Biofortification of Chicken Eggs with Vitamin K—Nutritional and Quality Improvements

peer reviewedNational nutrition surveys have shown that over half of all adults in Ireland, the United Kingdom (UK), and the United States of America (USA) have low vitamin K intakes. Thus, dietary strategies to improve vitamin K intakes are needed, and vitamin K biofortification of food may be one food-based approach. The primary aim of our study was to establish whether increasing the vitamin K3 content of hen feed can increase the vitamin K content of eggs, and the secondary aims were to examine the effects on hen performance parameters, as well as egg and eggshell quality parameters. A 12 week hen feeding trial was conducted in which Hyline chickens were randomized into four treatment groups (n = 32/group) and fed diets containing vitamin K3 (as menadione nicotinamide bisulfite) at 3 (control), 12.9, 23.7, and 45.7 mg/kg feed. Vitamin K1, menaquinone (MK)-4, MK-7, and MK-9 were measured in raw whole eggs via a liquid chromatography tandem mass spectrometry method. MK-4 was the most abundant form of vitamin K (91–98%) found in all eggs. Increasing the vitamin K3 content of hen feed over the control level significantly (p < 0.001) enhanced the MK-4 content of eggs (mean range: 46–51 µg/100 g, representing ~42–56% of US Adequate Intake values). Vitamin K biofortification also led to significant (p < 0.05) increases in the yellowness of egg yolk and in eggshell weight and thickness, but no other changes in egg quality or hen performance parameters. In conclusion, high-quality vitamin K-biofortified eggs can be produced with at least double the total vitamin K content compared to that in commercially available eggs

Soluble CD59 Expressed from an Adenovirus In Vivo Is a Potent Inhibitor of Complement Deposition on Murine Liver Vascular Endothelium

Inappropriate activation of complement on the vascular endothelium of specific organs, or systemically, underlies the etiology of a number of diseases. These disorders include atypical hemolytic uremic syndrome, membranoproliferative glomerulonephritis, atherosclerosis, age-related macular degeneration, diabetic retinopathy, and transplant rejection. Inhibition of the terminal step of complement activation, i.e. formation of the membrane attack complex, using CD59 has the advantage of retaining the upstream processes of the complement cascade necessary for fighting pathogens and retaining complement's crucial role in tissue homeostasis. Previous studies have shown the necessity of membrane targeting of soluble CD59 in order for it to prove an effective inhibitor of complement deposition both in vitro and in vivo. In this study we have generated an in vivo model of human complement activation on murine liver vascular endothelium. This model should prove useful for the development of anti-complement therapies for complement-induced pathologies of vascular endothelium. Using this model, we have demonstrated the viability of a non membrane-targeted soluble CD59 to significantly inhibit complement deposition on the endothelium of murine liver vasculature when expressed in vivo from an adenovirus. This result, unanticipated based on prior studies, suggests that the use of non membrane-targeted sCD59 as an anti-complement therapy be re-visited

A Non Membrane-Targeted Human Soluble CD59 Attenuates Choroidal Neovascularization in a Model of Age Related Macular Degeneration

Age related macular degeneration (AMD) is the most common cause of blindness amongst the elderly. Approximately 10% of AMD patients suffer from an advanced form of AMD characterized by choroidal neovascularization (CNV). Recent evidence implicates a significant role for complement in the pathogenesis of AMD. Activation of complement terminates in the incorporation of the membrane attack complex (MAC) in biological membranes and subsequent cell lysis. Elevated levels of MAC have been documented on choroidal blood vessels and retinal pigment epithelium (RPE) of AMD patients. CD59 is a naturally occurring membrane bound inhibitor of MAC formation. Previously we have shown that membrane bound human CD59 delivered to the RPE cells of mice via an adenovirus vector can protect those cells from human complement mediated lysis ex vivo. However, application of those observations to choroidal blood vessels are limited because protection from MAC- mediated lysis was restricted only to the cells originally transduced by the vector. Here we demonstrate that subretinal delivery of an adenovirus vector expressing a transgene for a soluble non-membrane binding form of human CD59 can attenuate the formation of laser-induced choroidal neovascularization and murine MAC formation in mice even when the region of vector delivery is distal to the site of laser induced CNV. Furthermore, this same recombinant transgene delivered to the intravitreal space of mice by an adeno-associated virus vector (AAV) can also attenuate laser-induced CNV. To our knowledge, this is the first demonstration of a non-membrane targeting CD59 having biological potency in any animal model of disease in vivo. We propose that the above approaches warrant further exploration as potential approaches for alleviating complement mediated damage to ocular tissues in AMD

Adeno-associated virus mediated delivery of a non-membrane targeted human soluble CD59 attenuates some aspects of diabetic retinopathy in mice.

Diabetic retinopathy is the leading cause of visual dysfunction in working adults and is attributed to retinal vascular and neural cell damage. Recent studies have described elevated levels of membrane attack complex (MAC) and reduced levels of membrane associated complement regulators including CD55 and CD59 in the retina of diabetic retinopathy patients as well as in animal models of this disease. We have previously described the development of a soluble membrane-independent form of CD59 (sCD59) that when delivered via a gene therapy approach using an adeno-associated virus vector (AAV2/8-sCD59) to the eyes of mice, can block MAC deposition and choroidal neovascularization. Here, we examine AAV2/8-sCD59 mediated attenuation of MAC deposition and ensuing complement mediated damage to the retina of mice following streptozotocin (STZ) induced diabetes. We observed a 60% reduction in leakage of retinal blood vessels in diabetic eyes pre-injected with AAV2/8-sCD59 relative to negative control virus injected diabetic eyes. AAV2/8-sCD59 injected eyes also exhibited protection from non-perfusion of retinal blood vessels. In addition, a 200% reduction in retinal ganglion cell apoptosis and a 40% reduction in MAC deposition were documented in diabetic eyes pre-injected with AAV2/8-sCD59 relative to diabetic eyes pre-injected with the control virus. This is the first study characterizing a viral gene therapy intervention that targets MAC in a model of diabetic retinopathy. Use of AAV2/8-sCD59 warrants further exploration as a potential therapy for advanced stages of diabetic retinopathy

Reducible PEG-POD/DNA Nanoparticles for Gene Transfer In Vitro and In Vivo: Application in a Mouse Model of Age-Related Macular Degeneration

Non-viral gene delivery systems are being developed to address limitations of viral gene delivery. Many of these non-viral systems are modeled on the properties of viruses including cell surface binding, endocytosis, endosomal escape, and nuclear targeting. Most non-viral gene transfer systems exhibit little correlation between in vitro and in vivo efficiency, hampering a systematic approach to their development. Previously, we have described a 3.5 kDa peptide (peptide for ocular delivery [POD]) that targets cell surface sialic acid. When functionalized with polyethylene glycol (PEG) via a sulfhydryl group on the N-terminal cysteine of POD, PEG-POD could compact plasmid DNA, forming 120- to 180-nm homogeneous nanoparticles. PEG-POD enabled modest gene transfer and rescue of retinal degeneration in vivo. Systematic investigation of different stages of gene transfer by PEG-POD nanoparticles was hampered by their inability to deliver genes in vitro. Herein, we describe functionalization of POD with PEG using a reducible orthopyridyl disulfide bond. These reducible nanoparticles enabled gene transfer in vitro while retaining their in vivo gene transfer properties. These reducible PEG-POD nanoparticles were utilized to deliver human FLT1 to the retina in vivo, achieving a 50% reduction in choroidal neovascularization in a murine model of age-related macular degeneration. Keywords: nanoparticles, age-related macular degeneration, PEGylation, cell penetrating peptide, gene transfer, reducible particles, laser-induced choroidal neovascularizatio

Adenovirus Vectors Targeting Distinct Cell Types in the Retina

Pseudotyping adenovirus serotype 5 vectors with Ad37 fiber enhances adenovirus tropism for Müller cells. Robust photoreceptor-specific transgene expression can be achieved in the context of adenovirus vectors with deletions in the RGD domain of penton base

Engineered Zinc Finger Nuclease–Mediated Homologous Recombination of the Human Rhodopsin Gene

Zinc finger nuclease–mediated homologous recombination is examined as a permanent genetic approach to treat retinitis pigmentosa

Aurintricarboxylic Acid Inhibits Complement Activation, Membrane Attack Complex, and Choroidal Neovascularization in a Model of Macular Degeneration

Citation: Lipo E, Cashman SM, KumarSingh R. Aurintricarboxylic acid inhibits complement activation, membrane attack complex, and choroidal neovascularization in a model of macular degeneration. Invest Ophthalmol Vis Sci. 2013;54:7107-7114. DOI:10.1167/iovs.13-12923 PURPOSE. Immunocytochemical and genetic data implicate a significant role for the activation of complement in the pathology of AMD. Individuals homozygous for a Y402H polymorphism in Factor H have elevated levels of membrane attack complex (MAC) in their choroidal blood vessels and RPE relative to individuals homozygous for the wild-type allele. An R95X polymorphism in C9, a protein necessary for the final assembly of MAC, is partially protective against the formation of choroidal neovascularization (CNV) in AMD patients. Aurintricarboxylic Acid (ATA) is a small molecule inhibitor of MAC. Our hypothesis was that attenuation of the formation of MAC on ocular tissues by ATA may protect mice against laser-induced CNV. METHODS. The ability of ATA to inhibit human complement-mediated cell lysis, inhibit formation of human MAC, and inhibit formation of tubes by endothelial cells was examined in vitro. Subsequently, the Bruch&apos;s membrane of adult mice was damaged using an argon laser, followed by intravitreal injection of ATA. One week later, choroidal flat mounts from these mice were stained for the presence of MAC, endothelial cells, and macrophages. RESULTS. ATA protects cells from human complement-mediated lysis, attenuates assembly of the MAC, and inhibits tube formation by endothelial cells in vitro. ATA also attenuates CNV, MAC deposition, and macrophage infiltration in a murine model of exudative AMD. CONCLUSIONS. ATA warrants further study as a potential drug for the treatment of exudative and nonexudative AMD. Keywords: macular degeneration, choroidal neovascularization, aurintricarboxylic acid, complement, membrane attack complex A ge-related macular degeneration (AMD) is the most common cause of blindness among the elderly, affecting approximately 1 in 3 people older than 65. 1-3 AMD begins with the appearance of lipoproteinaceous deposits between the RPE and Bruch&apos;s membrane. This stage of AMD is generally referred to as &apos;&apos;dry&apos;&apos; or nonexudative AMD that can progress toward a more advanced form known as geographic atrophy, involving degeneration and loss of RPE cells. Approximately 10% of moderate or advanced AMD patients progress toward &apos;&apos;wet&apos;&apos; or exudative AMD, involving the growth of new blood vessels from the choroid into the subretinal space, that is, choroidal neovascularization (CNV) (see Ref. 4 for review). The growth of these immature and leaky blood vessels results in macular edema and is associated with elevated levels of cytokines, such as VEGF. Inhibitors of VEGF are hence the current standard of care for wet AMD. 8-11 Activation of complement terminates with the formation of the membrane attack complex (MAC) on the surface of cells. 12 Low levels of MAC deposition are known to lead to cell mitogenesis and cytokine release and high levels of MAC are known to lead to cell lysis. 17 Geographic atrophy patients have reduced levels of CD59 (an inhibitor of MAC) on their RPE cell