P-21 Activated Kinase 2: Signal Transduction in Mast Cells, Megakaryocytes and in Vivo Homeostasis

Upon immune cell activation with antigen, growth factors, or other stimuli, the cytoskeleton undergoes extensive reorganization to elicit a cellular response. The cytoskeleton, consisting of microtubules and actin, is a highly organized network regulated by various signal transduction pathways. Specifically, Rho GTPases (RhoA, Rac1 and Cdc42) regulate the cytoskeleton, albeit through different pathways. p21-activated kinases (Pak) are serine/threonine kinases directly bound and activated by Rac1 and Cdc42. There are 6 Pak isoforms separated into 2 groups (groups I&II) in this family of kinases, and only recently have isoform specificities been identified by the use of genetically-engineered mouse models deleted for individual isoforms. In this dissertation we sought to identify if differences exist between Pak1 and Pak2 in immune function, in particular how they differ in regulation of the cytoskeleton reorganization required for immune cell function. Using primary bone marrow derived mast cells, an immune cell type responsible for anaphylaxis and allergic responses, we identified that Pak1 and Pak2 function in opposing manners with regard to antigen-induced degranulation. We identified key mechanisms involved in Pak2\u27s negative regulation of mast cell degranulation. These findings identify potential therapeutic side effects with the use of recently developed pan-Pak inhibitors in the clinic. Pak2 deletion was additionally investigated in an in vivo mouse model. We discovered that Pak2 is critical for homeostasis and survival in an adult animal. We identified macrothrombocytopenia, cause by an increase in circulating platelet half-life and clearance, as well as other defects in Pak2-deleted adult mice. Therefore, we evaluated the maturation process of the platelet-producing megakaryocyte and found that Pak2-null megakaryocytes have altered microtubules, proplatelet extensions and polyploidization. Various signaling pathways that regulate these functions were also suppressed with Pak2 deletion. Together, our findings identify Pak2 as the predominant isoform in hematopoietic compartment and immune cells, and suggest further analysis of critical immune cell side effects, which could occur in the patient with the use of pan-Pak inhibitors in the treatment of various cancers

The Latest Clinical Advances in Thrombocytopenia

Platelets are critical elements in the blood stream, supporting hemostasis (defense against bleeding), as well as performing even more complex tasks within networks of biological (immunity) and pathophysiological processes, such as cancer and ischemia/reperfusion injury. Changes in the number (and function) of platelets may have a substantial impact on any of these processes. The “simple” finding of a reduced platelet count (thrombocytopenia) has a history (origin) and a consequence (e.g., bleeding). This book brings together international experts to provide an up-to-date overview of the impact of thrombocytopenia from a clinical perspective

Treatment of Thrombotic Antiphospholipid Syndrome : The Rationale of Current Management - An Insight into Future Approaches

Vascular thrombosis and pregnancy morbidity represent the clinical manifestations of antiphospholipid syndrome (APS), which is serologically characterized by the persistent positivity of antiphospholipid antibodies (aPL). Antiplatelet and anticoagulant agents currently provide the mainstay of APS treatment. However, the debate is still open: controversies involve the intensity and the duration of anticoagulation and the treatment of stroke and refractory cases. Unfortunately, the literature cannot provide definite answers to these controversial issues as it is flawed by many limitations, mainly due to the recruitment of patients not fulfilling laboratory and clinical criteria for APS. The recommended therapeutic management of different aPL-related clinical manifestations is hereby presented, with a critical appraisal of the evidence supporting such approaches. Cutting edge therapeutic strategies are also discussed, presenting the pioneer reports about the efficacy of novel pharmacological agents in APS. Thanks to a better understanding of aPL pathogenic mechanisms, new therapeutic targets will soon be explored. Much work is still to be done to unravel the most controversial issues about APS management: future studies are warranted to define the optimal management according to aPL risk profile and to assess the impact of a strict control of cardiovascular risk factors on disease control

Renal Failure

The book "Renal Failure - The Facts" consists of some facts about diagnosis, etiopathogenis and treatment of acute and chronic renal failure. Acute, as well as chronic renal failure is great medical problems and their treatment is a burden for the budget of each government. The purpose of the chapters is to present some important issues of diagnosis and causes of AKI, as well as caused by snakes and arthropods, after cardiac surgery, as well as some therapeutic achievements in AKI. Well presented are the psychological condition in patients on haemodialysis, as well as the treatment of diabetic uremics. The book is aimed at clinicians with a special interest in nephrology, but it should also prove to be a valuable resource for any generalists who encounter a nephrological problems in their day-to-day practice

Identificación, clonaje, purificación y caracterización inmunológica de alérgenos únicos y de reactividad cruzada del mosquito Aedes aegypti

Tesis inédita de la Universidad Complutense de Madrid, Facultad de Ciencias Químicas, Departamento de Bioquímica y Biología Molecular I, leída el 09-05-2017Aedes aegypti es una especie de mosquito que se ha expandido en todos los continentes y se ha especializado en alimentarse de humanos. Se ha propuesto A. aegypti puede inducir diferentes tipos de respuestas alérgicas. En esta tesis estudiamos la composición alergénica de A. aegypti y evaluamos su potencial alergénico y reactividad cruzada. Usamos dos enfoques para identificar alérgenos. El estudio del Alergenoma de A. aegypti, mediante el análisis del espectro de proteínas separadas por electroforesis bidimensional, ensayos de unión a IgE y caracterización mediante espectrometría de masas. Se identificaron diez proteínas. Algunas presentaron variantes, o isoformas. La mezcla de tres de estos alérgenos fue suficiente para identificar casi todos los individuos sensibilizados. En un segundo enfoque, la tropomiosina natural de A. aegypti se purificó mediante cromatografía de exclusión y de intercambio iónico. Se encontró que en A. aegypti, la tropomiosina consiste en una mezcla de al menos 4 versiones (isoformas y variantes). Dos de estas versiones (Códigos UNIPROT: Q17H75 y Q17H80) se encontraron en mayores cantidades relativas. El análisis de la secuencia de aminoácidos y experimentos de inhibición indicaron que Q17H75 es potencialmente más alergénica y de mayor reactividad cruzada que Q17H80. Como resultado de estos estudios, seis nuevos alérgenos de A. aegypti fueron registrados en la base de datos de alérgenos: Aed a 5 (proteína sarcoplásmica de unión calcio), Aed a 6 (Porina 3), Aed a 7 (proteína de función no definida), Aed a 8 (Heat shock cognate70), dos variantes de tropomiosina (Aed a 10.0101 y Aed a 10.0201) y Aed a 11 (proteasa de aspártico lisosómica). Las versiones inmunológicamente activas de Aed a 10.0101, Aed a 10.0201 y Aed a 8 se expresaron en E. coli y se purificaron por afinidad. Los recombinantes mostraron capacidad de unión a anticuerpos, activación de basófilos e inmunogenicidad. rAed a 10.0101 y rAed a 10.0201 presentaron espectros de dicroísmo circular de proteínas alfa helicoidales. Ratones inmunizados con rAed a 8 produjeron niveles bajos de IgE y altos niveles de IgG, especialmente IgG2a, sugiriendo que podría tener propiedades inmuno reguladoras. Nuestros estudios demostraron que A. aegypti tiene alta reactividad cruzada con el camarón Litopenaeus vannamei, seguido por los ácaros (Dermatophagoides pteronyssinus, Blomia tropicalis y D. farinae) y la cucaracha Periplaneta americana. La frecuencia de sensibilización a A. aegypti es 65 por ciento. Más de 10 bandas de reactividad cruzada fueron detectadas. Cuatro fueron caracterizadas como: proteína de unión a odorante, citocromo C mitocondrial, ciclofilina (PPIasa) y una proteína de función biológica desconocida (AAEL001668PA). Se analizó la reactividad cruzada entre las tropomiosinas del ácaro (rDer p 10) y rAed a 10.0101 y rAed a 10.0201. Se encontró las tropomiosinas tienen reactividad cruzada con anticuerpos de humanos y ratones, aunque fue mayor entre rDer p 10 y rAed a 10.0101. También se observó que pueden activar los basófilos sensibilizados con rDer p 10. Las tres tropomiosinas indujeron la proliferación de esplenocitos de ratones inmunizados con rAed a 10.0101 o rAed a 10.0201. El mapeo de epítopos utilizando 28 péptidos solapantes que cubrían la secuencia de Aed a 10.0101 reveló cinco regiones de aminoácidos que contienen epítopos T implicadas en esta reactividad cruzada. En conclusión, A. aegypti es una fuente importante de alérgenos que pueden jugar un papel en la sensibilización y exacerbación de las alergias. Varios alérgenos están involucrados en estos fenómenos y algunos de ellos tienen reactividad cruzada con alérgenos de artrópodos. La reactividad cruzada puede ocurrir a nivel molecular y funcional, involucrando anticuerpos, basófilos y linfocitos T. Los alérgenos de los mosquitos pueden producirse como moléculas recombinantes similares a los alérgenos naturales. rAed a 8 indicaron puede tener un papel inmuno regulador intrínseco.Depto. de Bioquímica y Biología MolecularFac. de Ciencias QuímicasTRUEunpu

Resolvin E1 actions on polymorphonuclear neutrophils in diabetes

Dissertation (DScD) -- Boston University, Henry M. Goldman School of Dental Medicine, 2010 (Department of Periodontology and Oral Biology).Diabetes and periodontal disease exhibit a bidirectional relationship centered on an enhanced inflammatory response manifested both locally and systemically. The observation that hyperglycemia by itself, in the absence of additional inflammatory signals, promotes a proinflammatory environment indicates that diabetes is an independent risk factor for periodontal disease. Leukocyte pre-activation or priming in diabetes has been demonstrated. Excessive ROS release by leukocytes, upregulation of pro-inflammatory mediators and adhesion molecules are characteristic to T2DM-associated low-grade inflammation. However, the mechanisms by which chronic hyperglycemia leads to leukocyte activation are not fully understood. [TRUNCATED

The role of WAVE (WASP-family-verprolin homologousproteins) 1, 2 and 3 on cellular migration and invasion of colorectal cancer

Colorectal carcinoma (CRC) is the second most common cause of cancer deaths in the UK. More than half of patients are diagnosed at a late stage with around a quarter of patients having metastases at diagnosis (stage IV). Approximately 50% of diagnosed patients will progress to metastatic disease. The metastatic spread of malignant cells to distant sites in the body accounts for the majority of cancer-related death and significantly decreases patient survival. Whilst cell migration is a physiologically important process, when uncontrolled, it can be a contributing factor to the metastatic phenotype. Actin polymerisation enables the dynamic restructuring of the cytoskeleton which is fundamental to cell migration and is stimulated by the Arp (actin-related protein) 2/3 protein complex which in turn is activated by members of the WAVE (WASP Verprolin homologous protein) family. Clinico-pathological data was updated for a cohort of patients that had been involved in a previous colorectal tissue/carcinoma sampling study. The stored frozen tissue samples were analysed using histological and molecular biology techniques including conventional polymerase chain reaction, quantitative polymerase chain reaction, immunohistochemistry and in vitro gene knockdown studies. WAVE 1 and 3 expression was targeted separately in the RKO and CaCo2 cell lines utilising ribozyme transgene transfection to assess the effect knockdown on cell functions. A high WAVE 2 expression level is associated with more aggressive and higher stage primary tumours and also a shorter overall survival time and disease free survival time. WAVE 3 expression is higher in colorectal tissues compared to normal tissues but otherwise showed no significant difference. WAVE 1 did not show an increase in expression levels compared to normal colorectal tissues. The In vitro functional assays revealed a significant reduction in cell invasion and motility following WAVE 3 knockdown in CaCo2 cells. Knockdown of WAVE 1 in RKO cells resulted in a significant reduction in invasion and a moderate reduction in motility that was not significant. These results suggest WAVE1 and 3 proteins are involved in several metastatic traits and that WAVE 2 has significant correlation with higher stage disease. The data outlined here provides justification to further explore WAVE1, 2 and 3 as potential contributors of colorectal cancer progression