MRI-Based Tumour Targeting Enhancement with Magnetotactic Bacterial Carriers

RÉSUMÉ Le cancer constitue la première cause de mortalité au Québec, avec 20,000 décès estimés par année. Parmi tous les patients atteints du cancer, une grande proportion pourrait profiter de l’avancement technologique en ce qui concerne le transport de médicaments. En effet, l’un des meilleurs moyens d’augmenter l’efficacité d’un médicament contre le cancer, tout en réduisant sa toxicité sur les cellules saines, est de le diriger vers la tumeur et de le maintenir à cet endroit jusqu’à ce qu’un effet thérapeutique se produise. Le transport ciblé de médicaments vers la tumeur peut considérablement améliorer l’efficacité thérapeutique, surtout si le transporteur est capable d’atteindre les zones nécrotiques et se répartir uniformément dans la zone à traiter. Les bactéries, de par leur motilité, sont d’excellents candidats pour une telle application, surtout qu’elles peuvent aussi être facilement fonctionnalisées. Ainsi, la recherche sur le traitement du cancer utilisant des bactéries s’est imposée comme une approche prometteuse surtout qu’elle pallie à une limitation majeure de la chimiothérapie et de la radiothérapie en permettant le traitement des zones anaérobies. Alors que des laboratoires à travers le monde tentent de fabriquer des systèmes miniatures en se basant sur le modèle bactérien, nous avons opté pour l’utilisation des bactéries qui existent dans la nature. Notre stratégie a été de trouver un système biologique ayant les caractéristiques essentielles (e.x. diamètre total de moins de deux micromètres, force de poussée de plus de 4 pN, etc.) et de concentrer nos efforts à identifier une interface et une méthode permettant son contrôle pour des fins de ciblages thérapeutiques dans les lésions tumorales. Nous avons identifié les bactéries magnétotactiques de type MC-1 comme le meilleur transporteur potentiel de médicaments pour le ciblage du cancer. Les MC-1 sont à la fois dirigeables par champs magnétiques et anaérobies, ce qui leur donne un grand avantage par rapport aux bactéries traditionnellement utilisées pour le ciblage du cancer. Le ciblage du cancer avec des bactéries exploite le plus souvent l’affinité des bactéries anaérobies à la région nécrotique faible en oxygène de la tumeur. Certes, ce ciblage manque de spécificité et un des problèmes le plus reconnu est la nécessité d’injecter une forte dose de bactéries pour assurer une croissance de celles-ci à l’intérieur de la tumeur. Ceci n’est pas le cas avec les MC-1 car elles sont à la fois anaérobies et magnétotactiques grâce à une chaîne de nanoparticules d’environ 70 nanomètres de diamètre, formant une sorte de « nano-boussole » magnétique à----------ABSTRACT Magnetotactic Bacteria (MTB) are being explored as potential drug transporters to solid tumours. The MTB’s active motility combined with magnetotaxism (their ability to swim following the direction of a magnetic field) offer new and potentially more accurate solutions in delivering drugs to tumours. In fact, the flagella bundles of the MC-1 bacteria (with an overall ideal cell diameter of approximately 50% the diameter of the tiniest human blood vessels) provide 4.0 to 4.7pN of thrust force for propulsion (roughly 10 times the value of many other well-known flagellated bacteria). Since there are no existing methods or technologies capable of inducing an equivalent force on a carrier of appropriate size for traveling inside a tumour’s microvasculature, live microorganisms are considered as a viable option. Many of the parameters in a tumour microenvironment, such as malformed angiogenesis capillaries, heterogeneous blood flow, and high interstitial pressure, hinder the delivery of blood-borne drugs to the affected area. Active motility might prove to be helpful in bypassing these limitations and may facilitate the uniform distribution of the drug in the targeted area. An MTB navigation technique that allows targeting without prior knowledge of the exact architecture of the vessels network has been developed. This navigation technique exploits both the ability of the MTB to swim following an imposed magnetic field and their random, continuous motion at low magnetic fields. Firstly, a focused magnetic field on the target sets the overall direction of the bacteria. Then, as the bacteria approach the targeted zone, the intensity of the magnetic field is decreased, which allows better bacteria repartition by exploiting their free motion. An additional approach that enhances MTB targeting relies on modulating the magnetic field direction in time, while keeping the magnetic field lines pointed toward the target. Navigation experiments in complex micro-channel networks highlight this process, where the successful targeting of bacteria is demonstrated when an appropriate magnetic field algorithm is applied, especially when it takes into account the nature of the channel network. Tridimensional control and navigation of MTB is also possible with the same technique through proper powering of the magnetic coils. In fact, by controlling their magnetic environment, it is possible to form a swarm of MTB, control its size and position within a given volume using a computer program

Nanoparticle-based techniques for bladder cancer imaging: a review

Bladder cancer is very common in humans and is often characterized by recurrences, compromising the patient's quality of life with a substantial social and economic impact. Both the diagnosis and treatment of bladder cancer are problematic due to the exceptionally impermeable barrier formed by the urothelium lining the bladder; this hinders the penetration of molecules via intravesical instillation while making it difficult to precisely label the tumor tissue for surgical resection or pharmacologic treatment. Nanotechnology has been envisaged as an opportunity to improve both the diagnostic and therapeutic approaches for bladder cancer since the nanoconstructs can cross the urothelial barrier and may be functionalized for active targeting, loaded with therapeutic agents, and visualized by different imaging techniques. In this article, we offer a selection of recent experimental applications of nanoparticle-based imaging techniques, with the aim of providing an easy and rapid technical guide for the development of nanoconstructs to specifically detect bladder cancer cells. Most of these applications are based on the well-established fluorescence imaging and magnetic resonance imaging currently used in the medical field and gave positive results on bladder cancer models in vivo, thus opening promising perspectives for the translation of preclinical results to the clinical practice

Dinitropyrenes: Nitroreduction Processes and Evaluation of Indices of Exposure.

Investigations were conducted to identify biomarkers of exposure to dinitropyrenes (DNPs), which are mutagenic and carcinogenic residues of incomplete combustion. One possible biomarker, a DNP-protoporphyrin IX adduct, was not found in analytically useful quantities in liver tissue or in bile. Clastogenicity, a biomarker of effect, also was found to be absent in marrow cells following subchronic exposure of rats to DNPs by gavage. Solubility studies, used as a basis for dosing guidelines, revealed that DNPs are relatively insoluble in both aqueous and lipid tissue compartments. Calculated log P values indicated that the reduced metabolites of DNPs probably are more soluble in tissues than are DNPs. Thus, increasing solubility may drive nitroreduction. The dependence of expression of a DNP-related biomarker on nitroreductase activity led to efforts to quantify nitroreductase activity. A nitroreductase assay of hepatic cytosol surprisingly revealed that DNP nitroreduction proceeded according to log (FMN), as well as to enzyme content. Further experiments revealed that flavin-dependent nitroreduction occurred by chemical, rather than enzymatic activity. This reaction should have been unfavorable thermodynamically, and so electrochemical experiments were undertaken to re-measure the electrochemical half-wave reduction potential. Cyclic voltammograms of 1,6-DNP, using a platinum working electrode, vs. Ag/AgCl, yielded a reduction wave with a crest at $-$100 mV. Electrolysis of 1,6-DNP at potentials at least as positive as 0.00 mV vs. Ag/AgCl reference electrode, using platinum or mercury working electrodes, yielded 1-amino-6-nitropyrene and pyrene-1,6-diamine, proving that the wave in cyclic voltammetry was a nitroreduction wave. Thus, DNPs are reduced more easily than previously reported. Flavin-dependent nitroreduction has been studied extensively in certain nitroarene pharmaceuticals, and the nitroreduction mechanism is well understood. Extension of this chemistry to DNPs reveals that the oxidative damage and protein alkylation that accompany the nitroreduction reaction may act as tumor promoters. Thus, direct exposure of tissues to DNP nitroreduction may cause fixation of more mutations than might be expected of exposure to DNP metabolites only. This mechanism may be important to consider relative to tumorigenesis and biomarker studies, and to DNP risk assessment

Virulence of Salmonella typhimurium 1,4,[5],12:i:- : the new emergent strain

Tese de Doutoramento em Ciências Veterinárias na Especialidade Sanidade AnimalSalmonella serovar 1,4,[5],12:i:- is presently considered one of the major serovars responsible for human salmonellosis worldwide. A multidisciplinary approach, including the fields of epidemiology, spatial statistics, clinical and applied microbiology was used to perform an extensive characterization of Salmonella 1,4,[5],12:i:- isolates obtained by the National Health Institute Dr. Ricardo Jorge, which was lacking due to the recent emergence. It was observed that cases are reported in most districts, being more frequent in the Portuguese coastland. Spatial statistical analysis showed a significant geographic clustering, pointing out for the importance of evaluating these areas to identify risk factors, in order to establish adequate prevention programs. The most relevant antimicrobial profile in this serovar is the tetra-resistance pattern (R-type ASSuT), displaying resistance to ampicillin, streptomycin, sulphonamides and tetracyclines. A high occurrence of R-type ASSuT isolates was observed in the isolates under study, with the majority harboring the resistance genes frequently associated with the European clone, namely blaTEM, sul2, straA-straB, tetB. Additionally, resistance to quinolones and 3rd generation cephalosporin was also detected. In Portugal, the rapid spread of Salmonella 1,4,[5],12:i:- R-type ASSuT might be related with the diversity of pulsotypes and also the presence of a core of virulence factors, including biofilm production. Biofilm-forming ability varied between sample locations and collection year, and can be one of the virulence features related with the rise of this serovar. Furthermore, biofilm formation was evaluated in vitro using a simulated human intestinal environment. In such conditions was observed an impairment of biofilm production, revealing that conditions mimicking the human intestinal tract can influence the biofilm-forming ability of the isolates under study. This research highlight the critical importance of close surveillance of Salmonella 1,4,[5],12:i:- in Portugal, including R-type ASSuT isolates. Information gathered may unravel Salmonella 1,4,[5],12:i:- features, prevent the dissemination to other regions and also benefit the medical community in order to rationalize salmonellosis antimicrobial therapeutics.RESUMO - Virulência de Salmonella Typhimurium 1,4,[5],12:i:-, a nova estirpe pandémica* - Salmonella é uma bactéria Gram-negativa pertencente à família Enterobacteriaceae, sendo uma das principais responsáveis pela morbilidade e mortalidade associadas a toxinfecções alimentares. Pode manifestar-se num espectro de sintomatologia variado, incluindo a gastroenterite, a bacteriémia e a infecção focal. Este género incluí mais de 2600 serovares descritos, distribuídos por apenas duas espécies: Salmonella enterica que inclui todos os serovares patogénicos para os humanos e Salmonella bongori. Actualmente, um dos principais serovares responsáveis pela salmonelose humana em todo o mundo é o 1,4,[5],12:i:-. Este serovar é uma variante monofásica de Salmonella Typhimurium, muito semelhante a nível molecular, sendo caracterizado pela ausência da expressão do gene fljB. Devido à sua recente emergência, estudos que avaliem este serovar são escassos, particularmente em Portugal, o que definiu o âmbito desta investigação, que teve como objectivo a caracterização epidemiológica e microbiológica, tanto do ponto de vista fenotípico e genotípico, de isolados de Salmonella 1,4,[5],12:i:- obtidos em Portugal a partir de diferentes origens, incluindo amostras humanas, animais e ambientais. Numa primeira fase foi realizada uma caracterização demográfica, epidemiológica e espacial de todos os casos de Salmonelose 1,4,[5],12:i:- humana notificados em Portugal pelo Instituto Nacional de Saúde Dr. Ricardo Jorge (INSA), durante um período de 10 anos, desde 2001 a 2011. Foram recolhidos dados sobre a origem, ano e mês de amostragem, género, idade, distrito e município de residência dos pacientes. Foi realizada a análise estatística descritiva, bem como, a análise estatística espacial através do software SaTScan™, combinada com análise através de software de georeferenciação, o QGIS™, de forma a caracterizar a epidemiologia e identificar agrupamentos espaciais de risco superior de infecção por Salmonella 1,4,[5],12:i:- em Portugal. Globalmente, observou-se que em Portugal, a maioria dos distritos tem casos notificados de infecção por Salmonella 1,4,[5],12:i:-. Verificou-se também um aumento da incidência durante o intervalo de 2004 a 2011, com um maior número de casos na região litoral do país, incluindo distritos como Porto, Lisboa e Aveiro, o que pode ser explicado pela maior densidade populacional nestas áreas. A maioria das infecções ocorreu durante Maio e Outubro, e o menor número em Fevereiro, afectando principalmente indivíduos jovens.[...]*O autor escreve segundo o antigo Acordo OrtográficoThis work was supported by National Health Institute Doutor Ricardo Jorge (INSA) and funded by Centre for Interdisciplinary Research in Animal Health (CIISA)N/

Role of innate immunity and angiogenesis in osteosarcoma growth and metastasis, The

Department Head: Paul J. Laybourn.2010 Spring.Includes bibliographical references.Osteosarcoma is the most common primary bone tumor in dogs and humans. Novel therapeutics are required since a number of patients will succumb to metastatic disease. Currently, it is known that a neoplastic mass contains more than transformed cells, but requires the presence of immune cells to create a supportive environment, and endothelial cells to form blood vessels to deliver oxygen and nutrients. Not only do these cells play a role in primary tumor growth, but they create an atmosphere conducive to invasion and metastasis, the primary cause of death for osteosarcoma patients. Therefore, better understanding of how immune and endothelial cells support metastatic growth is crucial for better understanding osteosarcoama. Utilizing murine tumor models, we have been able to explore the observation that patients with post-surgical infections have increased time to metastasis and increased survival. We have determined that this effect is mediated by NK cells and monocytes, which inhibit tumor growth through restriction of angiogenesis. We have also investigated the role of an anti-inflammatory therapeutic, tepoxalin, which leads to tumor growth inhibition. These observations explain the paradox of inflammation, where the type and timing of inflammation may inhibit or promote an anti-tumor effect. Due to the importance of angiogenesis and metastasis in osteosarcoma, we sought to develop new models and techniques to assess these processes. Fine needle aspiration coupled with flow cytometry accurately measures angiogenesis in cutaneous tumors, thereby allowing for repeated assessment of angiogenesis in a minimally invasive manner. We have also developed a novel post-surgical model of luciferase transfected murine osteosarcoma that grows orthotopically and spontaneously metastasizes, allowing us to non-invasively investigate the development of metastases. These tools will allow for investigation into novel anti-angiogenic and anti-metastatic compounds. Novel prognostic markers are required to better determine the outcome of cancer patients. We have determined that monocyte and lymphocyte counts from a pre-treatment complete blood count are prognostic for disease free interval in dogs with osteosarcoma. These data describe the interactions between immune infiltrate and metastasis. The combination of the studies presented herein provides evidence for the interactions between the immune system and angiogenesis in the process of metastasis

Director\u27s report of research in Kansas 2010

This report contains the title, author, and publication information for manuscripts published by station scientists