Chronic hypotony and uveitis managed with pars plana vitrectomy, silicone oil tamponade, and permanent keratoprosthesis.

PURPOSE: We report a case of silicone oil placement after Boston Type 1 keratoprosthesis implantation for improvement and maintenance of intraocular pressure in a patient with chronic hypotony secondary to chronic uveitis. METHODS: Observational case report. RESULTS: A 54-year-old female with a history of bilateral chronic panuveitis and subsequent hypotony presented with progressive corneal decompensation and band keratopathy in her better-seeing left eye. Her CDVA in her left eye declined from count fingers at 3' to hand motion. In an effort to clear the visual axis and stabilize intraocular pressure, implantation of a Boston Type 1 Keratoprosthesis combined with pars plana vitrectomy and silicone oil injection was performed. Post operatively, the intraocular pressure improved to the mid-teens and vision improved to count fingers at 4'with a follow-up of 2 years. CONCLUSION: Pars plana vitrectomy and injection of silicone oil after placement of a Boston Type 1 keratoprosthesis can successfully improve and maintain intraocular pressure in eyes affected by chronic uveitis with ciliary body atrophy and chronic hypotony. This strategy effectively reverses such problems as hypotony maculopathy, disk swelling, scleral infolding, and corneal failure.

Peptide-Based Nanoparticles for Systemic Extrahepatic Delivery of Therapeutic Nucleotides.

Peptide-based nanoparticles (PBN) for nucleotide complexation and targeting of extrahepatic diseases are gaining recognition as potent pharmaceutical vehicles for fine-tuned control of protein production (up- and/or down-regulation) and for gene delivery. Herein, we review the principles and mechanisms underpinning self-assembled formation of PBN, cellular uptake, endosomal release, and delivery to extrahepatic disease sites after systemic administration. Selected examples of PBN that have demonstrated recent proof of concept in disease models in vivo are summarized to offer the reader a comparative view of the field and the possibilities for clinical application.

Traumatic Retinal Detachment in Patients with Self-Injurious Behavior: An International Multicenter Study.

PURPOSE: To describe the clinical characteristics, surgical outcomes, and management recommendations in patients with traumatic rhegmatogenous retinal detachment (RRD) resulting from self-injurious behavior (SIB). DESIGN: International, multicenter, retrospective, interventional case series. PARTICIPANTS: Patients with SIB from 23 centers with RRD in at least 1 eye. METHODS: Clinical histories, preoperative assessment, surgical details, postoperative management, behavioral intervention, and follow-up examination findings were reviewed. MAIN OUTCOME MEASURES: The rate of single-surgery anatomic success (SSAS) was the primary outcome. Other outcomes included new RRD in formerly attached eyes, final retinal reattachment, and final visual acuity. RESULTS: One hundred seven eyes with RRDs were included from 78 patients. Fifty-four percent of patients had bilateral RRD or phthisis bulbi in the fellow eye at final follow-up. The most common systemic diagnoses were autism spectrum disorder (35.9%) and trisomy 21 (21.8%) and the most common behavior was face hitting (74.4%). The average follow-up time was 3.3 ± 2.8 years, and surgical outcomes for operable eyes were restricted to patients with at least 3 months of follow-up (81 eyes). Primary initial surgeries were vitrectomy alone (33.3%), primary scleral buckle (SB; 26.9%), and vitrectomy with SB (39.7%), and 5 prophylactic SBs were placed. Twenty-three eyes (21.5%) with RRDs were inoperable. The SSAS was 23.1% without tamponade (37.2% if including silicone oil), and final reattachment was attained in 80% (36.3% without silicone oil tamponade). Funnel-configured RRD (P = 0.006) and the presence of grade C proliferative vitreoretinopathy (P = 0.002) correlated with re-detachment. The use of an SB predicted the final attachment rate during the initial surgery (P = 0.005) or at any surgery (P = 0.008. These associations held if restricting to 64 patients with ≥12 months followup. Anatomic reattachment correlated with better visual acuity (P < 0.001). CONCLUSIONS: RRD resulting from SIB poses therapeutic challenges because of limited patient cooperation, bilateral involvement, chronicity, and ongoing trauma in vulnerable and neglected patients. The surgical success rates were some of the lowest in the modern retinal detachment literature. The use of an SB may result in better outcomes, and visual function can be restored in some patients.

Pseudoflow with OCT Angiography in Eyes with Hard Exudates and Macular Drusen.

PURPOSE: To analyze "pseudoflow," a false positive flow-artifact observed with optical coherence tomography angiography (OCTA) of stationary hyperreflective structures corresponding to hard exudates and macular drusen. METHODS: Retrospective case series of patients with hard exudates (due to diabetic macular edema [DME] or retinal vein occlusion [RVO]) or macular drusen (due to nonneovascular, or dry, age-related macular degeneration [AMD]) studied with OCTA by using volume-based projection artifact removal (3D PAR). RESULTS: OCTA of 20 eyes (10 DME/10 RVO) with hard exudates were analyzed. All eyes exhibited pseudoflow corresponding to hard exudates. Seven eyes concurrently demonstrated hard exudates without pseudoflow that were noted in areas lacking vascular flow in the overlying retina. Eight eyes exhibited suspended scattering particles in motion. In 26 of 30 eyes with nonneovascular AMD, pseudoflow associated with macular drusen of any type was noted. Two of 11 eyes with small drusen, 16 of 17 eyes with medium or large drusen, 5 of 5 eyes with drusenoid pigment epithelial detachment, 12 of 16 eyes with ribbon-like subretinal drusenoid deposits, and 13 of 17 eyes with dot-like SDD exhibited pseudoflow. CONCLUSIONS: Pseudoflow due to projection artifact is common in eyes with hard exudates or macular drusen. 3D PAR reduces but does not eliminate pseudoflow, and pseudoflow may be detected within the foveal avascular zone, indicating that other factors, such as Z-axis micromotion, may also contribute to pseudoflow. TRANSLATIONAL RELEVANCE: This study provides insight into the etiology of pseudoflow noted on OCTA and will guide more accurate clinical interpretation and investigation of OCTA images.

Anti-JNK2 peptide-siRNA nanostructures improve plaque endothelium and reduce thrombotic risk in atherosclerotic mice.

BACKGROUND: A direct and independent role of inflammation in atherothrombosis was recently highlighted by the Canakinumab Antiinflammatory Thrombosis Outcome Study (CANTOS) trial, showing the benefit of inhibiting signaling molecules, eg, interleukins. Accordingly, we sought to devise a flexible platform for preventing the inflammatory drivers at their source to preserve plaque endothelium and mitigate procoagulant risk. METHODS: p5RHH-siRNA nanoparticles were formulated through self-assembly processes. The therapeutic efficacy of p5RHH-JNK2 siRNA nanoparticles was evaluated both in vitro and in vivo. RESULTS: Because JNK2 is critical to macrophage uptake of oxidized lipids through scavenger receptors that engender expression of myriad inflammatory molecules, we designed an RNA-silencing approach based on peptide-siRNA nanoparticles (p5RHH-siRNA) that localize to atherosclerotic plaques exhibiting disrupted endothelial barriers to achieve control of JNK2 expression by macrophages. After seven doses of p5RHH-JNK2 siRNA nanoparticles over 3.5 weeks in ApoE-/- mice on a Western diet, both JNK2 mRNA and protein levels were significantly decreased by 26% (P=0.044) and 42% (P=0.042), respectively. Plaque-macrophage populations were markedly depleted and NFκB and STAT3-signaling pathways inhibited by 47% (P<0.001) and 46% (P=0.004), respectively. Endothelial barrier integrity was restored (2.6-fold reduced permeability to circulating 200 nm nanoparticles in vivo, P=0.003) and thrombotic risk attenuated (200% increased clotting times to carotid artery injury, P=0.02), despite blood-cholesterol levels persistently exceeding 1,000 mg/dL. No adaptive or innate immunoresponses toward the nanoparticles were observed, and blood tests after the completion of treatment confirmed the largely nontoxic nature of this approach. CONCLUSION: The ability to formulate these nanostructures rapidly and easily interchange or multiplex their oligonucleotide content represents a promising approach for controlling deleterious signaling events locally in advanced atherosclerosis.

A role for peptides in overcoming endosomal entrapment in siRNA delivery - A focus on melittin.

siRNA has the possibility to revolutionize medicine by enabling highly specific and efficient silencing of proteins involved in disease pathogenesis. Despite nearly 20 years of research dedicated to translating siRNA from a research tool into a clinically relevant therapeutic, minimal success has been had to date. Access to RNA interference machinery located in the cytoplasm is often overlooked, but must be considered when designing the next generation of siRNA delivery strategies. Peptide transduction domains (PTDs) have demonstrated moderate siRNA transfection, which is primarily limited by endosomal entrapment. Strategies aimed at overcoming endosomal entrapment associated with peptide vectors are reviewed here, including osmotic methods, lipid conjugation, and fusogenic peptides. As an alternative to traditional PTD, the hemolytic peptide melittin exhibits the native capacity for endosomal disruption but causes cytotoxicity. However, appropriate packaging and protection of melittin with activation and release in the endosomal compartment has allowed melittin-based strategies to demonstrate both in vitro and in vivo safety and efficacy. These data suggest that melittin's membrane disruptive properties can enable safe and effective endosomolysis, building a case for melittin as a key component in a new generation of siRNA therapeutics.

Peptide-siRNA nanocomplexes targeting NF-κB subunit p65 suppress nascent experimental arthritis.

The NF-κB signaling pathway is implicated in various inflammatory diseases, including rheumatoid arthritis (RA); therefore, inhibition of this pathway has the potential to ameliorate an array of inflammatory diseases. Given that NF-κB signaling is critical for many immune cell functions, systemic blockade of this pathway may lead to detrimental side effects. siRNAs coupled with a safe and effective delivery nanoplatform may afford the specificity lacking in systemic administration of small-molecule inhibitors. Here we demonstrated that a melittin-derived cationic amphipathic peptide combined with siRNA targeting the p65 subunit of NF-κB (p5RHH-p65) noncovalently self-assemble into stable nanocomplexes that home to the inflamed joints in a murine model of RA. Specifically, administration of p5RHH-p65 siRNA nanocomplexes abrogated inflammatory cytokine expression and cellular influx into the joints, protected against bone erosions, and preserved cartilage integrity. The p5RHH-p65 siRNA nanocomplexes potently suppressed early inflammatory arthritis without affecting p65 expression in off-target organs or eliciting a humoral response after serial injections. These data suggest that this self-assembling, largely nontoxic platform may have broad utility for the specific delivery of siRNA to target and limit inflammatory processes for the treatment of a variety of diseases.

Mechanisms of nanoparticle-mediated siRNA transfection by melittin-derived peptides.

Traditional peptide-mediated siRNA transfection via peptide transduction domains exhibits limited cytoplasmic delivery of siRNA due to endosomal entrapment. This work overcomes these limitations with the use of membrane-destabilizing peptides derived from melittin for the knockdown of NFkB signaling in a model of adult T-cell leukemia/lymphoma. While the mechanism of siRNA delivery into the cytoplasmic compartment by peptide transduction domains has not been well studied, our analysis of melittin derivatives indicates that concurrent nanocomplex disassembly and peptide-mediated endosomolysis are crucial to siRNA transfection. Importantly, in the case of the most active derivative, p5RHH, this process is initiated by acidic pH, indicating that endosomal acidification after macropinocytosis can trigger siRNA release into the cytoplasm. These data provide general principles regarding nanocomplex response to endocytosis, which may guide the development of peptide/siRNA nanocomplex-based transfection.

RNA interference knockdown of DNA methyl-transferase 3 affects gene alternative splicing in the honey bee.

Studies of DNA methylation from fungi, plants, and animals indicate that gene body methylation is ancient and highly conserved in eukaryotic genomes, but its role has not been clearly defined. It has been postulated that regulation of alternative splicing of transcripts was an original function of DNA methylation, but a direct experimental test of the effect of methylation on alternative slicing at the whole genome level has never been performed. To do this, we developed a unique method to administer RNA interference (RNAi) in a high-throughput and noninvasive manner and then used it to knock down the expression of DNA methyl-transferase 3 (dnmt3), which is required for de novo DNA methylation. We chose the honey bee (Apis mellifera) for this test because it has recently emerged as an important model organism for studying the effects of DNA methylation on development and social behavior, and DNA methylation in honey bees is predominantly on gene bodies. Here we show that dnmt3 RNAi decreased global genomic methylation level as expected and in addition caused widespread and diverse changes in alternative splicing in fat tissue. Four different types of splicing events were affected by dnmt3 gene knockdown, and change in two types, exon skipping and intron retention, was directly related to decreased methylation. These results demonstrate that one function of gene body DNA methylation is to regulate alternative splicing.

Melittin derived peptides for nanoparticle based siRNA transfection.

Traditional transfection agents including cationic lipids and polymers have high efficiency but cause cytotoxicity. While cell penetrating peptide based transfection agents exhibit improved cytotoxicity profiles, they do not have the efficiency of existing lipidic agents due to endosomal trapping. As a consequence, we propose an alternative method to efficient peptide based siRNA transfection by starting with melittin, a known pore-forming peptide. By incorporating modifications to decrease cytotoxicity and improve siRNA binding, we have developed p5RHH, which can complex siRNA to form nanoparticles of 190 nm in diameter. p5RHH exhibits high efficiency with GFP knockdown at concentrations as low as 5 nM, with negligible cytotoxicity. To date, p5RHH has shown the ability to transfect B16 cells, Human Umbilical Vein Endothelial Cells, and RAW264.7 cells with high efficiency. These in vitro models demonstrate that p5RHH mediated transfection can block cancer cell proliferation, angiogenesis, and foam cell formation. Moreover, p5RHH/siRNA nanoparticles maintain their size and transfection efficiency in the presence of serum proteins suggesting the potential for use of p5RHH in vivo. These data suggest that our strategy for development of siRNA transfecting peptides can provide an avenue to safe and effective siRNA therapeutics.

Probing the surface-enhanced Raman scattering properties of Au-Ag nanocages at two different excitation wavelengths.

The surface-enhanced Raman scattering (SERS) properties of bimetallic Au-Ag nanocages has been thoroughly investigated by changing the nanocage composition, localized surface plasmon resonance (LSPR) peak position and excitation wavelength. We found a significant dependency on excitation wavelength for the Au-Ag nanocages, independent of the underlying LSPR, which can be extended to other bimetallic SERS substrates. While it is well-understood that plasmon damping can occur for Au nanoparticles when their LSPR peaks are close to interband transition frequencies and thereby attenuate SERS intensities, this study probes an additional aspect and shows that SERS intensities are reduced when the excitation light source is near interband transition frequencies regardless of the LSPR location.

The polarity protein Par1b/EMK/MARK2 regulates T cell receptor-induced microtubule-organizing center polarization.

Engagement of a T cell to an APC induces the formation of an immunological synapse as well as reorientation of the microtubule-organizing center (MTOC) toward the APC. How signals emanating from the TCR induce MTOC polarization is not known. One group of proteins known to play a critical role in asymmetric cell division and cell polarization is the partitioning defective (Par) family of proteins. In this study we found that Par1b, a member of the Par family of proteins, was inducibly phosphorylated following TCR stimulation. This phosphorylation resulted in 14-3-3 protein binding and caused the relocalization of Par1b from the membrane into the cytoplasm. Because a dominant-negative form of Par1b blocked TCR-induced MTOC polarization, our data suggest that Par1b functions in the establishment of T cell polarity following engagement to an APC.