Agregación de autoinmunidad en familias extremas con Síndrome de Sjogren: una aproximación a la medicina personalizada

Objetivo: investigar la agregación de autoinmunidad en familiares en primer grado (FPG) de probandos con Síndrome de Sjogren (SS) con poliautoinmunidad y autoinmunidad familiar. Metodología: estudio de corte transversal en el cual se incluyeron 14 familias de mujeres con SS. Se realizó un análisis genético a través del cálculo de riesgos relativos para cada enfermedad autoinmune (EAI). La agregación familiar se calculó para los FPG, teniendo en cuenta la prevalencia de la enfermedad en cada familia y en la población general. Resultados: Se analizaron un total de 112 individuos, con una media de edad de 51.7 años ± 12.9. Veintidós individuos de los FPG tuvieron al menos una EAI (28.2%). La prevalencia de poliautoinmunidad fue del 93% y 6.4% en probandos y FPG, respectivamente. Se observaron valores que soportaron la agregación familiar para varias EAIs, entre ellas púrpura trombocitopénica idiopática, granulomatosis con poliangeiitis, síndrome antifosfolipídico, lupus eritematoso sistémico y artritis reumatoide. Discusión: estos resultados indican que las EAIs se agregan entre familias con SS, sugiriendo un origen común entre ellas. La elección de fenotipos clínicamente relevantes representa una nueva aproximación a la medicina personalizada en autoinmunidad.

A gene expression signature for insulin resistance

Insulin resistance is a heterogeneous disorder caused by a range of genetic and environmental factors, and we hypothesize that its aetiology varies considerably between individuals. This heterogeneity provides significant challenges to the development of effective therapeutic regimes for long-term management of type 2 diabetes. We describe a novel strategy, using large-scale gene expression profiling, to develop a Gene Expression Signature (GES) that reflects the overall state of insulin resistance in cells and patients. The GES was developed from 3T3-L1 adipocytes that were made ‘insulin resistant’ by treatment with tumour necrosis factor-alpha (TNFα) and then reversed with aspirin and troglitazone (‘re-sensitized’). The GES consisted of five genes whose expression levels best discriminated between the insulin resistant and insulin re-sensitized states. We then used this GES to screen a compound library for agents that affected the GES genes in 3T3- L1 adipocytes in a way that most closely resembled the changes seen when insulin resistance was successfully reversed using aspirin and troglitazone. This screen identified both known and new insulin sensitizing compounds including non-steroidal anti inflammatory agents, β-adrenergic antagonists, beta-lactams and sodium channel blockers. We tested the biological relevance of this GES in participants in the San Antonio Family Heart Study (n = 1,240) and showed that patients with the lowest GES scores were more insulin resistant (according to HOMA_IR and fasting plasma insulin levels, P < 0.001). These findings show that GES technology can be used for both the discovery of insulin sensitizing compounds and the characterization of patients into subtypes of insulin resistance according to GES scores, opening the possibility of developing a personalized medicine approach to type 2 diabetes.

Service and cloud computing supporting genomic analysis of the mammalian species

 This research focused on building Software as a Service clouds to support mammalian genomic applications such as personalized medicine. Outcomes of this research included a Software as a Service cloud framework, the Uncinus research cloud and novel genomic analysis software. Results have been published in high ranking peer-reviewed international journals.

Biomarkers and clinical staging in psychiatry

Personalized medicine is rapidly becoming a reality in today's physical medicine. However, as yet this is largely an aspirational goal in psychiatry, despite significant advances in our understanding of the biochemical, genetic and neurobiological processes underlying major mental disorders. Preventive medicine relies on the availability of predictive tools; in psychiatry we still largely lack these. Furthermore, our current diagnostic systems, with their focus on well-established, largely chronic illness, do not support a pre-emptive, let alone a preventive, approach, since it is during the early stages of a disorder that interventions have the potential to offer the greatest benefit. Here, we present a clinical staging model for severe mental disorders and discuss examples of biological markers that have already undergone some systematic evaluation and that could be integrated into such a framework. The advantage of this model is that it explicitly considers the evolution of psychopathology during the development of a mental illness and emphasizes that progression of illness is by no means inevitable, but can be altered by providing appropriate interventions that target individual modifiable risk and protective factors. The specific goals of therapeutic intervention are therefore broadened to include the prevention of illness onset or progression, and to minimize the risk of harm associated with more complex treatment regimens. The staging model also facilitates the integration of new data on the biological, social and environmental factors that influence mental illness into our clinical and diagnostic infrastructure, which will provide a major step forward in the development of a truly pre-emptive psychiatry.

Estruturação ex-vivo de vasos sanguíneos a partir da diferenciação de células tronco de coelhos

Pós-graduação em Pesquisa e Desenvolvimento (Biotecnologia Médica) - FMB

Avaliação do polimorfismo INDEL no gene TYMS em associação a resposta quanto ao uso de fluoropirimidinas em pacientes portadores de neoplasias do trato gastrointestinal

Introdução: O câncer consiste em um problema de saúde publica mundial, com estimativa de 27 milhões de casos novos e 17 milhões de mortes por câncer no ano de 2030. No Brasil, as estimativas para o câncer no ano de 2014, apontam a ocorrência de aproximadamente 580 mil casos novos. As fluoropirimidinas são usadas nos principais esquemas quimioterápicos direcionados a tumores do trato gastrointestinal. Nos últimos anos muito se tem investigado sobre causas de respostas individuais diferenciadas em relação ao tratamento quimioterápico; dessa forma têm-se buscado uma terapia individualizada que possa maximizar a eficácia dos medicamentos e minimizar os efeitos adversos associados aos fármacos. Objetivamos buscar a associação do INDEL (rs16430) do gene TYMS com o padrão de resposta ao tratamento oncológico de fármacos com base em fluoropirimidinas, de maneira a contribuir para o desenvolvimento da medicina personalizada. Material: Estudadas 151 amostras de sangue periférico de pacientes oncológicos tratados com fluoropirimidinas, da população da região amazônica brasileira com elevado grau de mistura interétinica. Foi genotipado um polimorfismo INDEL (rs16430) no gene TYMS envolvido na resposta ao tratamento com uso de fluoropirimidinas. Resultados: A investigação relatou que a maioria dos pacientes tinha doença avançada no momento do diagnóstico; 32,7% receberam tratamento com intenção paliativa; e 22,8% tratamento neoadjuvante. Nossos resultados evidenciam que o polimorfismo INDEL no gene TYMS demonstrou ter um efeito de proteção à progressão tumoral (p=0,033). Pacientes tratados com fluoropirimidinas que eram homozigotos selvagens (INS/INS) apresentaram uma proteção à progressão tumoral de 24% comparado com outros genótipos desse polimorfismo. As estimativas de ancestralidade genômica global da amostra investigada foram: 62,4% europeia; 25,2% nativo americana e 12,4% africana. Foi possível estabelecer uma correlação inversa entre o aumento da ancestralidade ameríndia e a presença de metástase (p=0,024). Conclusão: Estudos farmacogenéticos podem proporcionar uma terapia personalizada reduzindo mortalidade por toxicidades e aumentando a eficácia terapêutica, desta forma proporcionando um tratamento oncológico com melhores resultados clínicos.

Il corpo e l'individuo tra proprietà ed autodeterminazione. Con particolare riferimento al fenomeno delle biobanche

La bioetica è il luogo ideale per cercare risposte ai grandi interrogativi concernenti la vita, la morte e la cura dell'essere umano. I recenti dibattiti sull'uso, ed il temuto abuso, del corpo umano in medicina hanno messo in evidenza la necessità di una discussione approfondita sul potere di scelta che l'individuo può esercitare sulla propria mente e sul proprio corpo. Spinta dal desidero di indagare l'estensione di tale potere di scelta ho voluto analizzare le tematiche riguardanti “il corpo”, “l'individuo”, “la proprietà” e “l'autodeterminazione”. L'analisi è stata condotta individuando alcuni dei differenti significati che questi termini assumono nei diversi ambiti che la bioetica lambisce e mostrando, in particolare, la visione di tale realtà attraverso le lenti del giurista. A chi appartiene il corpo? Chi ha il potere di decidere su di esso? Il potere di scelta valica gli antichi i confini legati al corpo del paziente e coinvolge tessuti, organi e cellule staccati dal corpo umano, parti che un tempo erano considerati scarti operatori sono oggi divenuti tesori inestimabili per la ricerca. L'importanza assunta dai campioni biologici ha portato alla creazione di biobanche nelle quali sono raccolti, catalogati e il DNA studiato in campioni biologici Le biobanche riflettono le tensioni della bioetica e del biodititto. Lo studio delle biobanche riguarda, tra l'altro, la riceca dell'equilibrio tra le diverse esigenze meritevoli di tutela: in primo luogo il diritto alla privacy, diritto a che le “proprie informazioni” non vengano divulgate ed il diritto a non essere discriminato ed in secondo luogo le necessità dettate dalla ricerca e dalla scienza medica. Nel 2009 la rivista Times messo biobanche tra le 10 idee in grado di cambiare il mondo anche in considarazione della medicina personalizzata e del fatto che costituiscono una la speranza per la ricerca contro le malattie attuali e future.

Nanotechnology control of self organized biomolecules and biomaterials for medical research

Nanotechnology entails the manufacturing and manipulation of matter at length scales ranging from single atoms to micron-sized objects. The ability to address properties on the biologically-relevant nanometer scale has made nanotechnology attractive for Nanomedicine. This is perceived as a great opportunity in healthcare especially in diagnostics, therapeutics and more in general to develop personalized medicine. Nanomedicine has the potential to enable early detection and prevention, and to improve diagnosis, mass screening, treatment and follow-up of many diseases. From the biological standpoint, nanomaterials match the typical size of naturally occurring functional units or components of living organisms and, for this reason, enable more effective interaction with biological systems. Nanomaterials have the potential to influence the functionality and cell fate in the regeneration of organs and tissues. To this aim, nanotechnology provides an arsenal of techniques for intervening, fabricate, and modulate the environment where cells live and function. Unconventional micro- and nano-fabrication techniques allow patterning biomolecules and biocompatible materials down to the level of a few nanometer feature size. Patterning is not simply a deterministic placement of a material; in a more extended acception it allows a controlled fabrication of structures and gradients of different nature. Gradients are emerging as one of the key factors guiding cell adhesion, proliferation, migration and even differentiation in the case of stem cells. The main goal of this thesis has been to devise a nanotechnology-based strategy and tools to spatially and temporally control biologically-relevant phenomena in-vitro which are important in some fields of medical research.

Analisi fosfo-proteomica di campioni di tumore: il problema della stratificazione dei pazienti ed eterogeneità intra-tumorale nell'era della terapia personalizzata

Nella presente tesi indaghiamo la potenzialità di LCM e Reverse Phase Protein microarray negli studi clinici. Si analizza la possibilità di creare una bio banca con line cellular primarie, al fine di conseguire drug test di sensibilità prima di decidere il trattamento da somministrare ai singoli pazienti. Sono stati ottenuti profili proteomici da biopsie pre e post terapia. I risultati dimostrano che questa piattaforma mostra il meccanismo di resistenza acquisito durante la terapia biologica. Questo ci ha portato ad analizzare una possibile stratificazione per pazienti con mCRC . I dati hanno rivelato distinti pathway di attivazione tra metastasi resecabile e non resecabili. I risultati mostrano inoltre due potenziali bersagli farmacologici. Ma la valutazione dell'intero tumore tramite singole biopsie sembra essere un problema a causa dell’eterogeneità intratumorale a livello genomico. Abbiamo indagato questo problema a livello dell'architettura del segnale in campioni di mCRC e ccRCC . I risultati indicano una somiglianza complessiva nei profili proteomici all'interno dello stesso tumore. Considerando che una singola biopsia è rappresentativa di un intera lesione , abbiamo studiato la possibilità di creare linee di cellule primarie, per valutare il profilo molecolare di ogni paziente. Fino ad oggi non c'era un protocollo per creare linee cellulari immortalizzate senza alcuna variazione genetica . abbiamo cosiderato, però, l'approccio innovativo delle CRCs. Ad oggi , non è ancora chiaro se tali cellule mimino il profilo dei tessuti oppure I passaggi in vitro modifichino i loro pathways . Sulla base di un modello di topo , i nostri dati mostrano un profilo di proteomica simile tra le linee di cellule e tessuti di topo LCM. In conclusione, i nostri dati dimostrano l'utilità della piattaforma LCM / RPPA nella sperimentazione clinica e la possibilità di creare una bio - banca di linee cellulari primarie, per migliorare la decisione del trattamento.

Entwicklung und Optimierung eines Mikroarray zur Erfassung der genetischen Prädisposition von Atherosklerose

Die Entstehung der Atherosklerose ist ein komplexer Vorgang, der sich durch Ablagerung von Lipiden an der Gefäßwand sowie durch immunologische und inflammatorische Prozesse auszeichnet. Neben konventionellen Risikofaktoren wie Alter, Geschlecht, Rauchen, HDL-Cholesterin, Diabetes mellitus und einer positiven Familienanamnese werden zur Bestimmung des atherosklerotischen Risikos neue Biomarker der inflammatorischen Reaktion untersucht. Ziel dieser Arbeit war die Entwicklung einer Methode zur Diagnostik des Atheroskleroserisikos. Es wurde eine neuartige Chip-Technologie eingesetzt, um das Risiko für eine potentiell drohende atherosklerotische Erkrankung abzuschätzen. Dabei wurde ausgenutzt, dass molekulare Veränderungen in Genen bestimmte Krankheitsbilder auslösen können. rnEs wurde ein molekularbiologischer Test entwickelt, welcher die Untersuchung von genetischen Variationen aus genomischer DNA ermöglicht. Dafür fand die Entwicklung einer Multiplex-PCR statt, deren Produkt mit der Chip-Technologie untersucht werden kann. Dazu wurden auf einem Mikroarray Sonden immobilisiert, mit deren Hilfe genspezifische Mutationen nachgewiesen werden können. So wurden mehrere Gene mit einem geringen Aufwand gleichzeitig getestet. rnDie Auswahl der entsprechenden Marker erfolgte anhand einer Literaturrecherche von randomisierten und kontrollierten klinischen Studien. Der Mikroarray konnte für zwölf Variationen in den acht Genen Prostaglandinsynthase-1 (PTGS1), Endotheliale NO-Synthase (eNOS), Faktor V (F5), 5,10-Methylentetrahydrofolsäure-Reduktase (MTHFR), Cholesterinester-Transferprotein (CETP), Apolipoprotein E (ApoE), Prothrombin (F2) und Lipoproteinlipase (LPL) erfolgreich etabliert werden. Die Präzision des Biochips wurde anhand der Echtzeit-PCR und der Sequenzierung nachgewiesen. rnDer innovative Mikroarray ermöglicht eine einfache, schnelle und kosteneffektive Genotypisierung von wichtigen Allelen. Viele klinisch relevante Variationen für Atherosklerose können nun in nur einem Test überprüft werden. Zukünftige Studien müssen zeigen, ob die Methode eine Vorhersage über den Ausbruch der Erkrankung und eine gezielte Therapie ermöglicht. Dies wäre ein erster Schritt in Richtung präventive und personalisierter Medizin für Atherosklerose.rn

Immune pathomechanism of drug hypersensitivity reactions

Drug hypersensitivity research has progressed enormously in recent years, and a greater understanding of mechanisms has contributed to improved drug safety. Progress has been made in genetics, enabling personalized medicine for certain drugs, and in understanding drug interactions with the immune system. In a recent meeting in Rome, the clinical, chemical, pharmacologic, immunologic, and genetic aspects of drug hypersensitivity were discussed, and certain aspects are briefly summarized here. Small chemicals, including drugs, can induce immune reactions by binding as a hapten to a carrier protein. Park (Liverpool, England) demonstrated (1) that drug haptens bind to protein in patients in a highly restricted manner and (2) that irreversibly modified carrier proteins are able to stimulate CD4(+) and CD8(+) T cells from hypersensitive patients. Drug haptens might also stimulate cells of the innate immune system, in particular dendritic cells, and thus give rise to a complex and complete immune reaction. Many drugs do not have hapten-like characteristics but might gain them on metabolism (so-called prohaptens). The group of Naisbitt found that the stimulation of dendritic cells and T cells can occur as a consequence of the transformation of a prohapten to a hapten in antigen-presenting cells and as such explain the immune-stimulatory capacity of prohaptens. The striking association between HLA-B alleles and the development of certain drug reactions was discussed in detail. Mallal (Perth, Australia) elegantly described a highly restricted HLA-B∗5701-specific T-cell response in abacavir-hypersensitive patients and healthy volunteers expressing HLA-B∗5701 but not closely related alleles. Expression of HLA-B∗1502 is a marker known to be necessary but not sufficient to predict carbamazepine-induced Stevens-Johnson syndrome/toxic epidermal necrolysis in Han Chinese. The group of Chen and Hong (Taiwan) described the possible "missing link" because they showed that the presence of certain T-cell receptor (TCR) clonotypes was necessary to elicit T-cell responses to carbamazepine. The role of TCRs in drug binding was also emphasized by Pichler (Bern, Switzerland). Following up on their "pharmacological interactions of drugs with immune receptors" concept (p-i concept), namely that drugs can bind directly to TCRs, MHC molecules, or both and thereby stimulate T cells, they looked for drug-binding sites for the drug sulfamethoxazole in drug-specific TCRs: modeling revealed up to 7 binding sites on the CDR3 and CDR2 regions of TCR Vα and Vβ. Among many other presentations, the important role of regulatory T cells in drug hypersensitivity was addressed.

Pharmacogenomic considerations in opioid analgesia

Translating pharmacogenetics to clinical practice has been particularly challenging in the context of pain, due to the complexity of this multifaceted phenotype and the overall subjective nature of pain perception and response to analgesia. Overall, numerous genes involved with the pharmacokinetics and dynamics of opioids response are candidate genes in the context of opioid analgesia. The clinical relevance of CYP2D6 genotyping to predict analgesic outcomes is still relatively unknown; the two extremes in CYP2D6 genotype (ultrarapid and poor metabolism) seem to predict pain response and/or adverse effects. Overall, the level of evidence linking genetic variability (CYP2D6 and CYP3A4) to oxycodone response and phenotype (altered biotransformation of oxycodone into oxymorphone and overall clearance of oxycodone and oxymorphone) is strong; however, there has been no randomized clinical trial on the benefits of genetic testing prior to oxycodone therapy. On the other hand, predicting the analgesic response to morphine based on pharmacogenetic testing is more complex; though there was hope that simple genetic testing would allow tailoring morphine doses to provide optimal analgesia, this is unlikely to occur. A variety of polymorphisms clearly influence pain perception and behavior in response to pain. However, the response to analgesics also differs depending on the pain modality and the potential for repeated noxious stimuli, the opioid prescribed, and even its route of administration.

Classification of acute and delayed contrast media-induced reactions: proposal of a three-step system

Physicians and scientists use a broad spectrum of terms to classify contrast media (CM)-induced adverse reactions. In particular, the designation of hypersensitivity reactions is quite varied. Consequently, comparisons of different papers dealing with this subject are difficult or even impossible. Moreover, general descriptions may lead to problems in understanding reactions in patients with a history of adverse CM-reactions, and in efficiently managing these patients. Therefore, the goal of this paper is to suggest an easy system to clearly classify these reactions. The proposed three-step systems (3SS) is built up as follows: step 1 exactly describes the clinical features, including their severity; step 2 categorizes the time point of the onset (immediate or nonimmediate); and step 3 generally classifies the reaction (hypersensitivity or nonhypersensitivity reaction). The 3SS may facilitate better understanding of the clinical manifestations of adverse CM reactions and may support the prevention of these reactions on the basis of personalized medicine approaches.