A double sandwich enzyme-linked immunosorbent assay (ELISA) was developed to quantify the proteinaceous inhibitor of tissue plasminogen activator (t-PA) which occur in the tissue of Erythrina caffra Thunb. Using the ELISA the t-PA inhibitor could be detected in nanogramme quantities on the micro titer plate. The concentration of the t-PA inhibitor was determined in different tissues of Erythrina caffra. t-PA inhibitor concentrations in the order of 1 000 microgrammes per gramme protein were found in the seeds. Relatively small quantities of t - PA inhibitor, in the order of 10 to 50 microgrammes per gramme protein, occurred in root, shoot, leaf and living bark material. The t-PA inhibitor was found to accumulate in a similar way to the storage proteins in developing seeds. The accumulation of the inhibitor is at a relatively low level during the early period of seed development but increases exponentially just before the seeds reach their maximum size. The t-PA inhibitor content of the cotyledons decreased drastically during the process of germination and subsequent seedling development. The disappearance of the inhibitor be the result of total degradation of the molecule can or partial proteolysis with the modified molecule still being present in the tissue. An attempt was made to increase the t-PA inhibitor content of excised leaves of Erythrina caffra with protein inducing substances such as polyamines, precursors of ethylene and phytic acid. The protein inducing compounds included cell wall hydrolysates of Erythrina caffra, the marine alga Ecklonia maxima Osbeck (Papenfuss) as well as Lycopersicon esculentum Mill which induced the, synthesis of proteinase inhibitors suggested to be involved in the defense mechanism of plants. None of the substances used, increased the t-PA inhibitor content of excised leaves or in vitro cultures of Erythrina caffra. It is suggested that the t-PA inhibitor is probably not involved in a defense mechanism of Erythrina caffra. A callus and suspension culture derived from shoot tissue was developed to determine the occurrence of the t-PA inhibitor in vitro. The optimal nutrient medium for the growth of callus was the salts and vitamins of MURASHIGE and SKOOG (1962). The medium was supplemented with 3 % sucrose, 0. 1 gramme per litre meso - inositol, 10 micromoles per litre benzyl adenine and 5 micromoles per litre 2,4- dichlorophenoxyacetic acid . Different auxins and cytokinins had a similar growth stimulatory effect on the growth of callus derived from a number of organs of Erythrina caffra. The callus from different organs did however, grow at different rates on the same nutrient medium. Callus derived from leaf, shoot, and cotyledonary tissue grew at similar rates on the nutrient media of MURASHIGE and SKOOG (1962), SCHENK and HILDEBRANDT (1972) and B5 (GAMBORG, MILLER and OJIMA, 1968) despite large differences in the concentration of the nutrients in the three nutri.ent media. The source of nitrogen and ratio of nitrate to ammonium was critical to the growth of callus cultures . The optimal concentration of nitrate and ammonium was 30 millimoles per litre . The growth of callus from different organs was significantly affected by the concentration of sucrose in the nutrient medium. A concentration of 3% was optimal for callus growth. Temperature had a significant effect on the growth of callus. The optimal temperature for callus growth was 25 °C. A shoot cell suspension culture was established and maintained at the same temperature and on the same medium as the callus cultures but with a ten times lower concentration of growth regulators. A low shake speed was essential for the growth of the suspension culture. Maximum growth was obtained at a shake speed of 60 rpm. Relatively low quantities of t-PA inhibitor, in the order of 1 to 5 microgrammes per gramme protein, was detected in the suspension cultures. An attempt was made to increase the t-PA inhibitor content of the suspension cultures with the pro te in i nduc i ng compounds used on excised leaves, but without success. However, the t-PA inhibitor content of the suspension culture was significantly increased with a ten times increase in the sulphate content of the nutrient medium. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 1990.

L’injection intraveineuse de l’activateur tissulaire du plasminogène (tPA) est le seul traitement aigu de l’ischémie cérébrale autorisé chez l’Homme. Cependant, la thrombolyse présente des limites d’utilisation, comme son étroite fenêtre thérapeutique, un risque hémorragique et une efficacité de recanalisation malgré tout relativement peu élevée. De plus, la littérature suggère fortement que non seulement le tPA endogène, mais aussi exogène (capable de traverser la barrière hémato-encéphalique), a des effets pro-excitotoxiques. Nous avons proposé que cet effet résulte du clivage de la sous unité NR1 du récepteur NMDA. Malgré un effort important de la communauté scientifique pour chercher de nouveaux traitements, tous les espoirs se sont avérés être des échecs. Sur ces bases, ces travaux de thèse ont consisté à : 1) Améliorer les approches précliniques en développant un nouveau modèle d’ischémie cérébrale chez la souris et en incluant dans les études un des principaux facteur de risque des AVC, le vieillissement ; 2) Développer une stratégie d’immunothérapie visant l’interaction tPA/ récepteur NMDA. J’ai ainsi montré qu’il existe une diminution du volume de lésion ischémique corrélée à l’âge et que cette diminution de tPA est due à une diminution d’expression du facteur de transcription D-Site Albumin Binding Protein (DBP). J’ai également développé un modèle innovant d’ischémie thrombo-embolique chez la souris, dans lequel la reperfusion par le tPA est bénéfique, si tant est qu’elle soit réalisée de manière précoce. Sur ce modèle, j’ai apporté par une stratégie d’immunisation active la preuve in vivo du clivage du domaine amino-terminal de la sous-unité NR1 des récepteurs NMDA. Enfin, j’ai produit un anticorps médicament, capable d’empêcher l’interaction du tPA avec la sous-unité NR1 des récepteurs NMDA, dont une injection unique permet de réduire les lésions ischémiques, mais aussi d’augmenter la fenêtre thérapeutique de la thrombolyse, conférant alors une récupération fonctionnelle à long terme. Cette stratégie pourrait donc accroître la proportion de patients traitables après un AVC ischémique aiguë / Reperfusion with tissue plasminogen activator (tPA) is the only approved treatment for ischemic stroke. However, thrombolysis has some limitations, including a narrow therapeutic window, an elevated risk of hemorrhage transformation and a low level of effective recanalization. Moreover, there is a growing body of evidence that both endogenous and exogenous tPA (able to cross the blood-brain barrier) could mediated pro-excitotoxic effects. We have proposed that this noxious effect results from the cleavage of the NR1 subunit of the NMDA receptor. My thesis work consisted in: 1) Improving pre-clinic approaches by developing a new model of thrombo-embolic ischemia in mice and by taking into account a major risk factor for stroke, aging; 2) Developing a strategy of immunotherapy targeting the interaction between tPA and NMDA receptor. I have thus shown that ischemic lesions decrease as a function of age, due to reduced levels of tPA. Moreover, I have identified DBP (D-site albumin Binding Protein), as being the transcription factor responsible for the control of tPA levels as a function of age. I have also developed a new model of thrombo-embolic ischemia in mice, in which tPA-induced thrombolysis is beneficial, provided it is performed soon enough. In this model, I have demonstrated by using a strategy of active immunization the in vivo occurrence of the cleavage of the NMDA receptor NR1 subunit by tPA. Finally, I have produced an antibody able to prevent the interaction between tPA and the NMDA receptor subunit, of which a single injection confers long lasting brain protection and neurological recovery and can also increase the therapeutic window of thrombolysis. This strategy could thus significantly increase the proportion of treatable ischemic stroke patients

Orientador: Alessandro Lia Mondelli / Resumo: Introdução: O Acidente Vascular Cerebral (AVC) é uma das principais causas de morte e sequelas neurológicas no mundo. O reconhecimento precoce e a pré-notificação hospitalar por serviços de atendimento pré-hospitalar têm sido relacionados com o aumento nas taxas de tratamento adequado. Sendo assim, a American Heart Association e a European Stroke Organisation recomendam o uso de escalas de avaliação pelas equipes do atendimento pré-hospitalar. Objetivo: Traduzir para o idioma português do Brasil, realizar a adaptação transcultural das escalas Cincinnati Prehospital Stroke Scale e Los Angeles Prehospital Stroke Screen e avaliar a confiabilidade e validade na população brasileira. Material e Métodos: Trata-se de um estudo metodológico, transversal e prospectivo realizado em 2 etapas: a primeira constituída pelos processos de tradução e adaptação transcultural das escalas e a segunda pela aplicação das mesmas, que ocorreu entre julho de 2016 e dezembro de 2017. Resultados e Discussão: A Cincinnati Prehospital Stroke Scale ficou denominada de “Escala de Avaliação pré-hospitalar do AVC – Cincinnati”. Os resultados mostraram um Coeficiente de alpha de Cronbach foi de 0,39 e uma alta confiabilidade interobservador do instrumento final, evidenciada pelo alto valor do índice de Kappa, principalmente nos itens “queda do braço” e “fala” que obtiveram o valor máximo. A escala apresentou acurácia de 93% (IC 95% 87,76, – 98,24%), sensibilidade de 92,42% (IC 95%, 86,03 – 98,80% / VPP = 71,7... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Introduction: Strokes are one of the leading causes of death and neurological disability in the world. Early recognition and prehospital notification may increase rates of thrombolysis with recombinant tissue plasminogen activator (rt-PA). Therefore, the American Heart Association and the European Stroke Organization recommend the use of assessment scales by prehospital care teams. Objective: To translate the Cincinnati Prehospital Stroke Scale (CPSS) and the Los Angeles Prehospital Stroke Screen to Portuguese, make a cross-cultural adaptation to Brazilian culture, and validate and verify its reliability in a Brazilian population. Material and Methods: This was a cross-sectional and prospective methodological study carried out in two stages: the first one consisted of the translation and crosscultural adaptation of the original scale and the second involved the application of the final instrument, which occurred between July 2016 and December 2017. Results and discussion: The final version of the Cincinnati Prehospital Stroke Scale was called the “Escala de Avaliação pré-hospitalar do AVC – Cincinnati”. The results showed Cronbach's alpha was 0,39 and a high interobserver reliability of the final instrument, evidenced by the high value of the Kappa index, especially in the items "arm drop" and "speech," which exhibited the maximum values. The scale showed accuracy of 93% (95% CI 87.76 - 98.24%), sensitivity of 92.42% (CI 95% 86.03 - 98.80% / PPV = 71.76) and specificity of 4%... (Complete abstract click electronic access below) / Doutor

<p>UVOD: Akutni ishemijski moždani udar (AIMU) je poremećaj moždane funkcije nastao usled vaskularnih o&scaron;tećenja, uzrokovane okluzijom ili embolijom krvnog suda. Za razliku od standardnog CT pregleda endokranijuma, CT perfuzija (CTP) je napredna dijagnostička procedura koja može u prvim satima od početka simptoma AIMU pružiti precizne informacije o lokalizaciji i veličini infarkta mozga, a u okviru infarkta, razlikovati srž i ishemijsku penumbru. Samim tim, CTP predstavlja svojevrsnu pomoć u selekciji pacijenata za intravensku primenu rekombinantnog tkivnog plazminogen aktivatora (rtPA). CILJ RADA: Cilj istraživanja je bio da se primenom CTP ispita koliko iznosi: optimalna veličina srži, zatim minimalni i optimalan odnos penumbre i srži infarkta pogodnih za primenu rtPA, maligni profil srži infarkta koji je nepogodan za primenu rtPA i % simptomatske hemoragije kao komplikacije nakon rtPA. MATERIJAL I METODE: Istraživanje je obavljeno u Specijalnoj bolnici &bdquo;Sveti Sava&ldquo; u Beogradu kao petogodi&scaron;nja retrospektivna studija. Studija je obuhvatila ukupno 130 pacijenata sa AIMU kod kojih je primenjena CTP. Eksperimentalnu grupu je sačinjavalo 100 pacijenata kojima je aplikovana rtPA, a kontrolnu grupu 30 pacijenata, koji nisu primili rtPA. Svim ispitanicima su načinjeni: standardni CT pregled glave i CT perfuzija odmah nakon prijema i kontrolni standardni CT pregled endokranijuma 24 h nakon prijema u bolnicu. Pregledi su obavljeni sa 16-slajsnom MSCT aparatu, pri čemu je pokrivenost CTP iznosila 2 cm. &bdquo;Mismatch&ldquo; postoji ukoliko je perfuziona lezija na CBF mapi veća od perfuzionog deficita na CBV mapi (srţ infarkta). REZULTATI: Rezultati studije su pokazali da pacijenti oboleli od AIMU sa mismatchom manjim od 20% nisu imali koristi od primenjene rtPA, a pacijenti sa 20% i vi&scaron;e mismatch-om su imali ili umeren ili značajan neurolo&scaron;ki oporavak. Optimalni mismatch, kojim se postiže visoka uspe&scaron;nost nakon rtPA je iznosio ˃ 101% penumbre. Pacijenti sa povr&scaron;inom perfuzionog deficita na CBV mapi manjom od 1175 mm2 su imali bolji neurolo&scaron;ki oporavak, u odnosu na pacijente savećim lezijama, a samim tim su bili pogodni za primenu rtPA. Perfuzioni deficit na CBV mapi veći od 3000 mm2 (60ml) je predstavljao maligni profil infarkta, usled povećanog rizika od nastanka simptomatske hemoragije i smrtnog ishoda. Simptomatska hemoragija u eksperimentalnoj grupi je iznosila svega 3%, &scaron;to predstavlja nizak procenat komplikacija nakon reperfuzione terapije. ZAKLJUČAK: CT perfuzija je urgentna i brza tehnika, koja nam daje jedinstvene informacije o AIMU, pomaže u dono&scaron;enju odluke o terapijskom pristupu i može poslužiti kao surogat biomarker u predikciji kliničkog ishoda.</p> / <p>INTRODUCTION: Acute ischemic stroke (AIS) is functional brain disorder, which is happened due to vascular damaged, caused by vessel occlusion or embolism. Unlike to noncontrast brain CT, CT perfusion (CTP) is advanced diagnostic procedure, which could give accurate information about localization and extent of AIS in the first hours of symptom onset, and also differentiate infarct cor and ischemic penumbra. Thus, CTP helps in selection patients for administration intravenous recombinant tissue plasminogen activator (rtPA) AIM: The aims of study were to estimate: the size of optimal infarct cor, minimal and optimal ratio between penumbra and infarct core which is suitable for rtPA, malignant profile of infarct core which is not suitable for rtPA and percentage of symptomatic intracerebral hemorrhage as compli-cation after rtPA using CTP. MATERIAL AND METHODS: The investigation was performed in Stroke hospital &bdquo;Sveti Sava&rdquo; in Belgrade as five years retrospective study. Study included 130 patients with AIS with performed CT perfusion. One hundred patients from experimental group were treated with rtPA and thirty patients from control group were not treated with rtPA. All patients underwent baseline noncontrast CT of brain, CT perfusion and control 24 hours follow-up noncontrast brain CT. Examinations were done on 16-slice MSCT and CTP covered an area of 20 mm of brain tissue. &bdquo;Mismatch &ldquo;was defined as a perfusion lesion (CBF lesion) larger than the core lesion (CBV). RESULTS: Results of study showed that the patients with AIS and mismatch less than 20%, had no benefit from rtPA, but patients with &ge; 20% of mismatch had good or excellent clinical outcome. Optimal mismatch, which provided favorable response after rtPA, was ˃101% of penumbra. Patients with cor perfusion lesion (CBV) less than 1175 mm2, are suitable for rtPAand had better clinical outcome, then the patients with larger lesions. Perfusion cor lesions (CBV) larger than 3000 mm2 (60ml) was malignantprofile, because of high risk of symptomatic intracerebral hemorrhage and mortality. Symptomatic hemorrhage in experimental group was only 3%, as a low percentage of complications after reperfusion therapy.<br />CONCLUSION: CTP is emergency and rapid technique, which provides unique information about AIS, helps with clinical decision making and could be surrogate biomarker in prediction of clinical outcome.</p>

Cell-free protein synthesis (CFPS) is a powerful protein expression platform where protein synthesis machinery is borrowed from living organisms. Target proteins are synthesized in a reaction tube together with cell extract, amino acids, energy source, and DNA. This reaction is versatile, and dynamic optimizations of the reaction conditions can be performed. The "œopen" nature of CFPS makes it a compelling candidate for many technologies and applications. This dissertation reports new and innovative applications of CFPS including 1) enzyme encapsulation in a virus-like particle, 2) detection of endocrine disrupting chemicals in the presence of blood and urine, and 3) expression of a multi-disulfide bond therapeutic protein. Two major limitations of enzymes are their instability and recycling difficulty. To overcome these limitations, we report the first enzyme encapsulation in the CFPS by immobilizing in a virus-like particle using an RNA aptamer. This technique allows simple and fast enzyme production and encapsulation We demonstrate, for the first time, the Rapid Adaptable Portable In vitro Detection biosensor platform (RAPID) for detecting endocrine disrupting chemicals (EDCs) in human blood and urine samples. Current living cell-based assays can take a week to detect EDCs, but RAPID requires only 2 hours. It utilizes the versatile nature of CFPS for biosensor protein complex production and EDC detection. Biotherapeutic protein expression in E. coli suffers from inclusion body formation, insolubility, and mis-folding. Since CFPS is not restricted by a cell wall, dynamic optimization can take place during the protein synthesis process. We report the first expression of full-length tissue plasminogen activator (tPA) using CFPS. These research works demonstrate the powerful and versatile nature of the CFPS.

No description available.

Previous work has shown that thrombin activatable fibrinolysis inhibitor (TAFI) was unable to prolong lysis of purified clots in the presence of Lys-plasminogen (Lys-Pg), indicating a possible mechanism for fibrinolysis to circumvent prolongation mediated by activated TAFI (TAFIa). Therefore, the effects of TAFIa on Lys-Pg activation and Lys-plasmin (Lys-Pn) inhibition by antiplasmin (AP) were quantitatively investigated using a fluorescently labeled recombinant Pg mutant which does not produce active Pn. High molecular weight fibrin degradation products (HMW-FDPs), a soluble fibrin surrogate that models Pn modified fibrin, treated with TAFIa decreased the catalytic efficiency (kcat/Km) of 5IAF-Glu-Pg cleavage by 417-fold and of 5IAF-Lys-Pg cleavage by 55-fold. A previously devised intact clot system was used to measure the apparent second order rate constant (k2) for Pn inhibition by AP over time. While TAFIa was able to abolish the protection associated with Pn modified fibrin in clots formed with Glu-Pg, it was not able to abolish the protection in clots formed with Lys-Pg. However, TAFIa was still able to prolong the lysis of clots formed with Lys-Pg. TAFIa prolongs clot lysis by removing the positive feedback loop for Pn generation. The effect of TAFIa modification of the HMW-FDPs on the rate of tissue type plasminogen activator (tPA) inhibition by plasminogen activator inhibitor type 1 (PAI-1) was investigated using a previously devised end point assay. HMW-FDPs decreased the k2 for tPA inhibition rate by 3-fold. Thus, HMW-FDPs protect tPA from PAI-1. TAFIa treatment of the HMW-FDPs resulted in no change in protection. Vitronectin also did not appreciably affect tPA inhibition by PAI-1. Pg, in conjunction with HMW-FDPs, decreased the k2 for tPA inhibition by 30-fold. Hence, Pg, when bound to HMW-FDPs, protects tPA by an additional 10-fold. TAFIa treatment of the HMW-FDPs completely removed this additional protection provided by Pg. In conclusion, an additional mechanism was identified whereby TAFIa can prolong clot lysis by increasing the rate of tPA inhibition by PAI-1 by eliminating the protective effects of Pn-modified fibrin and Pg. Because TAFIa can suppress Lys-Pg activation but cannot attenuate Lys-Pn inhibition by AP, the Glu- to Lys-Pg/Pn conversion is able to act as a fibrinolytic switch to ultimately lyse the clot. / Thesis (Master, Biochemistry) -- Queen's University, 2008-01-31 17:04:50.447

L’activateur tissulaire du plasminogène (tPA) est une sérine protéase qui est synthétisée principalementpar les cellules endothéliales des vaisseaux. Initialement découvert dans le compartiment vasculaire où sa fonctionprincipale est de participer à la fibrinolyse, le tPA est aussi exprimé dans le parenchyme cérébral par plusieurstypes cellulaires comme les neurones ou les oligodendrocytes. Le tPA est impliqué dans de nombreuses fonctionscérébrales comme la plasticité synaptique ou encore la potentialisation glutamatergique. Le tPA est aussi un acteurclé de la neuroinflammation. Il active la microglie et participe à l’ouverture de la barrière hémato-encéphalique pardes effets de type cytokine et via son domaine protéase en générant de la plasmine à partir du plasminogène. Uneactivité plus importante du tPA est retrouvée dans le liquide céphalorachidien des patients atteints de sclérose enplaques (SEP). De plus, le tPA possède des aspects délétères dans son modèle murin, l’encéphalomyélite autoimmune expérimentale (EAE). Dans le but de mieux comprendre le rôle du tPA dans la physiopathologie de l’EAE,nous nous sommes intéressés à son implication dans la réponse immunitaire pendant la maladie. Nos donnéesmontrent que les animaux tPA-/- ont des scores cliniques moins importants que les animaux WT pendant une EAE.Les nombres absolus de LT CD4+, de microglie activée et de macrophages infiltrés, ainsi que de cellulesdendritiques sont moins importants dans le parenchyme spinal des animaux tPA-/- en comparaison avec lesanimaux WT. En lien avec ces observations in vivo, le tPA augmente in vitro l’activation et la prolifération des LTainsi que la sécrétion d’IL-6 par un mécanisme dépendant du domaine protéase et de la génération de plasmine.Dans des expériences in vitro en collaboration avec l’équipe du Dr Diego Clemente, le tPA induit l’augmentation del’expression des molécules du CMH de classe II et des molécules de costimulation à la surface des cellulesdendritiques et des macrophages par un effet de type cytokine, suggérant une capacité plus importante pour cescellules à présenter des antigènes en présence de tPA. Notre étude apporte une meilleure compréhension du rôledu tPA dans la réponse immunitaire pendant l’EAE et ouvre de nouvelles perspectives dans l’étude de l’axe tPA/plasmine dans la physiopathologie de la maladie. / Tissue plasminogen activator (tPA) is a serine protease, mainly synthesized by endothelial cells of vessels.Initially discovered in the vascular compartment where its main function is to participate to fibrinolysis, tPA is alsopresent in the cerebral parenchyma, and expressed by several cell types like neurons or oligodendrocytes. tPA isinvolved in many physiological brain functions such as synaptic plasticity or glutamatergic potentiation. tPA is alsoa main actor of neuroinflammation. It activates microglia and participates in the opening of the blood-brain barrier(BBB) by cytokine-like effects and via its protease domain and plasmin generation from plasminogen. Interestingly,tPA activity is more important in cerebrospinal fluid of multiple sclerosis (MS) patients. In addition, tPA revealsdeleterious aspects in experimental autoimmune encephalomyelitis (EAE), the mouse model of MS. In order tobetter understand the role of tPA in EAE physiopathology, we focused on its involvement in the immune responseduring disease. tPA-/- EAE animals present less severe clinical scores than WT animals. Our results indicate alsothat absolute numbers of CD4 + T cells, activated microglia and infiltrated macrophages, as well as dendritic cellsare less important in the spinal parenchyma of tPA-/-. In connection with these in vivo observations, our in vitro datashow that tPA increases activation and proliferation of T cells, as well as IL-6 secretion by a protease-dependentmechanism and plasmin generation. In experiments in collaboration with Dr Diego Clemente's team, our data showthat tPA increases the expression of MHC class II and costimulatory molecules on the surface of dendritic cells andmacrophages in vitro by a cytokine-like effect, suggesting a more important ability for these cells to present antigenswith tPA. Our study provides a better understanding of the role of tPA in immune response during EAE, and opensup new perspectives in the study of the tPA / plasmin axis in the physiopathology of the disease.

Stroke is a common, multifactorial cardiovascular disease. A stroke event is the result of traditional risk factors (i.e. hypertension, diabetes, smoking), environmental exposures and genetic factors in a complex interplay. The genetic contribution is, as estimated by studies on the influence of family history on the risk of stroke, limited on the individual level, and overridden by, for example the excess risk associated with smoking. On the population level, and as a means to better understand the etiology of stroke, genetics can play a major role. Northern Sweden is well suited for studying the genetic aspects of stroke. The population shows signs of founder effects, and is relatively homogeneous. Large-scale cardiovascular health surveys, the MONICA Project and the Västerbotten Intervention Program, allow studies on risk factors in relation to stroke. Two prospective nested case-referent study samples, (113 cases and 226 controls; 275 cases and 549 controls), and a set of 56 families (117 affected) were collected for functional candidate gene association, and linkage, studies. The selected candidate genes included haemostatic factors and genes within the renin angiotensin system (RAS). Functional single nucleotide polymorphisms (SNPs) that influence the levels of PAI-1 (PAI-1 4G/5G), and tPA (tPA -7,351C&gt;T), have been identified. The angiotensin converting enzyme insertion/deletion polymorphism (ACE I/D) has been shown to be associated with ischaemic stroke. The angiotensin II receptor type 1 A1166C polymorphism (AT1R A1166C), less extensively studied, has been suggested to be associated with stroke, and to interact with the ACE I/D. We found that the PAI-1 4G/4G genotype was associated with an increased risk of future ischaemic stroke (OR 1.79, 95%CI 1.01-3.19), and this was replicated in a second study sample. Furthermore, levels of serum triglycerides modulated the effect of the genotype. In the study on tPA, no association between the tPA -7,351C&gt;T polymorphism and the risk of stroke was found in an analysis of the two study samples pooled. The two RAS polymorphisms were prospectively associated with ischaemic stroke independently of each other and other risk factors (OR 1.60, p=0.02 and OR 1.60, p=0.04, respectively). A candidate region linkage study, focusing on a previously reported stroke susceptibility locus on chromosome 5, was performed in a set of families. In addition, association between ischemic stroke and the positional candidate gene phosphodiesterase 4D (PDE4D) was tested. Linkage to 5q12 was replicated in this independent population, but not PDE4D association with stroke. This suggests that alternative genotypes in this stroke susceptibility locus contribute in different populations. In conclusion, the genetic component in the causation of stroke was investigated. The results of the functional candidate gene association studies showed (1) interaction between PAI-1 genotype and a putatively modifiable risk factor, triglycerides, (2) a prospective testing of the tPA SNP with no association detected, and (3) a novel, hypothesis-generating, finding in the case of AT1R polymorphism and the risk of ischaemic stroke. The replication of linkage to chromosome 5q12 in our northern Swedish population was interesting, and it will be further explored.

Exocrine pancreatic cancer is the fifth leading cause of death from malignant disease in Western society and it is one of the most aggressive human tumors. Once diagnosed, the 12-month patient survival is less than 5%. More than 90% of human exocrine tumors are classified as "ductal adenocarcinomas" on the basis of their microscopic appearance. The plasminogen system plays a critical role in intravascular thrombolysis as well as in other biological processes that require cellular migration, such as angiogenesis, inflammatory reactions, tissue remodelling, and tumor progression. There are two types of plasminogen activators that catalyze plasmin generation from plasminogen: tissue-type (tPA) and urokinase-type (uPA). Activation of plasminogen to plasmin results in progressive degradation of fibrin and other extracellular matrix components and may also lead to activation of metalloproteases, latent growth factors, and proteolysis of membrane glycoproteins. All these processes may contribute to tumor development and metastasis. There is extensive evidence supporting the notion that the uPA system, including its receptor and plasminogen activator inhibitor PAI-1, can contribute to tumorigenesis in a variety of tissue types but there is less evidence for such a role regarding tPA and annexin A2 (AnxA2), a putative tPA receptor. Previous studies of our group have shown that tPA is commonly expressed in pancreas cancer tissues and cell lines and appears to be selectively associated with the neoplastic phenotype. Using neutralizing antibodies or chemical inhibitors leads to reduced in vitro tumor invasion. Our results support that - in the pancreas - the tPA system plays an important role in tumor development and/or progression whereas the uPA system may play a more dominant role in pancreatitis. More recent studies have shown that tPA stimulates cell proliferation and angiogenesis in exocrine pancreatic tumors. These results allow new approaches to improve the treatment of this disease, but to do so it is necessary to use of mouse models of disease. In attempt to explore the role of tPA and its receptor, annexin A2, in pancreatic tumorigenesis, we have taken advantage of two well established transgenic mouse models: Ela1-TAg (1-127) and Ela1-myc. In these mice, transgenes are targeted to acinar cells using the Elastase-1 enhancer/promoter. We have also analyzed the pancreas of Ela1-CCK2 and MT-TGFa transgenic mice, as models of acinar-ductal transdifferentiation and ductal complex formation. Our results show that expression of tPA is undetectable in the non-neoplastic pancreatic epithelium and in metaplastic ducts and in acinar tumors. By contrast, tPA is overexpressed in neoplastic pancreatic ducts. This pattern expression is in agreement with the results described in humans, indicating that mouse models of pancreatic cancer may be useful for the study of human pathology. On the other hand, AnxA2 is undetectable in acinar tumors but it is detected in the apical membrane of normal and metaplastic duct epithelium. In addition, AnxA2 is strongly expressed in ductal tumor cells where it shows a non-polarized distribution. These results suggest that different molecular events may participate in the activation of tPA and its receptor, AnxA2, in non-neoplastic ducts. In order to analyze the role of tPA in the progression of pancreatic tumors, we mated Ela1-TAg and Ela1-myc transgenic mice to tPA-deficient mice. The proportion of tumors displaying pure acinar differentiation or mixed acinar/ductal components was similar in both mouse strains, indicating that tPA is not required for in acinar-ductal transdifferentiation. However, it was observed an increased survival in hybrid mice Ela1-myc:tPA-/- supporting a critical role for tPA in the progression of pancreatic ductal tumors. To get insight into the mechanism by wich tPA participates in this process we have analyzed factors related to tumor progression: tumors arising in a tPA-/- genetic background show a lesser vessel density and proliferation rate than those arising in wild type mice. These results indicate that tPA could play a role in angiogenesis stimulation and cell proliferation and suggest that the increase in survival observed in Ela1-myc tPA-/- mice could be a consequence of the inhibition of tumor angiogenesis and cell proliferation. In addition, we have analyzed the differential gene expression between Ela1-myc and Ela1-myc tPA-/- mice by microarrays. This analysis has led to the identification of related genes with tumor progression and invasion that can be a target for the action of tPA, although more work is necessary to determined their role. Finally, we have studied the direct effects of the expression of tPA in the pancreas, by the generation of two transgenic mice which tPA expression is targeted to acinar cells, using the Elastase-1 enhancer/promoter (Ela1-tPA), or to ductal cells using the Citokeratin 19 promoter (CK19-tPA). The results in Ela1-tPA mice show that overexpression of tPA in acinar cells does not affect normal mouse development. The effects on the pancreas analysed are currently being analyzed in greater detail. Altogether, the data described here support the relevant role of tPA in pancreas cancer progression and indicate that mouse models of pancreatic cancer may be useful for the preclinical evaluation of drugs targeting the tPA system.