Doctor of Medicine / Publications 1-49 represent studies that I have undertaken myself or conjointly over a 34 year period to investigate a variety of issues relating to diseases of the kidney and urinary tract in children. The studies were carried out at the Royal Alexandra Hospital for Children, Camperdown when I was Clinical Superintendent from 1968 - 1970; The Department of Paediatrics, University of Minnesota, Minneapolis, USA when I was Overseas Research Fellow of the Post Graduate Foundation in Medicine, University of Sydney, 1970 - 1972, then as Staff Physician in Nephrology at the Royal Alexandra Hospital for Children, Camperdown, 1972 - 1977, and then Head of that Department at the Hospital until 1995 and then as an Honorary Staff Specialist at that hospital. Some of the studies were done conjointly with members of the Renal Unit of Royal Prince Alfred Hospital where I hold an Honorary appointment and others conjointly with members of the Renal Unit of Prince Henry Hospital, Little Bay. I was appointed Clinical Associate Professor to the Department of Paediatrics and Child Health, University of Sydney in 1993. In 1966 paediatric nephrology was in the early phase of development as a medical subspecialty. There was no definitive textbook, the first was published in 1975 (Pediatric Nephrology, Ed. Mitchell I. Rubin. Williams and Wilkins.). In the preface to the 2nd edition of Renal Disease (Blackwell) in 1967 the editor D.A.K. Black noted that he had included a chapter on paediatric aspects which had been planned for the 1st edition in 1962 but ”it could not be arranged”. In the chapter on Renal Disease in Children the author, D.Macauly, comments that the mortality rate of acute renal failure in children was 50%. When I joined the resident staff of the Royal Alexandra Hospital for Children in 1966, children with renal disease were managed by general paediatricians. There was no active program for the treatment of children with acute or chronic renal failure. A small number of kidney biopsies had been performed by Dr Trefor Morgan who, together with Dr Denis Wade, had taught me the technique while I was a resident medical officer at the Royal Prince Alfred Hospital in the preceding year. With the guidance and support of Dr S.E.J. Robertson and Dr C. Lee, Honorary Medical Officers, and Dr R.D.K. Reye, Head of the Department of Pathology, I began performing kidney biopsies on children at the request of the paediatrician in charge. In the same year, encouraged again by Doctors Robertson and Lee, and by J.C.M. Friend and J. Brown, I introduced peritoneal dialysis for the treatment of children with acute renal failure, a technique which I had also been taught by Dr Trefor Morgan whilst I was a resident at Royal Prince Alfred Hospital. Dr Robertson encouraged me to present my experience in percutaneous renal biopsy in children at the Annual Meeting of the Australian Paediatric Association in 1968 and this study became the first paper I published in relation to disease of the urinary tract in children (1). In 1970 I was granted an Overseas Research Fellowship by the Post Graduate Foundation in Medicine, University of Sydney, to enable me to undertake a fellowship in the Department of Paediatrics at the University of Minnesota. I had the great fortune in undertaking studies in the new discipline of paediatric nephrology and related research under the guidance of Dr A. F. Michael, Dr R.L.Vernier and Dr A. Fish. I acquired the techniques of immunopathology and electron microscopy. On my return to Australia I established a Department of Nephrology at the Royal Alexandra Hospital for Children. I introduced immunofluorescent and electron microscopic studies for the kidney biopsies that I continued to perform and, with the support of Dr R.D.K. Reye, I provided the official reports of these studies until 1990. As a result these studies became part of the histopathologic service provided by the hospital. I continue to be consulted concerning the interpretation of some electron microscopic findings in renal tissue. With the assistance of Dr J.D. Harley I set up a laboratory in the Children’s Medical Research Foundation to continue and expand the studies I had commenced during my Fellowship. Establishing a dialysis and transplant program for children with end stage renal disease (ESRD) was extremely time consuming. At that time most children with ESRD died. The program was initially established jointly with the Renal Unit at Royal Prince Alfred Hospital in 1972 and eventually dialysis facilities were established at the Children’s Hospital using predominantly peritoneal dialysis. By 1978 the existence of the Unit was well known in the general community and articles appeared in the press. One prompted the late Sir Lorimer Dods, the first Professor of Paediatrics in Australia to write to me congratulating me on what I had achieved. He remarked “I have just read with special interest Shaun’s review in the SMH of some of your recent achievements in the field of renal failure in infancy and childhood and want to offer you my personal congratulations on all that you have achieved and are achieving in this area of paediatrics which, in my little world of yesterday, meant nothing more than progressive and unrelenting fatal illness”. Taking part in the development of a relatively new discipline led me to study a number of areas. I encouraged trainees to write reports concerning clinical observations and eventually I was joined by Fellows whom I encouraged and supported to study a number of different areas to ensure that children were being cared for in an environment of strong and open enquiry. This led to studies on investigations of chronic renal failure which Dr Elisabeth Hodson pursued and studies on urinary tract infection in small children for which Dr Jonathon Craig was awarded a PhD. As I had been a contributor and co-author in a number of these studies they have been included in my list of publications. As a result of this diversity I have listed the publications in 9 sections. The overall theme is to study diseases of the renal tract in children and treatments used to understand the processes and ensure the most effective treatment. Some published abstracts of papers presented at scientific meetings have been included to clarify invitations I received to prepare reviews and chapters on various subjects and my involvement in some conjoint studies. I was author or coauthor of several book chapters, reviews, editorials and certain published studies to which I was invited to contribute as a result of my primary studies and these I have included as “Derivative References”numbered 50-76.

<p>Antibiotic-resistant bacteria are increasingly a major healthcare problem but very few new classes of antibiotics have been discovered or launched in recent decades. Approaches to dealing with the problem include learning how bacteria evolve to resistance and improving dosing regimens with current antibiotics so as to reduce the selection of resistant bacteria. </p><p>This thesis presents studies examining whether antibiotic dosing at high levels can prevent the selection of fluoroquinolone-resistant mutants in <i>Escherichia coli</i>. It also addresses the genetics of fluoroquinolone resistance in <i>E. coli</i> in relation to fitness costs for the resistant bacteria, and the evolution of <i>E. coli</i> to reduce the costs of resistance.</p><p>The mutant prevention concentration (MPC) of ciprofloxacin was measured for a set of clinical urinary tract infection <i>E. coli</i> strains showing that MPC could not be predicted from the minimum inhibitory concentration (MIC). Results from an <i>in vitro</i> kinetic model showed that an AUC/MPC >22 for ciprofloxacin was the single best pharmacodynamic index that predicted prevention of resistance emergence in the wild-type. Simulating currently approved dosing regimens for three different fluoroquinolones it was found that only a few were effective in preventing the selection of a small sub-population of pre-existing mutants. </p><p>Step-wise selection of fluoroquinolone resistance showed that the accumulation of mutations usually reduced bacterial fitness<i> in vitro</i> and <i>in vivo</i>. Systematic construction of isogenic resistant strains confirmed this result and revealed that some combinations of resistance mutations mutually compensate and increase both resistance and fitness. It was discovered that mutations altering RNA polymerase could ameliorate the fitness costs of fluoroquinolone resistance. Thus, the major fitness cost of fluoroquinolone resistance is due to defective transcription. </p><p>The finding that fluoroquinolone resistance mutations can increase resistance while mutually compensating their fitness costs, shows that resistance to fluoroquinolones can continue to evolve in the absence of antibiotic selection.</p>

<p>The emergence of multidrug resistant bacteria world wide is a serious problem, and very few new drugs are under development. The selection of resistant bacteria is affected by factors such as mutation rate, biological fitness cost and the rate of fitness compensation. This thesis is focused on how mutation rate affects resistance to fluoroquinolones and on exploring a dosing strategy that might slow resistance development. </p><p>In a set of urinary tract <i>Escherichia coli</i> isolates MIC values above the breakpoint for the fluoroquinolones norfloxacin and ciprofloxacin carried at least three resistance-associated mutations. In these isolates the number of resistance mutations correlated with the mutation rate. During step-wise selection for decreased susceptibility to fluoroquinolones, the accumulation of mutations in <i>E. coli</i> was associated with an increasing biological cost both <i>in vitro</i> and <i>in vivo</i>. However, in some lineages an additional selection step for resistance was associated with a partial restoration of fitness. During step-wise selections we found, as expected, that reduced ciprofloxacin susceptibility frequently hitchhiked with a strong mutator phenotype. More surprisingly, we also found that reduced susceptibility was frequently associated with the emergence of rifampicin-resistant populations. We hypothesise that this correlation reflects selection for fitness-compensating mutations in RNA polymerase.</p><p>Mutant prevention concentration (MPC) dosing has been proposed as a strategy to reduce the selection of resistant bacterial populations. Based on limited data it had been thought that MPC might be a simple multiple of MIC, which can easily be determined. However, we showed for a collection of susceptible urinary tract <i>E. coli </i>that MPC could not be predicted from MIC and must be measured directly for relevant populations. Using an <i>in vitro</i> kinetic model we also showed that the pharmacodynamic index that best predicted prevention of resistance development in wild type <i>E. coli</i> was an AUC/MPC of > 22 for ciprofloxacin.</p>

The emergence of multidrug resistant bacteria world wide is a serious problem, and very few new drugs are under development. The selection of resistant bacteria is affected by factors such as mutation rate, biological fitness cost and the rate of fitness compensation. This thesis is focused on how mutation rate affects resistance to fluoroquinolones and on exploring a dosing strategy that might slow resistance development. In a set of urinary tract Escherichia coli isolates MIC values above the breakpoint for the fluoroquinolones norfloxacin and ciprofloxacin carried at least three resistance-associated mutations. In these isolates the number of resistance mutations correlated with the mutation rate. During step-wise selection for decreased susceptibility to fluoroquinolones, the accumulation of mutations in E. coli was associated with an increasing biological cost both in vitro and in vivo. However, in some lineages an additional selection step for resistance was associated with a partial restoration of fitness. During step-wise selections we found, as expected, that reduced ciprofloxacin susceptibility frequently hitchhiked with a strong mutator phenotype. More surprisingly, we also found that reduced susceptibility was frequently associated with the emergence of rifampicin-resistant populations. We hypothesise that this correlation reflects selection for fitness-compensating mutations in RNA polymerase. Mutant prevention concentration (MPC) dosing has been proposed as a strategy to reduce the selection of resistant bacterial populations. Based on limited data it had been thought that MPC might be a simple multiple of MIC, which can easily be determined. However, we showed for a collection of susceptible urinary tract E. coli that MPC could not be predicted from MIC and must be measured directly for relevant populations. Using an in vitro kinetic model we also showed that the pharmacodynamic index that best predicted prevention of resistance development in wild type E. coli was an AUC/MPC of &gt; 22 for ciprofloxacin.

BACKGROUND: An important characteristic of transitional cell carcinoma (TCC) is the formation of tumors in multiple sites throughout the whole urinary tracts. Two theories explain the pathophysiologic mechanisms of multifocal tumors: (1) intraluminal seeding, it indicates the multiple tumors come from a single transformed malignant cell with secondary seeding or migration at different sites, and (2) the field cancerization, carcinogens affect the urothelium at multiple sites, leading to numerous mutation and independent growth of multifocal polyclonal tumors. Multifocal urothelial carcinomas could come from intraluminal seeding or from field cancerization. However, the data of clinical behaviors between the two different tumor types are lacking. METHODS: Bilateral synchronous and metachronous primary TCC of the upper urinary tracts were derived from field cancerization. Recurrent bladder cancers following upper-tract tumors mostly come from intraluminal seeding. The recurrence, progression, and prognosis of the two different tumors were analyzed. RESULTS: Bilateral upper-tract urothelial carcinomas derived from field cancerization were frequently associated with renal insufficiency, which were more invasive and had poor prognosis than bladder tumors derived from intraluminal seeding. CONCLUSION: The clinical behaviors of the multiple urothelial tumors derived from field cancerization and from intraluminal seeding should be different.

Herbal teas and fruit juices used in our regular diet may have importance in the protective treatment of some infectious diseases. In this study, selected dietary beverages were investigated for their antioxidant capacities and antimicrobial activities against Proteus mirabilis, a well known bacteria in urinary tract infections. Herbal teas / sage (Salvia fruticosa Mill), anise (Pimpinella anisum L.), rosehip (Rosa canina L.), camomile (Anthemis arvensis L.) and fruit juices / grape (Vitis vinifera L.), orange (Citrus sinensis L.), peach (Prunus persica L.), and pomegranate (Punica granatum L.) were chosen as samples of regular diets. Selected fruit juices and aqueous infusion tea extracts, lyophilised to dryness, were used throughout this study. Antioxidant capacities of the extracts were carried out by using 2,2&rsquo / -azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) radical scavenging (ABTS) and 2,2-diphenyl-1-picrylhydrazyl radical scavenging (DPPH) methods along with the determination of total phenolic compounds in the extracts. Antimicrobial activities of extracts were determined by disc diffusion test, minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) methods. Among the herbal teas, sage infusion extract has displayed the highest radical scavenging capacity with ABTS EC50 value of 5.152 mg/mL, DPPH EC50 value of 0.072 mg/mL and with its high phenolic content of 0.411 mg/mg gallic acid equivalence. Among the fruit juices pomegranate has revealed significantly high DPPH EC50 and TEAC values 0.924 mg/mL and 0.552 mmol/g, respectively. Peach juice has been found with the highest total phenolic amount of 0.067 mg/mg gallic acid equivalent. Antimicrobial activities of herbal teas were correlating with antioxidant capacity studies, whereas sage infusion tea extract exhibited 3 mg/mL of minimum inhibitory concentration (MIC) and 6 mg/mL of minimum bactericidal concentration (MBC). Rosehip was also found as an effective antimicrobial agent with a minimum inhibitory concentration value of 3 mg/mL. In the meantime, there was no significant difference in the zone inhibition of herbal tea infusion extracts. In case of fruit juices grape and pomegranate may be effective antimicrobials in P. mirabilis infections with 0.75 mg/mL MIC and 6 mg/mL MBC, respectively at the same time both juices revealed significantly high inhibition zones with 11 mm.

Ph.D.

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

Šlapimo organų infekcija (ŠOI) vaikams yra antra pagal dažnį po kvėpavimo organų infekcijų. Tai dažniausia vaikų nefrologinė liga. ŠOI iki 3–6 mėn. amžiaus dažniau serga berniukai, vyresniame amžiuje – mergaitės. Priešmokykliniame amžiuje mergaitės simptomine šlapimo organų infekcija serga 6–20 kartų dažniau nei berniukai. Beveik kas trečia moteris iki 24 metų ŠOI yra sirgusi bent vieną kartą, o per gyvenimą ŠOI yra sirgusi beveik kas antra moteris. ŠOI linkusi pasikartoti. Kartojantis ŠOI, liga gali įgauti lėtinę eigą, sukelti inkstų randėjimą, lėtinį inkstų funkcijos nepakankamumą (IFN), nulemti hipertenzijos atsiradimą, o moterims – nėštumo komplikacijas. 29 % vaikų, kuriems buvo atliktos inkstų transplantacijos, inkstų pažeidimas buvo sąlygotas pielonefrito ar intersticinio nefrito. Lietuvoje vaikų lėtinio IFN priežastis 31,7 proc. obstrukcinė nefropatija ir lėtinis pielonefritas. Kol nebuvo taikomas profilaktinis gydymas, 60 proc. mergaičių ir 20 proc. berniukų ŠOI pasikartodavo jau pirmaisiais metais po pirmos ŠOI. Taikant profilaktinį gydymą, 1 m. laikotarpyje po persirgtos ŠOI. infekcijos pasikartojimas sumažėjo iki 15 proc. Mažo amžiaus vaikams ŠOI pasikartojimą dažniausiai lemia įgimtos šlapimo organų anomalijos. Dauguma jaunesnio mokyklinio amžiaus vaikų, kuriems yra pasikartojanti ŠOI, turi organiškai nepakitusius šlapimo organus. Pastaruoju metu atliekamos studijos, kurių tikslas nustatyti elgesio ir funkcinių sutrikimų vertę ŠOI pasikartojimui, tačiau duomenys... [toliau žr. visą tekstą] / Urinary tract infection (UTI) is one of the most common bacterial diseases in childhood. Recurrent UTI occurs in 20 – 86% of children. Recurrent UTI is relatively frequent in girls. At the age of 7, the prevalence of recurrent UTI in boys population is 1%, in girls population – 5%. Nearly one of three women will have at least one episode of UTI requiring antimicrobial therapy by the age of 24 years. Almost half of all women will experience one UTI during their lifetime. It is known, that in the group of young children the most common reason of recurrent UTI is anatomic abnormalities, such as vesicoureteral reflux (VUR), hydronephrosis. However, not all recurrent UTI can be explained by anatomic abnormalities. The vast majority of school age children with recurrent UTI have anatomically normal urinary tract. We found changes in urodynamic investigation for 91.4% of children with recurrent urinary tract infection at the age of 5–18. Having a history of previous recurrent UTI is a strong risk factor for having subsequent UTI. Antibacterial characteristics of urine and other host defence mechanisms may be important signs associated with UTI risk, but have not been clearly shown to be associated with UTI in healthy persons. Recent studies discuss about the role of behavioural and functional abnormalities (inadequate fluid intake, stool retention, infrequent voiding, etc.) that can predispose recurrent urinary tract infections. Influence of some these abnormalities for recurrent... [to full text]

<p>Urinary tract infections (UTIs) are the second most common type of infection identified in the clinical setting and disproportionately afflict women. UTIs most frequently manifest in the form of infection of the lower urinary tract, involving the bladder. Uropathogens, particularly uropathogenic E. coli, progressively colonize the urethra and ascend to the bladder, where they initiate cystitis. In some cases, infection further ascends through the ureters and reaches the kidneys, where it causes pyelonephritis. Infection of both the upper and lower urinary tract can have serious ramifications for the host, and this is in large part due not to infection itself but to host-directed responses to bacterial insults. </p><p> In this thesis, I will describe and discuss two distinct aspects of UTIs. In the first study, in vivo work in a mouse model of urinary tract infection revealed a novel role for mast cells, which are tissue-resident granulated innate immune cells, in directing the detachment and death of epithelial cells during cystitis, facilitating the clearance of bacteria from the bladder. An ex vivo porcine bladder infection model suggested a specific role for mast cell granules and the proteases contained therein, which was corroborated with in vitro experiments utlizing isolated mast cell granules and human epithelial cells to demonstrate granule-induced exfoliation and cell death. From this work, it is clear that mast cells play a highly targeted role in modulating urothelial integrity during bladder infection by mediating host-directed epithelial loss.</p><p> In the second study described in this dissertation, the synergistic roles of both pyelonephritis and vesico-ureteric reflux (VUR), a congenital urinary tract defect that results in the improper backflow of urine from the bladder to the kidney, in the development of reflux nephropathy, a fibrotic host response characterized by renal scar formation, were elucidated in a series of in vivo experiments. Specifically, the C3H mouse, which is naturally susceptible to VUR, was utilized to characterize the dynamics of kidney infection and the onset of reflux nephropathy. Renal scarring was dependent on the presence of sustained kidney infection and the accompanying inflammatory response due to VUR, while neither transient infection nor reflux alone were sufficient to provoke nephropathy. Thus, the development of reflux nephropathy is dependent upon the confluence of both infection and VUR. </p><p> This body of work reveals the double-edged sword of the host inflammatory response to urinary tract infection. In the bladder, mast cell activation and degranulation leads to granule-induced epithelial exfoliation and consequently a reduction in the bacterial burden in the bladder. However, the sustained inflammatory response that accompanies pyelonephritis in vesico-ureteric reflux-affected individuals results in significant damage to the kidney without any accompanying reduction in infection. These findings highlight the dueling roles of the host inflammatory response to infection in the upper and lower urinary tract and strongly suggest that differential clinical approaches to cystitis and pyelonephritis are necessary to promote an effective mast cell in the bladder in the former and facilitate the clearance of renal infection while mitigating tissue damage in the latter.</p> / Dissertation