Detection of urinary bladder cancer cells using redox ratio and double excitation wavelengths autofluorescence

The optical redox ratio as a measure of cellular metabolism is determined by an altered ratio between endogenous fluorophores NADH and flavin adenine dinucleotide (FAD). Although reported for other cancer sites, differences in optical redox ratio between cancerous and normal urothelial cells have not previously been reported. Here, we report a method for the detection of cellular metabolic states using flow cytometry based on autofluorescence, and a statistically significant increase in the redox ratio of bladder cancer cells compared to healthy controls. Urinary bladder cancer and normal healthy urothelial cell lines were cultured and redox overview was assessed using flow cytometry. Further localisation of fluorescence in the same cells was carried out using confocal microscopy. Multiple experiments show correlation between cell type and redox ratio, clearly differentiating between healthy cells and cancer cells. Based on our preliminary results, therefore, we believe that this data contributes to current understanding of bladder tissue fluorescence and can inform the design of endoscopic probes. This approach also has significant potential as a diagnostic tool for discrimination of cancer cells among shed urothelial cells in voided urine, and could lay the groundwork for an automated system for population screening for bladder cancer.

Discrimination of healthy and cancer cells of the bladder by metabolic state, based on autofluorescence

Bladder cancer is among the most common cancers worldwide (4th in men). It is responsible for high patient morbidity and displays rapid recurrence and progression. Lack of sensitivity of gold standard techniques (white light cystoscopy, voided urine cytology) means many early treatable cases are missed. The result is a large number of advanced cases of bladder cancer which require extensive treatment and monitoring. For this reason, bladder cancer is the single most expensive cancer to treat on a per patient basis. In recent years, autofluorescence spectroscopy has begun to shed light into disease research. Of particular interest in cancer research are the fluorescent metabolic cofactors NADH and FAD. Early in tumour development, cancer cells often undergo a metabolic shift (the Warburg effect) resulting in increased NADH. The ratio of NADH to FAD ("redox ratio") can therefore be used as an indicator of the metabolic status of cells. Redox ratio measurements have been used to differentiate between healthy and cancer breast cells and to monitor cellular responses to therapies. Here, we have demonstrated, using healthy and bladder cancer cell lines, a statistically significant difference in the redox ratio of bladder cancer cells, indicative of a metabolic shift. To do this we customised a standard flow cytometer to excite and record fluorescence specifically from NADH and FAD, along with a method for automatically calculating the redox ratio of individual cells within large populations. These results could inform the design of novel probes and screening systems for the early detection of bladder cancer.

Computational model of bladder tissue based on its measured optical properties

Urinary bladder diseases are a common problem throughout the world and often difficult to accurately diagnose. Furthermore, they pose a heavy financial burden on health services. Urinary bladder tissue from male pigs was spectrophotometrically measured and the resulting data used to calculate the absorption, transmission, and reflectance parameters, along with the derived coefficients of scattering and absorption. These were employed to create a "generic" computational bladder model based on optical properties, simulating the propagation of photons through the tissue at different wavelengths. Using the Monte-Carlo method and fluorescence spectra of UV and blue excited wavelength, diagnostically important biomarkers were modeled. Additionally, the multifunctional noninvasive diagnostics system "LAKK-M" was used to gather fluorescence data to further provide essential comparisons. The ultimate goal of the study was to successfully simulate the effects of varying excited radiation wavelengths on bladder tissue to determine the effectiveness of photonics diagnostic devices. With increased accuracy, this model could be used to reliably aid in differentiating healthy and pathological tissues within the bladder and potentially other hollow organs.

Changes in autofluorescence based organoid model of muscle invasive urinary bladder cancer

Muscle invasive urinary bladder cancer is one of the most lethal cancers and its detection at the time of transurethral resection remains limited and diagnostic methods are urgently needed. We have developed a muscle invasive transitional cell carcinoma (TCC) model of the bladder using porcine bladder scaffold and the human bladder cancer cell line 5637. The progression of implanted cancer cells to muscle invasion can be monitored by measuring changes in the spectrum of endogenous fluorophores such as reduced nicotinamide dinucleotide (NADH) and flavins. We believe this could act as a useful tool for the study of fluorescence dynamics of developing muscle invasive bladder cancer in patients.

Dynamic Compartmentalization of Persistent UPEC in the Superficial Bladder Epithelium

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.

Surgical technique to avoid bladder flap formation during cesarean section

Cesarean section (CS) is now the most common major surgical procedure performed on women worldwide. A CS can be performed by either suturing or not suturing of the visceral peritoneum. Creation of the bladder flap is an integral step of the standard cesarean section. The bladder flap is made by superficially incising and dissecting the peritoneal lining to separate the urinary bladder from the lower uterine segment. It’s still debated whether the formation of bladder flap is advantageous or not. If the uterine incision is made slightly above the vesicouterine peritoneal fold, the loose connective tissue between the uterus and the urinary bladder allows spontaneous descent of the bladder. Evidence on the role of the bladder flap in cesarean section is very limited. At present, it remains to be established whether there is any advantage in dissecting the bladder from the lower uterine segment during cesarean section.

Licochalcone A exerts antitumor activity in bladder cancer cell lines and mice models

Purpose: To investigate the effect of licochalcone A (LA) on the inhibition of cell proliferation and ERK1/2 phosphorylation in bladder carcinoma cell lines. Methods: Cell viability was investigated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) assay. Dye-binding method was used to examine the concentration of proteins. Lymphocytes were extracted from mice and after surface staining were subjected to BD fixation and permeabilization for intracellular staining. Flow cytometry was used to measure cellular fluorescence. Results: MTT results revealed a significant decrease in the proliferation of UM-UC-3, J82 and HT-1197 cell lines on treatment with LA. LA also induced reduction in phosphorylation of ERK1/2 in all three carcinoma cell lines. In the mouse model, licochalcone A treatment via intraperitoneal (ip) administration induced a significant decrease in the level of regulatory T cells (Tregs). Comparison of the mouse interferon-α (IFN-α)-treated and LA-treated groups revealed that LA treatment caused enhancement of cytotoxic T lymphocyte (CTL) activity similar to that of IFN-α. Administration of UM-UC-3 cells in C3H/HeN mice resulted in marked reduction in the counts for splenocytes and CD4+ CD25+ Foxp3+ T (regulatory T cells) cell proportion in LA-treated mice compared to untreated control group. Conclusion: Licochalcone A may be of therapeutic importance for the prevention of bladder carcinoma. However, studies are required to ascertain the compound’s usefulness in this regard.

Optical redox ratio and endogenous porphyrins in the detection of urinary bladder cancer:a patient biopsy analysis

Bladder cancer is among the most common cancers in the UK and conventional detection techniques suffer from low sensitivity, low specificity, or both. Recent attempts to address the disparity have led to progress in the field of autofluorescence as a means to diagnose the disease with high efficiency, however there is still a lot not known about autofluorescence profiles in the disease. The multi-functional diagnostic system "LAKK-M" was used to assess autofluorescence profiles of healthy and cancerous bladder tissue to identify novel biomarkers of the disease. Statistically significant differences were observed in the optical redox ratio (a measure of tissue metabolic activity), the amplitude of endogenous porphyrins and the NADH/porphyrin ratio between tissue types. These findings could advance understanding of bladder cancer and aid in the development of new techniques for detection and surveillance.

Challenges with in vitro and in vivo experimental models of urinary bladder cancer for novel drug discovery

Urinary bladder cancer (UBC) is the second most frequent malignancy of the urinary system and the ninth most common cancer worldwide, affecting individuals over the age of 65. Several investigations have embarked on advancing knowledge of the mechanisms underlying urothelial carcinogenesis, understanding the mechanisms of antineoplastic drugs resistance and discovering new antineoplastic drugs. In vitro and in vivo models are crucial for providing additional insights into the mechanisms of urothelial carcinogenesis. With these models, various molecular pathways involved in urothelial carcinogenesis have been discovered, allowing therapeutic manipulation.

Australian pelvic floor questionnaire : a validated interviewer-administered pelvic floor questionnaire for routine clinic and research

The aim of this study was to design and validate an interviewer-administered pelvic floor questionnaire that integrates bladder, bowel and sexual function, pelvic organ prolapse, severity, bothersomeness and condition-specific quality of life. Validation testing of the questionnaire was performed using data from 106 urogynaecological patients and a separately sampled community cohort of 49 women. Missing data did not exceed 2% for any question. It distinguished community and urogynaecological populations regarding pelvic floor dysfunction. The bladder domain correlated with the short version of the Urogenital Distress Inventory, bowel function with an established bowel questionnaire and prolapse symptoms with the International Continence Society prolapse quantification. Sexual function assessment reflected scores on the McCoy Female Sexuality Questionnaire. Cronbach’s α coefficients were acceptable in all domains. Kappa coefficients of agreement for the test–retest analyses varied from 0.5 to 1.0. The interviewer-administered pelvic floor questionnaire assessed pelvic floor function in a reproducible and valid fashion in a typical urogynaecological clinic.

Industrial design in endoscopy : the development of a tissue and organ extractor

Throughout history, developments in medicine have aimed to improve patient quality of life, and reduce the trauma associated with surgical treatment. Surgical access to internal organs and bodily structures has been traditionally via large incisions. Endoscopic surgery presents a technique for surgical access via small (1 Omm) incisions by utilising a scope and camera for visualisation of the operative site. Endoscopy presents enormous benefits for patients in terms of lower post operative discomfort, and reduced recovery and hospitalisation time. Since the first gall bladder extraction operation was performed in France in 1987, endoscopic surgery has been embraced by the international medical community. With the adoption of the new technique, new problems never previously encountered in open surgery, were revealed. One such problem is that the removal of large tissue specimens and organs is restricted by the small incision size. Instruments have been developed to address this problem however none of the devices provide a totally satisfactory solution. They have a number of critical weaknesses: -The size of the access incision has to be enlarged, thereby compromising the entire endoscopic approach to surgery. - The physical quality of the specimen extracted is very poor and is not suitable to conduct the necessary post operative pathological examinations. -The safety of both the patient and the physician is jeopardised. The problem of tissue and organ extraction at endoscopy is investigated and addressed. In addition to background information covering endoscopic surgery, this thesis describes the entire approach to the design problem, and the steps taken before arriving at the final solution. This thesis contributes to the body of knowledge associated with the development of endoscopic surgical instruments. A new product capable of extracting large tissue specimens and organs in endoscopy is the final outcome of the research.

Adverse health effects to air pollution and guidelines to prevent them

This is the first in a series of four articles which will explore different aspects of air pollution, its impact on health and challenges in defining the boundaries between impact and nonimpact on health. Hardly a new topic one might say. Indeed, it’s been an issue for centuries, millennia even! For example, Pliny the Elder (AD 23-79), a Roman officer and author of the ‘Natural History’ recommended that: “…quarry slaves from asbestos mines not be purchased because they die young”, and suggested: “…the use of a respirator, made of transparent bladder skin, to protect workers from asbestos dust.” Closer to modern times, a Danish Proverb states: "Fresh air impoverishes the doctor". While none of these statements are an air quality guideline in a modern sense, they do illustrate that, for a very long time, we have known that there is a link between air quality and health, and that some measures were taken to reduce the impact of the exposure to the pollutants. Obviously, we are much more sophisticated now!

CDKN2A mutation in a non-FAMMM kindred with cancers at multiple sites results in a functionally abnormal protein

The CDKN2A gene encodes p16 (CDKN2A), a cell-cycle inhibitor protein which prevents inappropriate cell cycling and, hence, proliferation. Germ-line mutations in CDKN2A predispose to the familial atypical multiple-mole melanoma (FAMMM) syndrome but also have been seen in rare families in which only 1 or 2 individuals are affected by cutaneous malignant melanoma (CMM). We therefore sequenced exons 1alpha and 2 of CDKN2A using lymphocyte DNA isolated from index cases from 67 families with cancers at multiple sites, where the patterns of cancer did not resemble those attributable to known genes such as hMLH1, hMLH2, BRCA1, BRCA2, TP53 or other cancer susceptibility genes. We found one mutation, a mis-sense mutation resulting in a methionine to isoleucine change at codon 53 (M531) of exon 2. The individual tested had developed 2 CMMs but had no dysplastic nevi and lacked a family history of dysplastic nevi or CMM. Other family members had been diagnosed with oral cancer (2 persons), bladder cancer (1 person) and possibly gall-bladder cancer. While this mutation has been reported in Australian and North American melanoma kindreds, we did not observe it in 618 chromosomes from Scottish and Canadian controls. Functional studies revealed that the CDKN2A variant carrying the M531 change was unable to bind effectively to CDK4, showing that this mutation is of pathological significance. Our results have confirmed that CDKN2A mutations are not limited to FAMMM kindreds but also demonstrate that multi-site cancer families without melanoma are very unlikely to contain CDKN2A mutations.