Performance of AAV8 vectors expressing human factor IX from a hepatic-selective promoter following intravenous injection into rats

Background: Vectors based on adeno-associated virus-8 (AAV8) have shown efficiency and efficacy for liver-directed gene therapy protocols following intravascular injection, particularly in relation to haemophilia gene therapy. AAV8 has also been proposed for gene therapy targeted at skeletal and cardiac muscle, again via intravascular injection. It is important to assess vector targeting at the level of virion accumulation and transgene expression in multiple species to ascertain potential issues relating to species variation in infectivity profiles. Methods: We used AAV8 vectors expressing human factor IX (FIX) from the liver-specific LP-1 promoter and administered this virus via the intravascular route of injection into 12 week old Wistar Kyoto rats. We assessed FIX levels in serum by ELISA and transgene expression at sacrifice by immunohistochemistry using anti-FIX antibodies. Vector DNA levels in organs we determined by real time PCR. Results: Administration of 1 × 1011 or 5 × 1011 scAAV8-LP1-hFIX vector particles/rat resulted in efficient production of physiological hFIX levels, respectively in blood assessed 4 weeks post-injection. This was maintained for the 4 month duration of the study. At 4 months we observed liver persistence of vector with minimal non-hepatic distribution. Conclusion: Our results demonstrate that AAV8 is a robust vector for delivering therapeutic genes into rat liver following intravascular injection

Biodistribution and inflammatory profiles of novel penton and hexon double-mutant serotype 5 adenoviruses

The use of adenovirus serotype 5 (Ad5) vectors in the clinical setting is severely hampered by the profound liver tropism observed after intravascular delivery coupled with the pronounced inflammatory and innate immune response elicited by these vectors. Liver transduction by circulating Ad5 virions is mediated by a high-affinity interaction between the capsid hexon protein and blood coagulation factor X (FX), whilst penton-α(v)integrin interactions are thought to contribute to the induction of anti-Ad5 inflammatory and innate immune responses. To overcome these limitations, we sought to develop and characterise for the first time novel Ad5 vectors possessing mutations ablating both hexon:FX and penton:integrin interactions. As expected, intravascular administration of the FX binding-ablated Ad5HVR5*HVR7*E451Q vector (AdT*) resulted in significantly reduced liver transduction in vivo compared to Ad5. In macrophage-depleted mice, increased spleen uptake of AdT* was accompanied by an elevation in the levels of several inflammatory mediators. However ablation of the penton RGD motif in the AdT* vector background (AdT*RGE) resulted in a significant 5-fold reduction in spleen uptake and attenuated the antiviral inflammatory response. A reduction in spleen uptake and inflammatory activation was also observed in animals after intravascular administration of Ad5RGE compared to the parental Ad5 vector, with reduced co-localisation of the viral beta-galactosidase transgene with MAdCAM-1+ sinus-lining endothelial cells. Our detailed assessment of these novel adenoviruses indicates that penton base RGE mutation in combination with FX binding-ablation may be a viable strategy to attenuate the undesired liver uptake and pro-inflammatory responses to Ad5 vectors after intravascular deliver

Surface Modified Vascular Tissue for Targeted Delivery

Thrombosis and restenosis are common problems associated with intravascular procedures such as anastomoses, balloon angioplasty, and carotid endarterectomies. Application of a molecular barrier at the site of injury to inhibit platelet deposition would be advantageous. Additional therapeutic benefit could be achieved if the modified surface provided a target for delivery of pharmaceuticals, vectors, or cells. This dissertation focuses on the development of an intravascular modification and targeted delivery system that possesses numerous potential applications in the treatment of vascular injury.Polyethylene glycol is commonly used for modification of molecules and surfaces to increase biocompatibility, reduce immunogenicity, and provide stealth characteristics. Protein-reactive polyethylene glycols could be used to modify vascular surfaces forming a molecular barrier. In addition, the polymer could be used as a target for delivery of agents by applying a recognizable tag to the terminus. Agents could be targeted to modified vascular tissue using, for instance, the biotin/avidin recognition system.The ability to modify vascular surfaces with protein-reactive polyethylene glycols was confirmed using quantitative flow cytometry and epi-fluorescence microscopy. Furthermore, in vitro perfusion studies with cultured cells and scrape-damaged arteries demonstrated preferential delivery of microspheres and cells to polyethylene glycol-biotin modified vascular surfaces.An in vivo rabbit model provided a more rigorous assessment of the polymer modification and targeted delivery system. Polymer modification of balloon injured rabbit femoral arteries persisted for a minimum of 72 hours. Targeted microspheres preferentially adhered to healthy and injured arteries modified with the reactive polymer as opposed to untreated controls. Furthermore, the ability to target microspheres to the modified arteries persisted for a minimum of 72 hours.In conclusion, it was shown that it is possible to modify vascular tissue with a protein-reactive polyethylene glycol and that this modification with signaling molecules can also provide a target for the site-specific delivery of vascular-infused agents. An intravascular targeted delivery system such as this might find numerous applications in the treatment of intravascular injury that is associated with angioplasty, stenting, and endarterectomy procedures

Safety, Efficacy and Evidence Base for Use of the Subcutaneous Implantable Cardioverter Defibrillator

The trans-venous implantable cardioverter defibrillator (TV-ICD) is effective in treating life-threatening ventricular arrhythmia and reduces mortality in high-risk patients. However, there are significant short- and long-term complications that are associated with intravascular leads. These shortcomings are mostly relevant in young patients with long life expectancy and low risk of death from non-arrhythmic causes. Drawbacks of trans-venous leads recently led to the development of the entirely subcutaneous implantable cardioverter defibrillator (S-ICD). The S-ICD does not require vascular access or permanent intravascular defibrillation leads. Therefore, it is expected to overcome many complications associated with conventional ICDs. This review highlights data on safety and efficacy of the S-ICD and is envisioned to help in identifying the role of this device in clinical practice

Intravascular Large B-Cell Lymphoma Presenting as Dementia and Hemolytic Anemia

Background: Intravascular lymphoma (IVL) is an uncommon disease characterized by atypical lymphoid cells growing inside the lumina of small vessels. The diversity of clinical presentation due to possible involvement of multiple organs often complicates its diagnosis. Case Report: Here, we report on a case of IVL with rapidly progressive dementia and Coombs-negative hemolytic anemia. Interestingly, the erythrocytes exhibited a decreased osmotic resistance. Bone marrow histopathology revealed increased erythropoiesis and, finally, a small monoclonal B lymphocyte population. Cerebral magnetic resonance imaging (MRI) demonstrated few micro-bleedings. Computed tomography (CT) showed bilateral ground-glass opacity of the lungs. Within a few days, the patient developed respiratory failure and died. On post-mortem examination, intravascular large B-cell lymphoma with almost complete infiltration of the brain and lungs was diagnosed. Conclusion: IVL should be considered early in situations of unexplained neuropsychiatric disease along with markedly elevated levels of lactic dehydrogenase, anemia, and hemolysis

The XIIIth Banff Conference on Allograft Pathology: The Banff 2015 Heart Meeting Report: Improving Antibody-Mediated Rejection Diagnostics: Strengths, Unmet Needs, and Future Directions.

The 13th Banff Conference on Allograft Pathology was held in Vancouver, British Columbia, Canada from October 5 to 10, 2015. The cardiac session was devoted to current diagnostic issues in heart transplantation with a focus on antibody-mediated rejection (AMR) and small vessel arteriopathy. Specific topics included the strengths and limitations of the current rejection grading system, the central role of microvascular injury in AMR and approaches to semiquantitative assessment of histopathologic and immunophenotypic indicators, the role of AMR in the development of cardiac allograft vasculopathy, the important role of serologic antibody detection in the management of transplant recipients, and the potential application of new molecular approaches to the elucidation of the pathophysiology of AMR and potential for improving the current diagnostic system. Herein we summarize the key points from the presentations, the comprehensive, open and wide-ranging multidisciplinary discussion that was generated, and considerations for future endeavors

Automation Process for Morphometric Analysis of Volumetric CT Data from Pulmonary Vasculature in Rats

With advances in medical imaging scanners, it has become commonplace to generate large multidimensional datasets. These datasets require tools for a rapid, thorough analysis. To address this need, we have developed an automated algorithm for morphometric analysis incorporating A Visualization Workshop computational and image processing libraries for three-dimensional segmentation, vascular tree generation and structural hierarchical ordering with a two-stage numeric optimization procedure for estimating vessel diameters. We combine this new technique with our mathematical models of pulmonary vascular morphology to quantify structural and functional attributes of lung arterial trees. Our physiological studies require repeated measurements of vascular structure to determine differences in vessel biomechanical properties between animal models of pulmonary disease. Automation provides many advantages including significantly improved speed and minimized operator interaction and biasing. The results are validated by comparison with previously published rat pulmonary arterial micro-CT data analysis techniques, in which vessels were manually mapped and measured using intense operator intervention

Shape-driven segmentation of the arterial wall in intravascular ultrasound images

Segmentation of arterial wall boundaries from intravascular images is an important problem for many applications in the study of plaque characteristics, mechanical properties of the arterial wall, its 3D reconstruction, and its measurements such as lumen size, lumen radius, and wall radius. We present a shape-driven approach to segmentation of the arterial wall from intravascular ultrasound images in the rectangular domain. In a properly built shape space using training data, we constrain the lumen and media-adventitia contours to a smooth, closed geometry, which increases the segmentation quality without any tradeoff with a regularizer term. In addition to a shape prior, we utilize an intensity prior through a non-parametric probability density based image energy, with global image measurements rather than pointwise measurements used in previous methods. Furthermore, a detection step is included to address the challenges introduced to the segmentation process by side branches and calcifications. All these features greatly enhance our segmentation method. The tests of our algorithm on a large dataset demonstrate the effectiveness of our approach

Routine Changing of Intravenous Administration Sets Does Not Reduce Colonization or Infection in Central Venous Catheters

Objective: To determine the effect of routine intravascular administration-set changes on central venous catheter (CVC) colonization and catheter related bacteremia (CRB). Design: Prospective, randomised controlled trial Setting: 18-bed ICU in a University-affiliated, tertiary referral hospital. Participants: 404 chlorhexidine and silver sulfadiazine coated multi-lumen CVCs from 251 intensive care unit (ICU) patients. Interventions: After ethical approval, CVCs inserted in ICU and in situ on Day 4 were randomised to have their administration-sets changed on Day 4 (n = 203) or not at all (n = 201). Fluid container and blood product administration-set use was limited to 24 hours. CVCs were removed (Day 7, not required or suspected infection), and cultured for colonization ( 15 cfu). Medical and laboratory staff were blinded. CRB was diagnosed by a blinded intensivist using strict definitions. Data was collected on; catheter life, CVC site, APACHE II score, patient age, diagnosis, hyperglycemia, hypoalbuminemia, immune status, number of fluid containers and intravenous injections, propofol, blood, TPN or lipid infusion. Results: There were 10 colonized CVCs in the set change group and 19 in the no change group. This was not a statistically significant difference on Kaplan Meier survival analysis (Effect Size = 0.09, Log Rank = 0.87, df = 1, p = 0.35). There were 3 cases of CRB per group. Logistic regression found that burns diagnosis and increased ICU stay were the only factors that significantly predicted colonization (p < 0.001). Conclusions: Intravenous administration-sets can be used for 7-days. Routine administration-set changes are unnecessary before this time