3D cultures of prostate cancer cells cultured in a novel high-throughput culture platform are more resistant to chemotherapeutics compared to cells cultured in monolayer

Despite monolayer cultures being widely used for cancer drug development and testing, 2D cultures tend to be hypersensitive to chemotherapy and are relatively poor predictors of whether a drug will provide clinical benefit. Whilst generally more complicated, three dimensional (3D) culture systems often better recapitulate true cancer architecture and provide a more accurate drug response. As a step towards making 3D cancer cultures more accessible, we have developed a microwell platform and surface modification protocol to enable high throughput manufacture of 3D cancer aggregates. Herein we use this novel system to characterize prostate cancer cell microaggregates, including growth kinetics and drug sensitivity. Our results indicate that prostate cancer cells are viable in this system, however some non-cancerous prostate cell lines are not. This system allows us to consistently control for the presence or absence of an apoptotic core in the 3D cancer microaggregates. Similar to tumor tissues, the 3D microaggregates display poor polarity. Critically the response of 3D microaggregates to the chemotherapeutic drug, docetaxel, is more consistent with in vivo results than the equivalent 2D controls. Cumulatively, our results demonstrate that these prostate cancer microaggregates better recapitulate the morphology of prostate tumors compared to 2D and can be used for high-throughput drug testing.

Mechanisms of cisplatin resistance: DNA repair and cellular implications

Cisplatin (cis-diamminedichloroplatinum (II)), is a platinum based chemotherapeutic employed in the clinic to treat patients with lung, ovarian, colorectal or head and neck cancers. Cisplatin acts to induce tumor cell death via multiple mechanisms. The best characterized mode of action is through irreversible DNA cross-links which activate DNA damage signals leading to cell death via the intrinsic mitochondrial apoptosis pathway. However, the primary issue with cisplatin is that while patients initially respond favorably, sustained cisplatin therapy often yields chemoresistance resulting in therapeutic failure. In this chapter, we review the DNA damage and repair pathways that contribute to cisplatin resistance. We also examine the cellular implications of cisplatin resistance that may lead to selection of subpopulations of cells within a tumor. In better understanding the mechanisms conferring cisplatin resistance, novel targets may be identified to restore drug sensitivity.

Selection and characterisation of two attenuated vaccine lines of Eimeria tenella in Australia

Objective To attenuate two strains of Eimeria tenella by selecting for precocious development and evaluate the strains in characterisation trials and by field evaluation, to choose one precocious line for incorporation into an Australian live coccidiosis vaccine for poultry. Design Two strains from non-commercial flocks were passaged through chickens while selecting for precocious development. Each strain was characterised for drug sensitivity, pathogenicity, protection against homologous and heterologous challenge, and oocyst output in replicated experiments in which the experimental unit was a cage of three birds. Oocyst output and/or body weight gain data collected over a 10 to 12 day period following final inoculation were measured. Feed conversion ratios were also calculated where possible. Results Fifteen passages resulted in prepatent periods reduced by 24 h for the Redlands strain (from 144 h to 120 h)and 23 h for the Darryl strain (from 139 h to 116 h). Characterisation trials demonstrated that each precocious line was significantly less pathogenic than its parent strain and each effectively induced immunity that protected chickens against challenge with both the parent strain and other virulent field strains. Both lines had oocyst outputs that, although significantly reduced relative to the parent strains, remained sufficiently high for commercial vaccine production, and both showed susceptibility to coccidiostats. Conclusion Two attenuated lines have been produced that exhibit the appropriate characteristics for use in an Australian live coccidiosis vaccine.

Pharmacogenetics of MicroRNAs and MicroRNAs Biogenesis Machinery in Pediatric Acute Lymphoblastic Leukemia

Despite the clinical success of acute lymphoblastic leukemia (ALL) therapy, toxicity is frequent. Therefore, it would be useful to identify predictors of adverse effects. In the last years, several studies have investigated the relationship between genetic variation and treatment-related toxicity. However, most of these studies are focused in coding regions. Nowadays, it is known that regions that do not codify proteins, such as microRNAs (miRNAs), may have an important regulatory function. MiRNAs can regulate the expression of genes affecting drug response. In fact, the expression of some of those miRNAs has been associated with drug response. Genetic variations affecting miRNAs can modify their function, which may lead to drug sensitivity. The aim of this study was to detect new toxicity markers in pediatric B-ALL, studying miRNA-related polymorphisms, which can affect miRNA levels and function. We analyzed 118 SNPs in pre-miRNAs and miRNA processing genes in association with toxicity in 152 pediatric B-ALL patients all treated with the same protocol (LAL/SHOP). Among the results found, we detected for the first time an association between rs639174 in DROSHA and vomits that remained statistically significant after FDR correction. DROSHA had been associated with alterations in miRNAs expression, which could affect genes involved in drug transport. This suggests that miRNA-related SNPs could be a useful tool for toxicity prediction in pediatric B-ALL.

An integrated approach to the prediction of chemotherapeutic response in patients with breast cancer.

BACKGROUND: A major challenge in oncology is the selection of the most effective chemotherapeutic agents for individual patients, while the administration of ineffective chemotherapy increases mortality and decreases quality of life in cancer patients. This emphasizes the need to evaluate every patient's probability of responding to each chemotherapeutic agent and limiting the agents used to those most likely to be effective. METHODS AND RESULTS: Using gene expression data on the NCI-60 and corresponding drug sensitivity, mRNA and microRNA profiles were developed representing sensitivity to individual chemotherapeutic agents. The mRNA signatures were tested in an independent cohort of 133 breast cancer patients treated with the TFAC (paclitaxel, 5-fluorouracil, adriamycin, and cyclophosphamide) chemotherapy regimen. To further dissect the biology of resistance, we applied signatures of oncogenic pathway activation and performed hierarchical clustering. We then used mRNA signatures of chemotherapy sensitivity to identify alternative therapeutics for patients resistant to TFAC. Profiles from mRNA and microRNA expression data represent distinct biologic mechanisms of resistance to common cytotoxic agents. The individual mRNA signatures were validated in an independent dataset of breast tumors (P = 0.002, NPV = 82%). When the accuracy of the signatures was analyzed based on molecular variables, the predictive ability was found to be greater in basal-like than non basal-like patients (P = 0.03 and P = 0.06). Samples from patients with co-activated Myc and E2F represented the cohort with the lowest percentage (8%) of responders. Using mRNA signatures of sensitivity to other cytotoxic agents, we predict that TFAC non-responders are more likely to be sensitive to docetaxel (P = 0.04), representing a viable alternative therapy. CONCLUSIONS: Our results suggest that the optimal strategy for chemotherapy sensitivity prediction integrates molecular variables such as ER and HER2 status with corresponding microRNA and mRNA expression profiles. Importantly, we also present evidence to support the concept that analysis of molecular variables can present a rational strategy to identifying alternative therapeutic opportunities.

Application of Machine Learning Techniques to Acute Myeloid Leukemia

Thesis (Master's)--University of Washington, 2016-03

Nouveaux paramètres d'exploration de la fonction plaquettaire en clinique : thrombose tardive, profilage micro-membranaire et détection de sous-populations cellulaires

Les plaquettes sanguines sont les principaux acteurs de l’hémostase primaire et de la thrombose, deux éléments majeurs de la physiopathologie vasculaire. Plusieurs médicaments régulent les fonctions plaquettaires mais peu de tests sont validés pour suivre leur efficacité en fonction de l’évolution clinique des patients. Mon doctorat a eu pour but de développer de nouvelles approches d’évaluation de la fonction plaquettaire. Deux essais cliniques réalisés sur des patients atteints de syndrome coronarien stable ont constitué la première partie de mon doctorat. La première étude met en évidence la nécessité d'une standardisation des tests biologiques pour la détection de patients répondant moins au clopidogrel, un inhibiteur du récepteur plaquettaire de l'ADP P2Y12. L’étude suivante montre le potentiel thérapeutique, chez ces patients, de l’inhibition conjointe de P2Y12 et du second récepteur plaquettaire de l'ADP P2Y1, sur la fonction plaquettaire. De plus, le suivi en temps réel par vidéomiscroscopie a permis de distinguer des effets précoces et tardifs des antiplaquettaires sur la formation du thrombus en chambre de perfusion. La seconde partie de mon doctorat concerne les microdomaines membranaires de type « lipid rafts » qui tiennent une place fondamentale dans les fonctions cellulaires et plaquettaires. Ainsi plusieurs récepteurs dépendent de ces microdomaines, régulant la fonction plaquettaire et les effets des médicaments antiplaquettaires. Cependant, les techniques d’étude de ces microdomaines sont complexes et peu adaptées aux études cliniques. Profitant de nouvelles sondes fluorescentes sensibles au niveau d’ordre liquide membranaire (OLM), nous avons développé une méthode de mesure de l’OLM par cytométrie de flux spectrale. Grâce à cette approche, nous avons montré que l’activation plaquettaire diminue l’OLM alors qu’il est augmenté chez des patients traités par un inhibiteur de la synthèse du cholestérol ou par le clopidogrel. Nous avons également mis en évidence, en condition de forces de cisaillement élevées correspondant à celles retrouvées au niveau de sténoses artérielles, une sous-population plaquettaire présentant un OLM plus bas. Le passage dans le domaine clinique de ces approches fondamentales qui privilégient l’étude dynamique des plaquettes pourrait permettre d’améliorer le diagnostique et le suivi de traitement de pathologies cardiovasculaires.

Diferencias microbiológicas en pacientes con exacerbación severa de EPOC con o sin consolidación neumónica

La EPOC es una causa importante de morbilidad y mortalidad en el mundo y su prevalencia en Bogotá alcanza hasta 8,5%. Las exacerbaciones están asociadas a deterioro funcional y de la calidad de vida por lo que se consideran un factor cardinal de la enfermedad. En la literatura se ha descrito que las infecciones por bacterias y/o virus son las responsables del 78% de las exacerbaciones. Estos datos han sido descritos en poblaciones diferentes y no hay datos en la literatura que muestren cual es la epidemiología local de las exacerbaciones de EPOC y menos aún de aquellas que se asocian a consolidaciones neumónicas. Objetivo: Comparar la microbiología de las exacerbaciones severas de la EPOC que requieren ingreso a UCI con y sin infiltrados alveolares. Materiales y métodos: Estudio de corte transversal en el que se estudiaron pacientes con EPOC que ingresaron a la UCI Médica de la FCI-IC por exacerbación severa, asociada o no a infiltrados alveolares. Se tomaron muestras de microbiología, serológicas y radiografía de tórax para evaluar la etiología de la exacerbación, si se asocia a coinfección viral y a consolidación neumónica o no. Resultados: No se encontró una diferencia estadísticamente significativa en la microbiología de los diferentes grupos evaluados. Se encontró un resistencia global del 24% y llama la atención que hay una alta prevalencia de Serratia Marcescens AMPc entre los 2 grupos, germen que no está descrito como patógeno común en la literatura. Se encontraron diferencias en cuanto a factores de riesgo para presentar neumonía asociada como lo son un mayor índice de paquetes/año (55.1.6 vs. 36.3 paq/año, sig.=0.021). Así mismo se demostró que los pacientes con neumonía asociada presentan mayor necesidad de IOT (48.9 vs. 23.9, sig.=0.013). No hay diferencia significativa en desenlaces como mortalidad (20.5 vs. 13.0, sig.=0.346). Conclusiones: A pesar de no haber diferencia microbiológica entre los 2 grupos se encontraron variables como factores de riesgo y variables clínicas que pueden ayudar a proponer planes de manejo en los dos escenarios. El hecho de encontrar un paciente con neumonía asociada al cuadro de exacerbación no debe afectar en la toma de decisiones en relación al tratamiento antibiótico.

Characteristics of Yersinia pseudotuberculosis isolated from animals in Brazil

Strains (105) of Yersinia pseudotuberculosis isolated in Brazil between 1982 and 1990 mere bio-serotyped. They were also studied for plasmid profile, autoagglutination and calcium dependence at 37 degrees C, Congo red uptake, pyrazinamidase activity, esculin hydrolysis, salicin fermentation and drug sensitivity: 95.24% were biotype 2, serogroup O:3; 2.86% were biotype 1, serogroup O:1; and 1.90% were biotype 2, non-agglutinable. Plasmids were found in 77.14% of the strains (one in each strain). There was total correlation between the presence of the virulence plasmid and autoagglutination, calcium dependence at 37 degrees C and Congo red uptake. The esculin, salicin and pyrazinamidase tests were not efficient in differentiating pathogenic from non-pathogenic Y, pseudotuberculosis isolates. All strains were highly sensitive to the drugs used. These results indicate that Y. pseudotuberculosis is a potential pathogen for humans in Brazil, especially because the bio-serogroups detected among animals are those most frequently associated with human diseases.