também o faça, porém ainda não foi reproduzida tal associação a nível morfológico. O objetivo deste estudo é avaliar se dieta hipercolesterolêmica promove alterações estruturais vesicais em ratos, especialmente no que diz respeito à remodelação colágena. Método: Foram utilizadas 45 ratas da raça Wistar, de quatro semanas de idade, divididas em três grupos: 1) controle com dieta comum padrão para roedores (DN); 2) modelo de OIV com DN e 3) controle com dieta de alto teor lipídico (DATL 1,25% colesterol). Análise sérica do colesterol e fração LDL e medição do peso corporal foram realizadas em todos os animais inicialmente e no final do estudo. Com quatro semanas de estudo, as ratas dos grupos 1 e 3 foram submetidas à cirurgia simulada, enquanto os animais do grupo 2 foram efetivamente submetidos à cirurgia de OIV parcial. Após dissecção da uretra, fez-se uma ligadura parcial com Nylon 5-0, com um lúmen residual de aproximadamente 1 mm. Após seis semanas, todos os animais foram submetidos à remoção de suas bexigas e então sacrificados. Análise morfológica foi realizada através da coloração de Picrosirius vermelho e de imuno-histoquímica para os colágenos tipos I e III. As variáveis categóricas fora expressas em médias ± desvio padrão e a comparação entre grupos realizada pelo método ONEWAY-ANOVA e pela análise de comparações múltiplas de Tukey, quando houve diferença. A significância estatística foi definida como p < 0,05. Resultados: Este estudo demonstrou que a DATL em ratas Wistar proporcionou aumento significativo das taxas de LDL-colesterol (p < 0,001) e do peso corporal (p = 0,017) em relação a ratas alimentadas com DN no período de dez semanas. Além disto, induziu alterações morfológicas significativas da matriz extracelular, no que diz respeito à remodelação das fibras colágenas imaturas e do colágeno tipo III em relação ao grupo controle (p = 0,002 e p = 0,016, respectivamente), de forma semelhante ao que ocorre no modelo experimental de OIV parcial crônica. Conclusão: A dieta hipercolesterolêmica administrada a ratas Wistar promoveu, além de aumento do peso corporal e elevação da fração LDL-colesterol, alterações significativas na composição colágena da MEC vesical.
Purpose: Preserved bladder function is defined as the adequate storage and emptying of its urinary content. Compliance is an important factor for these functions and is directly related to the extracellular matrix composition. Its abnormalities can lead to bladder dysfunctions. The collagen represents 50% of bladder stroma, playing an important role in the bladder adaptation to specific pathologic conditions. Types I and III collagens are the most prevalent in bladder wall whereas type III collagen is the first synthesized in reparation and fibrosis processes. Bladder outlet obstruction (BOO) promotes this process and hypercholesterolemia is also believed to create conditions for it, although no morphologic association has already been demonstrated. In this study we aimed to verify if hypercholesterolemic diet promotes structural bladder wall modifications, regarding the collagen remodeling. Methods: Forty-five female heterogenic Wistar 4 weeks-old rats were divided into three groups: 1) control fed on a normal diet (ND); 2) BOO model fed on a ND and 3) control fed on a hypercholesterolemic diet (HCD 1.25% cholesterol). Initially, serum cholesterol, LDL-cholesterol and body weight were measured. Four weeks later groups 1 and 3 underwent a sham operation while group 2 underwent a partial BOO operation. After the urethra was dissected a 5-zero nylon suture was passed and tied loosely around the urethra with a 22G needle besides it. Six weeks later the bladders of all animals were removed, serum cholesterol and LDL-cholesterol analysis was performed, body weight was measured and then they were sacrificed. Morphological analysis was performed by Picrosirius red staining and immunohistochemistry for types I and III collagen. Statistical analysis was done comparing groups by the Oneway-Anova method and Tukey multiple comparisons when needed. Significance was considered when p < 0.05. Results: Wistar rats fed on a HC diet had a significant increase of LDL-cholesterol levels (p < 0.001) and body weight (p = 0.017), when compared to the control group fed on a normal diet in the period of ten weeks. Moreover, HC diet induced significant morphological alterations of the extracellular matrix of the bladder wall, regarding immature collagen fibers and type III collagen remodeling, when compared to the control group (p = 0.002 and p = 0.016, respectively), resembling the process promoted in the BOO model. Conclusions: A hypercholesterolemic diet in Wistar rats promoted, besides the body weight and LDL-cholesterol increase, morphological alterations of the bladder extracellular matrix, regarding collagen remodeling.

This study was prompted by the inconsistent reports and apparent controversies that exist in the biomedical literature on the responses of diabetic bladder strips to cholinergic nerve stimulation or exogenous administration of muscarinic agonists, especially acetylcholine (ACh), in vitro. In the present study, acetylcholine-induced contractions of urinary bladders isolated from normoglycaemic (normal) and streptozotocin-treated, diabetic Wistar rats were examined under physiological conditions. Mechanical contractile changes of the isolated urinary bladders of STZ-treated, diabetic rats in response to bath-applied acetylcholine were compared with those obtained from isolated urinary bladders of normal, age-matched, control rats. Results obtained show that urinary bladders from diabetic rats consistently weighed more, and were always more spontaneously active after mounting, than those of the age-matched normal, control rats. ft A Acetylcholine (ACh, 10" -10" M) provoked concentration-related, atropine-sensitive contractions of the isolated urinary bladders of both diabetic and age-matched normal, control rats. However, acetylcholine always induced more powerful and greater contractions of the diabetic bladders compared with bladders from the age-matched normal, control rats. The enhanced contractile responses of the diabetic bladder strips to bath-applied ACh were detected soon after induction of diabetes, and the magnitude and/or intensity of the enhanced contractile responses to ACh continued to increase as the diabetic state of the animals progressed. Although this preliminary study could not establish the mechanism of the increased contractile responsiveness of the diabetic bladders to the muscarinic agonist (ACh) used, the results tend to suggest that alterations in diabetic urinary bladder synaptosomal, vesicle-bound neurotransmitter (ACh) concentrations and the compensatory increase in the density of muscarinic M3-receptor population in diabetic bladders are two of the most attractive plausible mechanisms of the increased diabetic bladder responsiveness to bath-applied acetylcholine.
Thesis (M.Sc.Pharm.)-University of KwaZulu-Natal, Westville, 2006.

碩士
國立成功大學
藥理學研究所
84
It is well known that the rat urinary bladder possesses an non- adrenergic, non-cholinergic ( NANC) innervation. However, the neurotransmitter for this NANC neurotransmission is still contro- versial and is the main interest of our research.The rat detrusor muscle strips were studied in organ bath in vitro and contraction were induced by electric-field stimulation (EFS). The amplitude of contraction increased as the frequency was increased.The frequency-dependent contractions were almost completely abolished by tetrodotoxin (TTX), indicating that the response to EFS was predominantly neurogenic. The NANC contraction was obtained in the presence of the four autonomic nerve blockers (atropine, guanethidine, phentolamine and propranolol). The NANC component constituted a greater portion of the contraction at low frequency than that at high frequency. Desensitization induced by a.b-MeATP , a P2x receptor agonist, reduced the NANC contractions evoked by a constant stimulating frequency 3Hz (EF50).Meanwhile, this NANC response was abolished mostly in ATP desensitized sample. Adenosi -ne has a similar activity asATP. In addition, this NANCcontrac -tion was also abolished mostly by P2 receptor blockers suramin and P2x receptor blocker PPADS, at concentrations sufficient to block a.b-MeATP-induced contraction. It is suggested that this NANC contraction in urinary bladder might be one of the pur- inergic actions of ATP. Dipyridamole, a adenosine uptake inhibitor ,inhibited NANC contraction at concentrations which influenced the action of a, b-MeATP. DPCPX, a A1 adenosine receptor blocker, did not affect NANC contraction even at high concentration. The adenyl purines released by EFS from nerve were detected by HPLC and the amount of ATP was more than adenosine.Therefore,combining measurement of adenyl purines release and the results of fun- ctional studies,the present study suggests that ATP plays an important role in NANC neurotransmission of the rat bladder.

碩士
國立臺灣大學
生理學研究所
89
Abstract Oxidative stress resulting from bladder overdistension and relief (OD/RE) may produce micturition dysfunction. Hypoxia adaptation produced protection against ischemic insults in some organs. Whether this benefit extends to ameliorate OD/RE induced bladder damage is not yet defined. We compared rats kept at sea level（SL）and chronically hypoxia rats（high altitude; HA, induced by exposure to an altitude chamber at 5,500 m for 15 days/day）. The bladders of SL rats and HA rats were subjected to 1-h or 2-h of saline overdistension (1 ml/100 gw body weight) and followed by relief. We analyzed bladder function by transcystometrogram and determined the amount of reactive oxygen species (ROS) from the whole blood in vitro and bladder surface in vivo by a chemiluminescence method. Results showed that OD/RE reduced the bladder intravesical pressure in SL and HA rats. However, the functional recovery of urinary bladder 2-hr after relief from bladder overdistention was significantly better in HA rats than in SL rats. In addition, ROS release from the blood samples and the bladder surface was increased in SL rats. The enhanced ROS release reached the peak 5 min after bladder relief. ROS release in HA rats was also increased, but less than that of the SL rats. The expression of Cu/Zn superoxide dismutase (Cu/Zn SOD) and heat shock protein 70 (HSP70) was upregulated in the hypoxia adapted bladder. Our results suggest that the increased Cu/Zn SOD and HSP 70 might be related to the acquisition of bladder protection against OD/RE.

碩士
國立成功大學
藥理學研究所
86
Abstract In the urinary bladder, muscarinic cholinoceptors (mAChRs) play an important role in the control of bladder contraction during micturition. Activation of mAChR mainly causes urinary bladder emptying. Many reports have demonstrated that there are two types of mAChRs in the urinary bladder, M2 and M3-mAChR. Besides, bladder dysfunction is one of the common complications in diabetic mellitus (DM). Therefore, in this study, using muscle contractile studies, Western-blotting and Northern-blotting analyse, mAChRs in both normal and DM rats' bladders were elucidated and compared. The present study aimed to compare the responses of detrusor muscle strips to cholinergic agonists in the Wistar rats and streptozotocin (STZ)-induced diabetic rats with or without electric-field stimulation (EFS) in vitro. The selective agonist for M2 receptor, arecaidine propargyl ester hydrobromide (APE), was cumulatively added from 10-7 M to 10-5 M .APE induced contraction was more marked in diabetic rats. Contraction induced by EFS was also more pronounced in diabetic rats than that in Wistar rats. In addition, the levels of mAChR protein and mRNA expression were enhanced in diabetic rats . The results suggested that M2-mAChR was highly expressed in diabetic rats resulting in hyperactivity of bladder. Therefore, three possible factors were further investigated, including hyperglycemia, bladder distension and sorbitol (the glucose metabolite) in this study. Hyperglycemia of the Wistar rat was induced with glucose administration for 1 week (1 g/kg, 12 time s per day, p.o.)；and bladder distension by urethral ligation for 1 day. Besides, the contractile responses were also evaluated after administration of sorbitol for 1 week (1 g/kg, 12 times per day, i.p.). The data showed that in urethra ligation rats the contractile response was similar to that in Wistar rats. On the other hand, the glucose and sorbitol feeding groups showed similar response to those in diabetic rats. Meanwhile, the protein and mRNA level were also measured in Wistar rats. The obtained results suggested that the hyperactivity of bladder might be correlated to higher expression of M2-mAChR and/or hyperglycemia in diabetic rats. The diabetic rats were intraperitoneally injected with aldose reductase inhibitor, ONO-2235 for 14 days (100mg/kg/day). The contractile responses were between those of diabetic and Wistar rats；and so were the M2-mAChR protein and mRNA. The results suggested that the hyperactivity of bladder in diabetic rat might be reversed by inhibition of sorbitol formation. The bladder neck were treated in Wistar rats and diabetic rats with a1-, a2-, b-adrenoceptor agonists. They were methoxamine, clonidine and isoproterenol, respectively. The contractile responses showed no significant differences between Wistar and diabetic rats.

碩士
國立臺灣大學
生醫電子與資訊學研究所
101
Spinal cord injury is often accompanied with detrusor external urethral sphincter (EUS) dyssynergia, which may lead to low efficiency of micturition function and incomplete voiding, and increase the risk of urinary tract infection in the patients. Being under this symptom for a long period, a patient may also develop ureteral edema, renal damage, and even renal failure. 　　　Electrical neural moderation is one of the new treatment directions. It can provide the function that the damaged nerve originally does and improve the voiding efficiency. So it is critical to detect the status of bladder and give the muscle electrical stimulation at the right time. Nowadays clinical detection of the bladder status depends on urodynamic study, such as using external ultrasound to estimate the volume of the bladder, or placing a catheter through the urinary tract to measure the intravesical pressure. But external ultrasound can-not give the electrical neural moderation an immediate feedback; neither is it suitable for patients to catheterize for a long period. The objective of this study is to use EUS-electromyography (EMG) as detector of the bladder status. EMG signal is filtered and amplified by a 5-stage circuit, and processed by using a LabVIEW algorithm. A moving window method is used to decrease time delay and maintain moderated sensitivity of the algorithm, realizing a real-time detection of the bladder status. The algorithm is applied on normal rats and rats with spinal cord injury experimentally. 　　　From experimental result analyzed by ROC method, using EUS-EMG to detect the bladder status compared with real voiding status, the accuracies of voiding detection exhibit 97% in normal rats and 90.3% in those with spinal cord injury. The accuracies of both results are better than that from using intravesical pressures as detector. Those findings demonstrate the feasibility of this study. Signal output function of the DAQ card can be used to trigger functional electrical stimulation, which improves voiding function of patients with spinal cord injury.

The mammalian urinary bladder is a highly specialized organ that must be able to withstand considerable amounts of osmotic pressure at its mucosal surface, in addition to maintaining an impenetrable barrier against potential pathogens. The lower urinary tract's virtually inevitable exposure to external microbial pathogens warrants efficient tissue-specialized defenses to maintain sterility. The observation that the bladder can become chronically infected with uropathogenic E.coli (UPEC) in combination with clinical observations that antibody responses following bladder infections are not detectable, suggest defects in the formation of adaptive immunity and immunological memory. We have identified a broadly immunosuppressive transcriptional program specific to the bladder, but not the kidney, during infection of the urinary tract that is dependent on tissue-resident mast cells. This mast cell-dependent phenomenon involves localized production of IL-10 and results in suppressed humoral and cell-mediated responses and bacterial persistence. Therefore, in addition to the previously described role of mast cells orchestrating the early innate immune responses in the bladder during infection, they subsequently play a tissue-specific immunosuppressive role. These findings may explain the prevalent recurrence of bladder infections and suggest the bladder as a site exhibiting an intrinsic degree of mast cell-maintained immune privilege.

Interestingly, though the bladder is not capable of initiating an effective adaptive immune response during bladder infections, we have generated data showing that it was possible to circumvent the immune limitations of the bladder to provoke a strong adaptive and protective immune response by vaccinating against UPEC at an alternate mucosal site. We reasoned that by immunizing the nasal regions of mice with a vaccine formulation comprising of FimH adhesin, a highly conserved adhesive moiety of type 1 fimbriae expressed on UPEC, and an effective mucosal adjuvant we would evoke protective immunity against UPEC infections. We found that a FimH vaccine coupled with either a mast cell activating adjuvant c48/80 or CpG oligodeoxynucleotide, a TLR9 agonist, evoked high levels of FimH specific IgG antibody in the serum and IgA in the urine of immunized mice. We also observed that following UPEC challenge, these FimH/adjuvant immunized mice exhibited significantly reduced bacterial load in the bladders compared to mice challenged with just FimH. These studies reveal that immunization of nasal regions with a FimH vaccine is an effective strategy to overcome the limitation in adaptive immunity observed in the bladder.

Dissertation

Urinary tract infections (UTIs) are typically caused by bacteria that colonize different regions of the urinary tract, mainly the bladder and the kidney. Approximately 25% of women that suffer from UTIs experience a recurrent infection within 6 months of the initial bout, making UTIs a serious economic burden resulting in more than 10 million hospital visits and $3.5 billion in healthcare costs in the United States alone. Type-1 fimbriated Uropathogenic E. coli (UPEC) is the major causative agent of UTIs, accounting for almost 90 % of bacterial UTIs. The unique ability of UPEC to bind and invade the superficial bladder epithelium allows the bacteria to persist inside epithelial niches and survive antibiotic treatment. Persistent, intracellular UPEC are retained in the bladder epithelium for long periods, making them a source of recurrent UTIs. Hence, the ability of UPEC to persist in the bladder is a matter of major health and economic concern, making studies exploring the underlying mechanism of UPEC persistence highly relevant.

In my thesis, I will describe how intracellular Uropathogenic E.coli (UPEC) evade host defense mechanisms in the superficial bladder epithelium. I will also describe some of the unique traits of persistent UPEC and explore strategies to induce their clearance from the bladder. I have discovered that the UPEC virulence factor Alpha-hemolysin (HlyA) plays a key role in the survival and persistence of UPEC in the superficial bladder epithelium. In-vitro and in-vivo studies comparing intracellular survival of wild type (WT) and hemolysin deficient UPEC suggested that HlyA is vital for UPEC persistence in the superficial bladder epithelium. Further in-vitro studies revealed that hemolysin helped UPEC persist intracellularly by evading the bacterial expulsion actions of the bladder cells and remarkably, this virulence factor also helped bacteria avoid t degradation in lysosomes.

To elucidate the mechanistic basis for how hemolysin promotes UPEC persistence in the urothelium, we initially focused on how hemolysin facilitates the evasion of UPEC expulsion from bladder cells. We found that upon entry, UPEC were encased in “exocytic vesicles” but as a result of HlyA expression these bacteria escaped these vesicles and entered the cytosol. Consequently, these bacteria were able to avoid expulsion by the cellular export machinery.

Since bacteria found in the cytosol of host cells are typically recognized by the cellular autophagy pathway and transported to the lysosomes where they are degraded, we explored why this was not the case here. We observed that although cytosolic HlyA expressing UPEC were recognized and encased by the autophagy system and transported to lysosomes, the bacteria appeared to avoid degradation in these normally degradative compartments. A closer examination of the bacteria containing lysosomes revealed that they lacked V-ATPase. V-ATPase is a well-known proton pump essential for the acidification of mammalian intracellular degradative compartments, allowing for the proper functioning of degradative proteases. The absence of V-ATPase appeared to be due to hemolysin mediated alteration of the bladder cell F-actin network. From these studies, it is clear that UPEC hemolysin facilitates UPEC persistence in the superficial bladder epithelium by helping bacteria avoid expulsion by the exocytic machinery of the cell and at the same time enabling the bacteria avoid degradation when the bacteria are shuttled into the lysosomes.

Interestingly even though UPEC appear to avoid elimination from the bladder cell their ability to multiple in bladder cells seem limited.. Indeed, our in-vitro and in-vivo experiments reveal that UPEC survive in superficial bladder epithelium for extended periods of time without a significantly change in CFU numbers. Indeed, we observed these bacteria appeared quiescent in nature. This observation was supported by the observation that UPEC genetically unable to enter a quiescence phase exhibited limited ability to persist in bladder cells in vitro and in vivo, in the mouse bladder.

The studies elucidated in this thesis reveal how UPEC toxin, Alpha-hemolysin plays a significant role in promoting UPEC persistence via the modulation of the vesicular compartmentalization of UPEC at two different stages of the infection in the superficial bladder epithelium. These results highlight the importance of UPEC Alpha-hemolysin as an essential determinant of UPEC persistence in the urinary bladder.

Dissertation

碩士
國立清華大學
電機工程學系
88
The electromyogram (EMG) activity of external urethral sphincter (EUS) and cystometrogram (CMG) of the urinary bladder can reflect the clinical information and status for the physician about the function of the bladder and urethra. The traditional linear methods cannot well describe complex nature phenomena . There is no exception for our cases. In this thesis, we investigate the time series of EUS EMG signals of normal and SCI Wistar rats with room temperatures from the viewpoint of nonlinear dynamics. The techniques of modern nonlinear dynamical systems are used to analyze the time series of external urethral sphincter electromyogram (EUS EMG) and the cystometrogram (CMG) of the urinary bladder of Wistar rat. They include singular value decomposition (SVD), recurrence plot analysis (RPA), and recurrence quantification analysis (RQA). These methods are good tools to recognize the different phases of micturition and the differences caused by spinal cord injury in Wistar rats. SVD indicates that SCI rats in bursting phase have more principal directions in the embedding space than normal rats. RPA displays the important features in bursting phase for both normal and SCI rats and RQA shows the differences between normal rats, SCI rats bursting with voiding and SCI rats bursting without voiding. Although the dynamics of micturation are complex, they still have some regularity in our study.

Due to its close proximity to the gastrointestinal tract, the human urinary tract is

subjected to constant barrage by gut-­associated bacteria. However, for the most part, this tract has resisted infection by various microbes. The impregnability of the urinary tract to microbial colonization is attributable to the ability of the bladder to promptly sense and mount robust responses to microbial challenge. A powerful mechanism for the elimination of invading bacteria was recently described in bladder epithelial cells, involving non-­lytic ejection of intracellular bacteria back into the extracellular milieu. In spite of the effectiveness of this defense strategy, much of the underlying mechanisms surrounding how this powerful cellular defense activity detects intracellular UPEC and shuttles them from their intracellular location to the plasma membrane of BECs to be exported remains largely a mystery.

Here, we describe uropathogenic E.coli (UPEC) expelled from infected bladder

epithelium cells (BECs) within membrane-­bound vesicles as a distinct cellular defense

response. Examination of the intracellular UPEC revealed that intracellular bacteria were

initially processed via autophagy, the conventional degradative pathway, then delivered

into multivesicular bodies (MVBs) and encapsulated in nascent intraluminal vesicle membrane. We further show the bacterial expulsion is triggered when intracellular UPEC follow the natural degradative trafficking pathway and reach lysosomes and attempt to neutralize its pH to avoid degradation. This pathogen-­mediated activity is detected by mucolipin TRP channel 3 (TRPML3), a transient receptor potential cation channel localized on lysosomes, which spontaneously initiates lysosome exocytosis resulting in expulsion of exosome-­encased bacteria. These studies reveal a cellular default system for lysosome homeostasis and also, how it is coopted by the autonomous defense program to clear recalcitrant pathogens.

Dissertation

The urinary tract is one of the most intractable mucosal surfaces for pathogens to colonize. In addition to the natural barriers at this site, potential pathogens have to contend with the vigorous local innate immune response that is initiated by engagement of surveillance molecule TLRs. TLR4 appears to be not only exclusively expressed on superficial BECs but also critical to triggering robust local innate immune responses. TLR4 recognizes Gram-negative bacterial component LPS and initiates a series of intracellular NF-kappaB associated signaling events resulting in a cytokine response. We examined intracellular signaling events in human BECs leading to the production of IL-6, a major urinary cytokine, following activation by E. coli and isolated LPS, and observed that, in addition to the classical NF-kappaB associated pathway, BEC TLR4 triggers a distinct and more rapid signaling response involving, sequentially, Ca2+, AC3 generated cAMP, and the transcriptional factor CREB. This capacity of BECs to mobilize secondary messengers and evoke a more rapid IL-6 response might be critical in their role as first responders to microbial challenge in the urinary tract.

Here, we also report two additional distinct TLR4-mediated defense mechanisms in BECs. First, BEC TLR4 inhibits bacterial invasion, a necessary step for successful infection. TLR4-mediated suppression of bacterial invasion was linked to increased intracellular cAMP levels which negatively impacted Rac-1 mediated mobilization of the cytoskeleton. Additionally, we found that BECs continue to fight UPEC even after bacterial invasion by triggering bacterial exocytosis through a distinct TLR4-mediated mechanism following activation by LPS. In addition, we reveal that Caveolin-1, Rab27b, PKA, and MyRIP are components of the exocytic compartment and that they form a complex involved in the exocytosis of bacteria. The ability of TLR4 to mediate the rapid cytokine response, the inhibition of bacterial invasion, and the expulsion of intracellular bacteria from infected cells represents three previously unrecognized functions for this innate immune receptor.

Dissertation

Background and Purpose: Primary nocturnal enuresis (PNE) is a heterogeneous disorder with various underlying pathophysiological mechanisms. Results of our recent studies focused on the relationship of bladder function, sleep and brain function demonstrated a simultaneous occurrence of bladder and brain dysfunction in children with severe refractory PNE. We therefore proposed to use an animal model with altered bladder function to evaluate if abnormalities in bladder function induce functional derangement in brainstem micturition centers and/or sleep-arousal centers.
Materials and methods: In general, the study was divided in to 6 parts. Male Wistar rats (~ 1.5 months) were used for the study.
Study I: Establishment of animal model —— Male Wistar rats (200-220 g) underwent either Sham surgery or surgical reduction of bladder volume (RBV). Animals were used for further Cystometry, EEG, MRS and Cognitive function studies 4-5 weeks postoperatively.
Study II: Conventional Fill Cystometry (CFC) to evaluate bladder functional changes in response to surgical bladder volume reduction —— Twenty-four rats (RBV=12, SHAM 12) were used for the study. CFC was performed under conscious condition for evaluating the functional changes in response to surgical bladder capacity reduction.
Study III: Radiotelemetered EEG study to assess the impact of bladder dysfunction on sleep architecture and cortical arousals in rats —— Twenty-four rats (RBV=12, SHAM 12) were used for the study. Radiotelemeters were implanted in both groups 4 weeks post-operatively. The EEG biopotential and bladder pressure were monitored for 48 hours. Sleep architecture and cortical arousals were then evaluated manually.
Study IV: Evaluation of cognitive function following surgical bladder volume reduction —— Ninety eight rats (RBV=50, SHAM =48) were used for the study.
Morris Water Maze task: A circular plastic translucent pool half-filled with 26 ± 2ºC water, was used in the Morris Animals were given 9 consecutive training (2/day) sessions of Morris Water Maze (MWM) at 4 weeks postoperatively.
8-arm Radial Maze: Food pellets were randomly placed inside each arm of the maze and the rats were allowed to explore the maze freely for 5 minutes. The rat was allowed to explore the maze for 5 minutes. Total time spent in each arm, total distance traveled in the maze was recorded.
Study V: Magnetic Resonance Spectroscopy to detect functional changes in brain in response to bladder dysfunction elicited by surgical bladder volume reduction —— Proton magnetic resonance spectroscopy was employed to examine brain metabolic changes in 24 rats (RBV=12, SHAM=12). Single voxel 1 H MRS experiments were performed using a 7 T MRI scanner. MR spectra were then processed using the jMRUI software.
Phase VI: Enzyme -linked immunosorbent assay for the assessment of associated changes in neurotransmitters —— Animals were euthanized after MRS study and brain samples were collected. Serotonin and dopamine levels were assessed in 10 mg of tissue extracts from brainstem and cortex, with ELISA kits.
Results: Study I: Bladder reduction surgery did not affect the increase in body weight post -operatively. Average body weight of the RBV and the sham groups were 340.2 ± 47.2 g and 340.5 ± 67.9 g respectively at 4 weeks post operatively.
Study II: Compared to sham group, the maximum cystometric capacity in animals with RBV was remarkably reduced at week 4 (0.78 ± 0.12 ml vs. 1.46 ± 0.22 ml, RBV vs. Sham respectively; p<0.005). Moreover, maximum detrusor pressure during voiding was significantly increased in RBV group at week 4 post operatively (32.4± 2.14 vs.23.27±1.2 5 cm H2O, RBV vs. Sham respectively).
Study III: Light non-repaid eye movement sleep occurred significantly more in RBV rats compared to sham group (61.8% vs 35%). Deep sleep and rapid eye movement sleep occurred significantly less in RBV group compared to that of sham group (30.7% vs 53.4%).
Study IV: Results showed that the RBV group used a significantly longer latency to locate the platform compared to Sham group (24.4s vs 17.19s, RBV vs. Sham respectively, p<0.001).. Moreover, significantly more animals from the RBV group could not complete the visit of the 8 arms of radial maze than that of the sham group.
Study V: Seven metabolites were detected and quantified. The results demonstrated significant changes in the lactate (Lac) metabolism in some specific regions of rat brain. At 4 weeks post - operatively, level of lactate significantly decreased in the hippocampus (43%, P<0.001) cingulate and retrosplenial cortex (29%, p<0.05) of RBV rats compared to that of sham rats.
Study VI: Results demonstrated a significant increase in Serotonin level in the brainstem of RBV rats compared to that of SHAM rats (23.726 + 0.88 ng/ml vs. 1.88 + 0.302 ng/ml). Dopamine levels decreased significantly in brainstem samples of RBV group compared to sham group (2.85 + 0.10 ng/ml vs. 6.85 + 0.84 ng/ml).
Conclusion: Surgical bladder volume reduction of bladder capacity can induce functional changes in the central nervous system. An alteration of the sleep architecture occurred in response to surgical reduction of bladder volume in rats, suggesting that there exists a potential for central consequences of bladder dysfunction. Bladder disorder chronically altered brain energy metabolism. Furthermore, bladder disorder altered the central neurotransmission in the brainstem and cortex. The finding of bladder dysfunction induced significant impairments in cognitive function in RBV rats, suggesting that the alteration in brain energy metabolism may contribute to the behavioral and attention problems, impaired learning and cognitive performance.
研究背景： 原發性夜間遺尿症（PNE）是一種異質性疾病，涉及多種潛在的病理生理機制。我們最近的研究主要集中在膀胱功能，睡眠和腦功能的關係，結果顯示膀胱和腦功能障礙同時出現在患有嚴重難治性的PNE的兒童。因此，我們建議採用一種已改變膀胱功能的動物模型來評估膀胱功能異常會否引起腦幹排尿中心和/或睡眠 - 覺醒中心的功能紊亂
研究工具和方法： 研究被分成6個部分。雄性Wistar大鼠（約1.5個月）被用於研究。
研究I： 動物模型的建立 —— 雄性Wistar大鼠（200-220克），會先接受假手術或手術降低膀胱容量（RBV）。手術後4至5週，動物會進行進一步的膀胱測壓，腦電圖，MRS和認知功能研究。
研究II： 以常規填充膀胱測壓（CFC）評估減少膀胱容量手術對膀胱功能的變化 —— 二十四隻大鼠（RBV=12，對照=12）被用於研究。 CFC是用以評估在有意識的條件下，膀胱因膀胱容量減少的手術而引起的功能變化。
研究III： Radiotelemetered腦電圖研究，以評估在大鼠膀胱功能失調對睡眠結構和皮質覺醒的影響 —— 二十四隻大鼠（RBV=12，對照=12）被用於研究。膀胱容量減少的手術4週後，Radiotelemeters被植入在兩個組別的大鼠，並監測其腦電生物電勢和膀胱內壓48小時，然後手動評估睡眠結構和皮層覺醒。。
研究IV： 評估在膀胱容量減少的手術後對認知功能的影響 —— 103個大鼠（RBV=56，對照= =47）被用於研究。
Morris水迷宮任務： 一個圓形的塑料半透明池盛載半滿的水，溫度介乎26 - ±2℃，手術4週後，該池被用在莫里斯動物進行連續9次Morris水迷宮（MWM）培訓（每天2次）。
八臂迷宮： 食物顆粒被隨機放置在迷宮的每個臂內，大鼠可以自由地探索迷宮5分鐘。大鼠被允許探索迷宮5分鐘。在每個手臂所用的總時間，以及在迷宮行走的總距離都會被記錄。
研究V： 以磁共振波譜檢測膀胱容量減少的手術所引起的膀胱功能障礙對腦功能的改變 —— 以質子磁共振波譜研究24隻大鼠腦內的代謝變化(RBV=12，對照==12）。以7 T MRI掃描儀進行磁共振波譜實驗，然後使用jMRUI軟件處理MR譜。
第六期： 以酶聯免疫吸附測定法評估神經遞質的相關變化 —— 動物在進行MRS研究後實施安樂死，並收集其腦樣品。從腦幹和皮層提取10毫克組織提取物，使用ELISA試劑盒，以評估羥色胺和多巴胺水平。
結果： 研究I： 膀胱容量減少手術並沒有影響體重增加。手術4週後，利巴韋林和對照實驗組的平均體重分別為340.2±47.2克和340.5±67.9克。
研究II： 相比起對照實驗組的動物，RBV組的最大膀胱容量顯著降低（0. 0.78 ± 0.12毫升對1.46±0.22毫升），排尿頻率顯著增加（2.53±0.30 對.0.53±0.05/hr）。此外，排尿時最大逼尿肌壓力亦顯著升高（32.0.8±2.19 比.20.37±1.2 5厘米水分子）
研究III： 相比起對照實驗組的動物，光非快速動眼期睡眠顯著地較多發生於RBV大鼠身上（61.8％對35.6％），深層睡眠和快速動眼期睡眠顯著地較少發生在RBV組（32.3％對52.8％）
研究IV： 結果表明，RBV組使用了顯著較長的時間來定位平台（24.4s vs. vs.17.19s）。而且，在RBV組，顯著地較多動物無法完成行走8臂的放射狀迷宮。
研究V： 進行檢測和定量七種代謝物。結果顯示乳酸（LAC）代謝在大鼠大腦的某些特定區域出現顯著變化。在手術4週後，相比起對照實驗組的動物，RBV組大鼠在海馬體（43％，P <0.001），扣帶和夾肌皮質（29％，P <0.05）的乳酸水平均顯著減少。
研究VI： 結果顯示RBV大鼠腦幹的血清素水平較對照實驗組的顯著增加（23.726+0.88納克/毫升與1.88±0.302ng/ml）。RBV大鼠腦幹的多巴胺水平則較對照實驗組的顯著下降（2.850.10納克/毫升與6.85+0.84毫微克/毫升）。
結論： 外科膀胱容量減少可誘導中樞神經系統的功能變化。以外科手術減少膀胱容量的大鼠亦引起睡眠結構改變，這顯示膀胱功能障礙對中樞有潛在影響。膀胱疾病長期改變大腦的能量代謝。此外，膀胱疾病亦改變了在腦幹和大腦皮層的中樞神經遞質傳遞。研究發現膀胱功能障礙顯著地損害RBV大鼠的認知功能，顯示改變大腦的能量代謝亦可導致行為和專注力的問題，從而損害學習和認知能力。
Yeung, Chung Kwong.
Thesis Ph.D. Chinese University of Hong Kong 2014.
Includes bibliographical references (leaves 199-230).
Abstracts also in Chinese.
Title from PDF title page (viewed on 14, September, 2016).
Yeung, Chung Kwong.
Detailed summary in vernacular field only.
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