Aptamers in oncology: a diagnostic perspective

Nucleic acid sequences can produce a wide variety of three-dimensional conformations. Some of these structural forms are able to interact with proteins and small molecules with high affinity and specificity. These sequences, comprising either double or single stranded oligonucleotides, are called 'aptamers' based on the Greek word aptus, which means 'to fit'. Using an efficient selection process, randomised oligonucleotide libraries can be rapidly screened for aptamers with the appropriate binding characteristics. This technology has spawned the development of a new class of oligonucleotide therapeutic products. However, while interest among pharmaceutical companies continues to grow with some candidates already in clinical trials and one in the market, there appears to be some reluctance to fully explore the diagnostic potential of this technology. This article will review aptamer developments in diagnostics, compare them with other oligonucleotide therapeutics and highlight both potentials and pitfalls of technological development in this area

Plasma-based assays distinguish hyperfibrinolysis and shutdown subgroups in trauma-induced coagulopathy

BACKGROUND Trauma patients with abnormal fibrinolysis have increased morbidity and mortality. Knowledge of mechanisms differentiating fibrinolytic phenotypes is important to optimize treatment. We hypothesized that subjects with abnormal fibrinolysis identified by whole blood viscoelastometry can also be distinguished by plasma thrombin generation, clot structure, fibrin formation, and plasmin generation measurements. METHODS Platelet-poor plasma (PPP) from an observational cross-sectional trauma cohort with fibrinolysis shutdown (% lysis at 30 minutes [LY30] \u3c 0.9, n = 11) or hyperfibrinolysis (LY30 \u3e 3%, n = 9) defined by whole blood thromboelastography were studied. Noninjured control subjects provided comparative samples. Thrombin generation, fibrin structure and formation, and plasmin generation were measured by fluorescence, confocal microscopy, turbidity, and a fluorescence-calibrated plasmin assay, respectively, in the absence/presence of tissue factor or tissue plasminogen activator (tPA). RESULTS Whereas spontaneous thrombin generation was not detected in PPP from control subjects, PPP from hyperfibrinolysis or shutdown patients demonstrated spontaneous thrombin generation, and the lag time was shorter in hyperfibrinolysis versus shutdown. Addition of tissue factor masked this difference but revealed increased thrombin generation in hyperfibrinolysis samples. Compared with shutdown, hyperfibrinolysis PPP formed denser fibrin networks. In the absence of tPA, the fibrin formation rate was faster in shutdown than hyperfibrinolysis, but hyperfibrinolysis clots lysed spontaneously; these differences were masked by addition of tPA. Tissue plasminogen activator–stimulated plasmin generation was similar in hyperfibrinolysis and shutdown samples. Differences in LY30, fibrin structure, and lysis correlated with pH. CONCLUSION This exploratory study using PPP-based assays identified differences in thrombin generation, fibrin formation and structure, and lysis in hyperfibrinolysis and shutdown subgroups. These groups did not differ in their ability to promote tPA-triggered plasmin generation. The ability to characterize these activities in PPP facilitates studies to identify mechanisms that promote adverse outcomes in trauma

Extracellular annexin A5: Functions of phosphatidylserine-binding and two-dimensional crystallization

AbstractIn normal healthy cells phosphatidylserine is located in the inner leaflet of the plasma membrane. However, on activated platelets, dying cells and under specific circumstances also on various types of viable leukocytes phosphatidylserine is actively externalized to the outer leaflet of the plasma membrane. Annexin A5 has the ability to bind in a calcium-dependent manner to phosphatidylserine and to form a membrane-bound two-dimensional crystal lattice. Based on these abilities various functions for extracellular annexin A5 on the phosphatidylserine-expressing plasma membrane have been proposed. In this review we describe possible mechanisms for externalization of annexin A5 and various processes in which extracellular annexin A5 may play a role such as blood coagulation, apoptosis, phagocytosis and formation of plasma membrane-derived microparticles. We further highlight the recent discovery of internalization of extracellular annexin A5 by phosphatidylserine-expressing cells

Tissuue Engineering in Maxillar Sinus Lifting: A Comparation of Differents Grafts and Confocal Laser Scanning Microscopic Evaluation

Bone is a specialized connective tissue, most prominently characterized by its mineralized organic matrix that imparts the physical properties that allow bone tissue to resist load, to support functional organs, and to protect highly sensitive body parts. Bone loss and bone damage may occur as a result of genetic conditions, infectious diseases, tumours, and trauma. Bone healing and repair, involves integrative activity of native tissues and living cells, and lends itself to the incorporation of naturally derived or biocompatible synthetic scaffolds, aimed at replacing missing or damaged osseous tissues. There are several modalities of bone regeneration including tissue engineering, guided bone regeneration, distraction ontogenesis, and bone grafting. This book concentrates on such procedures that may well be counted among the recent outstanding breakthroughs in bone regenerative therapy

Myosin II Isoforms in Breast Cancer: Role of Contractility in Matrix Organization, Cell Migration, and Endothelial Barrier Function

Breast cancer is a devastating and complicated disease. A large proportion of breast cancer related deaths are due to metastasis. Metastasis is when cancer cells leave the primary tumor, travel through the bloodstream, and proliferate in distant sites. Cell contractility is involved in a number of cellular processes associated with metastasis including matrix organization, tumor cell migration, and endothelial cell barrier function. The main regulator of contractility in nonmuscle cells is nonmuscle myosin II. There are three isoforms of this protein: myosin IIA, IIB, and IIC. The specific roles of the individual isoforms in cancer progression and metastasis have yet to elucidated. This works aims to determine if myosin II isoforms play separate roles in three functions associated with cancer progression: tumor cell organization of a collagen matrix, tumor cell migration, and endothelial cell barrier function. In Study 1, the ability of breast cancer cells lacking myosin II isoforms to organize a collagen matrix was assessed. In addition, the migration potential of these cells in 3D matrices was measured. Myosin II isoforms were found to play distinct roles in cancer cell matrix organization and cell migration. In Study 2, the barrier function of endothelial cells lacking myosin II isoforms was tested. This cell function is involved in metastasis when cancer cells cross the blood vessel wall to enter and exit the bloodstream. Both myosin II isoforms were found to be involved in endothelial cell contractility, and the IIA isoform was found to be necessary for barrier function. Collectively, these studies indicate that myosin II isoforms play distinct and non-redundant roles in cell processes associated with cancer metastasis

NEW GUIDED BONE REGENERATION PROCEDURE USING LEUKOCYTE AND PLATELET RICH FIBRIN (L PRF) IN ORAL SURGERY

Non-transfusional hemocomponents are autogenous products used in several surgical fields obtained by the centrifugation of a blood sample from a patient and able to promote hard and soft tissue regeneration, local haemostasis, and the acceleration of wound healing. The aim of this PhD thesis was the validation of an innovative protocol for the Guided Bone Regeneration (GBR) technique using second-generation autologous platelet concentrates, Leukocytes and Platelet-rich Fibrin (L-PRF), in post-extraction sockets of patients in need of dental avulsions and successive implant prosthetic rehabilitation, evaluating its ability to prevent alveolar bone resorption and promote bone regeneration. The secondary aim was the design and development of a multicomposite loaded with antimicrobic and antioxidant agents, to be applied wrapped in the L-PRF membrane, enhancing its regenerative properties. This project cohering with the National Operational Program (PON) “Research and Innovation” (R&I) 2014-2020 funding by the Italian Ministry of Education, University and Research, aimed to promote the research and the innovation of the country, with a particular interest on the Health Specialization Area

A Microfluidic Approach For Evaluating Novel Antithrombotic Targets

Microfluidic systems allow precise control of the anticoagulation/pharmacology protocols, defined reactive surfaces, hemodynamic flow and optical imaging routines, and thus are ideal for studies of platelet function and coagulation response. This thesis describes the use of a microfluidic approach to investigate the role of the contact pathway factors XII and XI, platelet-derived polyphosphate, and thiol isomerases in thrombus growth and to evaluate their potential as safer antithrombotic drug targets. The use of low level of corn trypsin inhibitor allowed the study of the contact pathway on collagen/kaolin surfaces with minimally disturbed whole blood sample and we demonstrated the sensitivity of this assay to antithrombotic drugs. On collagen/tissue factor surfaces, we found the relative contributions of the extrinsic pathway, the contact pathway, and the thrombin feedback pathway vary with tissue factor surface concentration. Platelet-derived polyphosphate potentiated the thrombin feedback pathway at low tissue factor level but enhanced fibrin fiber structure regardless of tissue factor level. At locations with low tissue factor level, thrombosis may be druggable by contact pathway and polyphosphate inhibition, although thrombolytic susceptibility may benefit from polyphosphate antagonism regardless of tissue factor level. We developed a peptide-based platelet-targeting thiol reduction sensor to visualize thrombus-incorporated thiol reductase activity. Although distribution of thiol reductase activity was shown to be correlated with the level of platelet activation, protein disulfide isomerase inhibition showed a limited effect on platelet aggregation in microfluidic thrombosis assay. We also used the microfluidic system to explore the injury patch size limit for triggering clotting. We observed a full clotting response of platelet deposition, thrombin generation and fibrin polymerization on one of the smallest biological units of a single collagen fiber presenting tissue factor and von Willebrand factor suggesting the lack of physiological injury patch size limit. Finally, we made the first estimation of thrombin flux from growing thrombus under flow using the microfluidic thrombosis assay in combination with enzyme-linked immunosorbent measurement of thrombin-antithrombin complex. We found thrombin is robustly generated within clots by the extrinsic pathway, followed by late-stage factor XIa contributions, with fibrin localizing thrombin via its antithrombin activity as a self-limiting hemostatic mechanism

Mechanical Flow Restoration in Acute Ischemic Stroke: A Model System of Cerebrovascular Occlusion: A Dissertation

Stroke is the third most common cause of death and a leading cause of disability in the United States. The existing treatments of acute ischemic stroke (AIS) involve pharmaceutical thrombolytic therapy and/or mechanical thrombectomy. The Food and Drug Administration (FDA)-approved recombinant tissue plasminogen activator (tPA) administration for treatment of stroke is efficacious, but has a short treatment time window and is associated with a risk of symptomatic hemorrhage. Other than tPA, the Mechanical Embolus Removal in Cerebral Ischemia (MERCI) retriever system and the Penumbra Aspiration system are both approved by the FDA for retrieval of thromboemboli in AIS patients. However, the previous clinical studies have shown that the recanalization rate of the MERCI system and the clinical outcome of the Penumbra system are not optimal. To identify the variables which could affect the performance of the thrombectomy devices, much effort has been devoted to evaluate thrombectomy devices in model systems, both in vivo and in vitro, of vascular occlusion. The goal of this study is to establish a physiologically realistic, in vitro model system for the preclinical assessment of mechanical thrombectomy devices. In this study, the model system of cerebrovascular occlusion was mainly composed of a human vascular replica, an embolus analogue (EA), and a simulated physiologic mock circulation system. The human vascular replica represents the geometry of the internal carotid artery (ICA)/middle cerebral artery (MCA) that is derived from image data in a population of patients. The features of the vasculature were characterized in terms of average curvature (AC), diameter, and length, and were used to determine the representative model. A batch manufacturing was developed to prepare the silicone replica. The EA is a much neglected component of model systems currently. To address this limitation, extensive mechanical characterization of commonly used EAs was performed. Importantly, the properties of the EAs were compared to specimens extracted from patients. In the preliminary tests of our model system, we selected a bovine EA with stiffness similar to the thrombi retrieved from the atherosclerotic plaques. This EA was used to create an occlusion in the aforesaid replica. The thrombectomy devices tested included the MERCI L5 Retriever, Penumbra system 054, Enterprise stent, and an ultrasound waveguide device. The primary efficacy endpoint was the amount of blood flow restored, and the primary safety endpoint was an analysis of clot fragments generated and their size distribution. A physiologically realistic model system of cerebrovascular occlusion was successfully built and applied for preclinical evaluation of thrombectomy devices. The recanalization rate of the thrombectomy device was related to the ability of the device to capture the EA during the removal of the device and the geometry of the cerebrovasculature. The risk of the embolic shower was influenced by the mechanical properties of the EA and the design of the thrombectomy device