Effective intravitreal gene delivery to retinal pigment epithelium with hyaluronic acid nanospheres.

Inherited retinal degeneration (IRD) can cause a wide range of different forms of vision loss and blindness, and in spite of extensive advancements in gene therapy research, therapeutic approaches for targeting IRDs are still lacking. We have recently developed an approach for the intravitreal co-delivery of hyaluronic-acid nanospheres (HA-NSs) with sulfotyrosine (ST), effectively reaching the outer retina from the vitreal cavity. Here, our goal was to understand whether DNA-filled HA-NSs could generate gene expression in the outer retina. TxRed-labeled HA-NSs were compacted with plasmid DNA carrying a GFP reporter gene and intravitreally injected into the mouse retina. Follow-up at 4 weeks showed widespread gene expression in the outer retina and reduced, albeit present, expression at 8 weeks post-injection. Further analysis revealed this expression to be largely localized to the retinal pigment epithelium (RPE). These data show that intravitreal delivery of HA-NSs is a promising non-viral platform for the delivery of therapeutic genes and can generate pan-tissue, persistent gene expression in the RPE.

Downregulation of rhodopsin is an effective therapeutic strategy in ameliorating peripherin-2-associated inherited retinal disorders.

Given the absence of approved treatments for pathogenic variants in Peripherin-2 (PRPH2), it is imperative to identify a universally effective therapeutic target for PRPH2 pathogenic variants. To test the hypothesis that formation of the elongated discs in presence of PRPH2 pathogenic variants is due to the presence of the full complement of rhodopsin in absence of the required amounts of functional PRPH2. Here we demonstrate the therapeutic potential of reducing rhodopsin levels in ameliorating disease phenotype in knockin models for p.Lys154del (c.458-460del) and p.Tyr141Cys (c.422 A > G) in PRPH2. Reducing rhodopsin levels improves physiological function, mitigates the severity of disc abnormalities, and decreases retinal gliosis. Additionally, intravitreal injections of a rhodopsin-specific antisense oligonucleotide successfully enhance the physiological function of photoreceptors and improves the ultrastructure of discs in mutant mice. Presented findings shows that reducing rhodopsin levels is an effective therapeutic strategy for the treatment of inherited retinal degeneration associated with PRPH2 pathogenic variants.

Reduction of Outdoor and Indoor PM2.5 Source Contributions via Portable Air Filtration Systems in a Senior Residential Facility in Detroit, Michigan.

Background: The Reducing Air Pollution in Detroit Intervention Study (RAPIDS) was designed to evaluate cardiovascular health benefits and personal fine particulate matter (particulate matter < 2.5 µm in diameter, PM2.5) exposure reductions via portable air filtration units (PAFs) among older adults in Detroit, Michigan. This double-blind randomized crossover intervention study has shown that, compared to sham, air filtration for 3 days decreased 3-day average brachial systolic blood pressure by 3.2 mmHg. The results also showed that commercially available HEPA-type and true HEPA PAFs mitigated median indoor PM2.5 concentrations by 58% and 65%, respectively. However, to our knowledge, no health intervention study in which a significant positive health effect was observed has also evaluated how outdoor and indoor PM2.5 sources impacted the subjects. With that in mind, detailed characterization of outdoor and indoor PM2.5 samples collected during this study and a source apportionment analysis of those samples using a positive matrix factorization model were completed. The aims of this most recent work were to characterize the indoor and outdoor sources of the PM2.5 this community was exposed to and to assess how effectively commercially available HEPA-type and true HEPA PAFs were able to reduce indoor and outdoor PM2.5 source contributions. Methods: Approximately 24 h daily indoor and outdoor PM2.5 samples were collected on Teflon and Quartz filters from the apartments of 40 study subjects during each 3-day intervention period. These filters were analyzed for mass, carbon, and trace elements. Environmental Protection Agency Positive Matrix Factorization (PMF) 5.0 was utilized to determine major emission sources that contributed to the outdoor and indoor PM2.5 levels during this study. Results: The major sources of outdoor PM2.5 were secondary aerosols (28%), traffic/urban dust (24%), iron/steel industries (15%), sewage/municipal incineration (10%), and oil combustion/refinery (6%). The major sources of indoor PM2.5 were organic compounds (45%), traffic + sewage/municipal incineration (14%), secondary aerosols (13%), smoking (7%), and urban dust (2%). Infiltration of outdoor PM2.5 for sham, HEPA-type, and true HEPA air filtration was 79 ± 24%, 61 ± 32%, and 51 ± 34%, respectively. Conclusions: The results from our study showed that intervention with PAFs was able to significantly decrease indoor PM2.5 derived from outdoor and indoor PM2.5 sources. The PAFs were also able to significantly reduce the infiltration of outdoor PM2.5. The results of this study provide insights into what types of major PM2.5 sources this community is exposed to and what degree of air quality and systolic blood pressure improvements are possible through the use of commercially available PAFs in a real-world setting.

Expression of the human usherin c.2299delG mutation leads to early-onset auditory loss and stereocilia disorganization.

Usher syndrome (USH) is the leading cause of combined deafness and blindness, with USH2A being the most prevalent form. The mechanisms responsible for this debilitating sensory impairment remain unclear. This study focuses on characterizing the auditory phenotype in a mouse model expressing the c.2290delG mutation in usherin equivalent to human frameshift mutation c.2299delG. Previously we described how this model reproduces patient's retinal phenotypes. Here, we present the cochlear phenotype, showing that the mutant usherin, is expressed during early postnatal stages. The c.2290delG mutation results in a truncated protein that is mislocalized within the cell body of the hair cells. The knock-in model also exhibits congenital hearing loss that remains consistent throughout the animal's lifespan. Structurally, the stereocilia bundles, particularly in regions associated with functional hearing loss, are disorganized. Our findings shed light on the role of usherin in maintaining structural support, specifically in longer inner hair cell stereocilia, during development, which is crucial for proper bundle organization and hair cell function. Overall, we present a genetic mouse model with cochlear defects associated with the c.2290delG mutation, providing insights into the etiology of hearing loss and offering potential avenues for the development of effective therapeutic treatments for USH2A patients.

Comparative study of PRPH2 D2 loop mutants reveals divergent disease mechanism in rods and cones.

Mutations in the photoreceptor-specific tetraspanin gene peripherin-2 (PRPH2) lead to widely varying forms of retinal degeneration ranging from retinitis pigmentosa to macular dystrophy. Both inter- and intra-familial phenotypic heterogeneity has led to much interest in uncovering the complex pathogenic mechanisms of PRPH2-associated disease. Majority of disease-causing mutations in PRPH2 reside in the second intradiscal loop, wherein seven cysteines control protein folding and oligomerization. Here, we utilize knockin models to evaluate the role of three D2 loop cysteine mutants (Y141C, C213Y and C150S), alone or in combination. We elucidated how these mutations affect PRPH2 properties, including oligomerization and subcellular localization, and contribute to disease processes. Results from our structural, functional and molecular studies revealed that, in contrast to our understanding from prior investigations, rods are highly affected by PRPH2 mutations interfering with oligomerization and not merely by the haploinsufficiency associated with these mutations. On the other hand, cones are less affected by the toxicity of the mutant protein and significantly reduced protein levels, suggesting that knockdown therapeutic strategies may sustain cone functionality for a longer period. This observation provides useful data to guide and simplify the current development of effective therapeutic approaches for PRPH2-associated diseases that combine knockdown with high levels of gene supplementation needed to generate prolonged rod improvement.

The usherin mutation c.2299delG leads to its mislocalization and disrupts interactions with whirlin and VLGR1.

Usher syndrome (USH) is the leading cause of combined deafness-blindness with type 2 A (USH2A) being the most common form. Knockout models of USH proteins, like the Ush2a-/- model that develops a late-onset retinal phenotype, failed to mimic the retinal phenotype observed in patients. Since patient's mutations result in the expression of a mutant protein and to determine the mechanism of USH2A, we generated and evaluated an usherin (USH2A) knock-in mouse expressing the common human disease-mutation, c.2299delG. This mouse exhibits retinal degeneration and expresses a truncated, glycosylated protein which is mislocalized to the photoreceptor inner segment. The degeneration is associated with a decline in retinal function, structural abnormalities in connecting cilium and outer segment and mislocaliztion of the usherin interactors very long G-protein receptor 1 and whirlin. The onset of symptoms is significantly earlier compared to Ush2a-/-, proving expression of mutated protein is required to recapitulate the patients' retinal phenotype.

Riboflavin deficiency leads to irreversible cellular changes in the RPE and disrupts retinal function through alterations in cellular metabolic homeostasis.

Ariboflavinosis is a pathological condition occurring as a result of riboflavin deficiency. This condition is treatable if detected early enough, but it lacks timely diagnosis. Critical symptoms of ariboflavinosis include neurological and visual manifestations, yet the effects of flavin deficiency on the retina are not well investigated. Here, using a diet induced mouse model of riboflavin deficiency, we provide the first evidence of how retinal function and metabolism are closely intertwined with riboflavin homeostasis. We find that diet induced riboflavin deficiency causes severe decreases in retinal function accompanied by structural changes in the neural retina and retinal pigment epithelium (RPE). This is preceded by increased signs of cellular oxidative stress and metabolic disorder, in particular dysregulation in lipid metabolism, which is essential for both photoreceptors and the RPE. Though many of these deleterious phenotypes can be ameliorated by riboflavin supplementation, our data suggests that some patients may continue to suffer from multiple pathologies at later ages. These studies provide an essential cellular and mechanistic foundation linking defects in cellular flavin levels with the manifestation of functional deficiencies in the visual system and paves the way for a more in-depth understanding of the cellular consequences of ariboflavinosis.

Co-Injection of Sulfotyrosine Facilitates Retinal Uptake of Hyaluronic Acid Nanospheres Following Intravitreal Injection.

Gene and drug delivery to the retina is a critical therapeutic goal. While the majority of inherited forms of retinal degeneration affect the outer retina, specifically the photoreceptors and retinal pigment epithelium, effective targeted delivery to this region requires invasive subretinal delivery. Our goal in this work was to evaluate two innovative approaches for increasing both the persistence of delivered nanospheres and their penetration into the outer retina while using the much less invasive intravitreal delivery method. We formulated novel hyaluronic acid nanospheres (HA-NS, 250 nm and 500 nm in diameter) conjugated to fluorescent reporters and delivered them intravitreally to the adult Balb/C mouse retina. They exhibited persistence in the vitreous and along the inner limiting membrane (ILM) for up to 30 days (longest timepoint examined) but little retinal penetration. We thus evaluated the ability of the small molecule, sulfotyrosine, to disrupt the ILM, and found that 3.2 µg/µL sulfotyrosine led to significant improvement in delivery to the outer retina following intravitreal injections without causing retinal inflammation, degeneration, or loss of function. Co-delivery of sulfotyrosine and HA-NS led to robust improvements in penetration of HA-NS into the retina and accumulation along the interface between the photoreceptors and the retinal pigment epithelium. These exciting findings suggest that sulfotyrosine and HA-NS may be an effective strategy for outer retinal targeting after intravitreal injection.

Gene Therapy to the Retina and the Cochlea.

Vision and hearing disorders comprise the most common sensory disorders found in people. Many forms of vision and hearing loss are inherited and current treatments only provide patients with temporary or partial relief. As a result, developing genetic therapies for any of the several hundred known causative genes underlying inherited retinal and cochlear disorders has been of great interest. Recent exciting advances in gene therapy have shown promise for the clinical treatment of inherited retinal diseases, and while clinical gene therapies for cochlear disease are not yet available, research in the last several years has resulted in significant advancement in preclinical development for gene delivery to the cochlea. Furthermore, the development of somatic targeted genome editing using CRISPR/Cas9 has brought new possibilities for the treatment of dominant or gain-of-function disease. Here we discuss the current state of gene therapy for inherited diseases of the retina and cochlea with an eye toward areas that still need additional development.

Upper-Extremity Perceptual-Motor Training Improves Whole-Body Reactive Agility Among Elite Athletes With History of Sport-Related Concussion.

CONTEXT: Sport-related concussion (SRC) elevates risk for subsequent injury, which may relate to impaired perceptual-motor processes that are potentially modifiable. OBJECTIVE: To assess a possible upper-extremity (UE) training effect on whole-body (WB) reactive agility performance among elite athletes with history of SRC (HxSRC) and without such history of SRC. DESIGN: Cohort study. SETTING: Residential training center. PARTICIPANTS: Elite athletes (12 males and 8 females), including 10 HxSRC and 10 without such history of SRC. INTERVENTION: One-minute training sessions completed 2 to 3 times per week over a 3-week period involved verbal identification of center arrow direction for 10 incongruent and 10 congruent flanker test trials with simultaneous reaching responses to deactivate illuminated buttons. MAIN OUTCOME MEASURES: Pretraining and posttraining assessments of UE and WB reactive responses included flanker test conflict effect (incongruent minus congruent reaction time) and WB lateral average asymmetry derived from reaction time, speed, acceleration, and deceleration in opposite directions. Discrimination was assessed by receiver operating characteristic analysis, and training effect was assessed by repeated-measures analysis of variance. RESULTS: Pretraining discrimination between HxSRC and without such history of SRC was greatest for conflict effect ≥80 milliseconds and WB lateral average asymmetry ≥18%. Each athlete completed 6 training sessions, which improved UE mean reaction time from 767 to 646 milliseconds (P < .001) and reduced mean conflict effect from 96 to 53 milliseconds (P = .039). A significant group × trial interaction was evident for WB lateral average asymmetry (P = .004), which was reduced from 24.3% to 12.5% among those with HxSRC. CONCLUSIONS: Suboptimal perceptual-motor performance may represent a subtle long-term effect of concussion that is modifiable through UE training, which appears to improve WB reactive capabilities.

Concussion History and Neuromechanical Responsiveness Asymmetry.

CONTEXT: Detection of subtle changes in brain sensorimotor processes may enable clinicians to identify athletes who would derive the greatest benefit from interventions designed to reduce the risk for future injury and progressive neurologic or musculoskeletal dysfunction. OBJECTIVE: To develop a generalizable statistical model for identifying athletes who possess subtle alterations in sensorimotor processes that may be due to previous concussion. DESIGN: Cross-sectional study. SETTING: Residential Olympic Training Center sports medicine clinic. PATIENTS OR OTHER PARTICIPANTS: A primary cohort of 35 elite athletes and a secondary cohort of 40 elite athletes who performed identical tests the preceding year. INTERVENTION(S): Two upper extremity tests of visual-motor reaction time and 2 tests of whole-body reactive agility were administered. The whole-body tests required lateral or diagonal responses to virtual-reality targets, which provided measures of reaction time, speed, acceleration, and deceleration. MAIN OUTCOME MEASURE(S): Sport-related concussion history, which was reported by 54% (n = 19) of the athletes in the primary cohort and 45% (n = 18) of the athletes in the secondary cohort. RESULTS: Univariable analyses identified 12 strong predictors of sport-related concussion history, which we combined to create a composite metric with maximum predictive value. Composite lateral asymmetry for whole-body reactive movements and persisting effects of previous musculoskeletal injury yielded a logistic regression model with exceptionally good discrimination (area under the curve = 0.845) and calibration (predicted-observed probabilities within 7 subgroups: r = 0.959, P = .001). Application of the derived model to compatible data acquired from another cohort of elite athletes demonstrated very good discrimination (area under the curve = 0.772) and calibration (within 8 subgroups: r = 0.849, P = .008). CONCLUSIONS: Asymmetry in whole-body reactive movement capabilities may be a manifestation of a subtle abnormality in the functional connectivity of brain networks that might be relevant to previously reported associations between sport-related concussion history and musculoskeletal injury occurrence.

Reduction of personal PM2.5 exposure via indoor air filtration systems in Detroit: an intervention study.

The adverse health effects of fine particulate matter (PM < 2.5 µm in diameter [PM2.5]) air pollution are well-documented. There is a growing body of evidence that high-efficiency particulate arrestance (HEPA) filtration can reduce indoor PM2.5 concentrations and deliver some health benefits via the reduction of exposure to PM. However, few studies have tested the ability of portable air filtration systems to lower overall personal-level PM2.5 exposures. The Reducing Air Pollution in Detroit Intervention Study (RAPIDS) was designed to evaluate cardiovascular health benefits and personal PM2.5 exposure reductions via indoor portable air filtration systems among senior citizens in Detroit, Michigan. We evaluated the utility of two commercially available high-efficiency (HE: true-HEPA) and low-efficiency (LE: HEPA-type) indoor air filtration to reduce indoor PM2.5 concentrations and personal PM2.5 exposures for 40 participants in a double-blinded randomized crossover intervention. Each participant was subjected to three intervention scenarios: HE, LE, or no filter (control) of three consecutive days each, during which personal, indoor, and outdoor PM2.5 concentrations were measured daily. For mean indoor PM2.5 concentrations, we observed 60 and 52% reductions using HE and LE filters, respectively, relative to no filtration. Personal PM2.5 exposures were reduced by 53 and 31% using HE and LE filters, respectively, when compared with the control scenario. To our knowledge, this is the first indoor air filtration intervention study to examine the effectiveness of both HE and LE filters in reducing personal PM2.5 exposures.

Granulomatosis with polyangiitis presenting as facial nerve palsy in a teenager.

Granulomatosis with polyangiitis (GPA, previously known as Wegener's granulomatosis) is an autoimmune systemic small-vessel vasculitis, associated with the presence of anti-neurophil cytoplasmic antibodies with a cytoplasmic staining pattern (c-ANCA). It is characterized by necrotizing granulomas, usually affecting the airways and kidneys. GPA should be considered when patients do not improve despite adequate treatment of otologic symptoms, when patients have unspecific symptoms suggesting systemic disease (e.g. fever, malaise), or when other organs are involved (kidney, lungs, etc.). We present an interesting case of a 14-year-old female with eight-weeks of bilateral otalgia, unilateral facial nerve palsy, decreased appetite, and fatigue refractory to steroid, anti-viral, and antibiotic treatment ultimately diagnosed with GPA.

Middle Ear Obliteration with Blind-Sac Closure of the External Auditory Canal for Spontaneous CSF Otorrhea.

Outcome Objectives To (1) identify unique features of patients who underwent middle ear/mastoid obliteration with blind-sac closure of the external auditory canal for spontaneous cerebrospinal fluid (CSF) otorrhea and (2) explore outcomes. Study Design Case series with chart review. Setting Tertiary care center. Subjects and Methods Adults treated for spontaneous cerebrospinal fluid otorrhea from 2007 through 2015 were reviewed and stratified into 2 groups based on the surgery performed: (1) 11 patients underwent middle ear/mastoid obliteration with blind-sac closure of the external auditory canal and (2) 26 patients underwent other procedures. Demographics, body mass index, revised cardiac risk index, Duke Activity Status Index scores, and anticoagulation use were documented. Audiologic data were gathered from pre- and postoperative visits. The primary outcome measure was leak recurrence. Complications were tabulated. Results Poor preoperative hearing was a relative indication for obliteration. Obliteration patients had higher body mass index (43.2 vs 34.9 kg/m2; P < .05), incidence of super-morbid obesity (45% vs 7.6%; P = .015), anticoagulation usage (36% vs 0%; P = .004), cardiac risk scores (1.2 vs 0.1 dB; P < .0004), and Duke Activity Status Index scores. There was 1 leak recurrence (9%). Major and minor complication rates were 9% and 36%, respectively. Mean follow-up was 30.8 ± 8.6 months. Conclusion Middle ear and mastoid obliteration with blind-sac closure of the external auditory canal is effective for treating spontaneous CSF otorrhea. The small cohort reviewed did not experience any major perioperative morbidity. The technique may be best suited for patients with poor hearing, the infirm, and those in whom craniotomy is contraindicated.

Analysis of Audiometric Outcomes following Combined Middle Cranial Fossa/Transmastoid Approaches for Spontaneous Cerebrospinal Fluid Otorrhea.

Outcome Objectives To (1) explore audiometric outcomes following use of a combined transmastoid/middle cranial fossa (TM-MCF) approach in the treatment of spontaneous cerebrospinal fluid (CSF) otorrhea and (2) determine the influence of dehiscence location and reconstructive methodology on audiometric outcomes. Study Design Case series with chart review. Setting Tertiary care center. Subjects and Methods Adults with spontaneous CSF otorrhea were reviewed from 2007 to 2016 if they underwent combined TM-MCF. Definitive audiometric evaluations were performed at least 3 months postoperatively. The primary outcomes measures were pre- to postoperative changes in pure-tone average (PTA) and air-bone gap (ABG). Two subset analyses were performed for audiometric outcomes comparisons: first, patients with skull base dehiscences anterior to the ossicular chain were compared with those with posterior dehiscences. Second, patients undergoing skull base resurfacing plus abdominal fat graft mastoid obliteration (AFGMO) were compared with those undergoing resurfacing alone. Results A total of 28 patients and 31 ears were reviewed. There was 1 recurrent leak (3.5%). The cohort demonstrated significant improvement in mean postoperative ABG ( P = .008) but not PTA. On subset analysis, ears with posterior dehiscences demonstrated significant improvements in PTA ( P = .03) and ABG ( P = .05), while ears with anterior dehiscences did not. In addition, ears undergoing resurfacing plus AFGMO achieved significant improvements on all parameters ( P = .01). Only 3 of 15 ears undergoing resurfacing plus AFGMO experienced worsened postoperative hearing. Conclusion Use of the combined TM-MCF approach for treating spontaneous CSF otorrhea achieved good audiometric outcomes. Patients with skull base dehiscences posterior to the ossicles and those undergoing skull base resurfacing plus AFGMO achieved the most favorable results.

Pleomorphic rhabdomyosarcoma presenting as a hypopharyngeal mass.

BACKGROUND: Rhabdomyosarcoma (RMS) is a rare malignancy derived from skeletal muscle with approximately 40% of cases involving the head and neck. The pleomorphic variant, however, most commonly occurs in the extremities and has never, to our knowledge, been described in the pharynx. METHODS: A 46-year-old man with no significant medical history presented to the emergency department complaining of hemoptysis. A CT scan of the head and neck revealed a hypopharyngeal mass originally favored to be a benign process. RESULTS: Operative endoscopy revealed a previously unseen mucosal ulceration, and subsequent biopsy resulted in the final diagnosis of pleomorphic RMS. CONCLUSION: Although quite rare, RMS should be considered in the differential diagnosis of a hypopharyngeal mass.

Steroids for treatment of sudden sensorineural hearing loss: a meta-analysis of randomized controlled trials.

OBJECTIVE: To systematically review the available evidence regarding steroid treatment for sudden sensorineural hearing loss (SSHL) and to update prior analyses when possible. DATA SOURCES: OVID Medline. REVIEW METHODS: An electronic database search (OVID Medline) was performed with the objective of identifying all randomized controlled trials examining the use of steroids for the treatment of SSHL. The search was limited to English-language publications between January 1980 and June 2013. Reference lists were examined for additional articles. RESULTS: A total of 15 articles including 1,166 subjects were included in three separate analyses. Three articles (181 subjects) were included in the steroid versus placebo analysis, which resulted in an odds ratio of 1.52 (95% confidence interval [CI]: 0.83-2.77). Six articles (702 subjects) were included in the systemic versus intratympanic steroids analysis, which resulted in an odds ratio of 1.14 (95% CI: 0.82-1.59). Six articles (283 subjects) were included in the salvage treatment analysis, which resulted in an odds ratio of 6.04 (95% CI: 3.26-11.2). CONCLUSIONS: A meta-analysis of randomized controlled trials does not support the use of steroids over placebo in this condition, a finding consistent with previous analyses. Although systemic or intratympanic steroid administration does not have a significant treatment effect, steroids for salvage treatment of patients failing traditional therapy appear to have an effect. However, this result should be interpreted with caution given the poor quality of component trials. Implications for clinical practice and future trial design are discussed.