Magnetic field-guided cell delivery with nanoparticle-loaded human corneal endothelial cells.

To improve the delivery and integration of cell therapy using magnetic cell guidance for replacement of corneal endothelium, here we assess magnetic nanoparticles' (MNPs') effects on human corneal endothelial cells (HCECs) in vitro. Biocompatible, 50 nm superparamagnetic nanoparticles endocytosed by cultured HCECs induced no short- or long-term change in viability or identity. Assessment of guidance of the magnetic HCECs in the presence of different magnet shapes and field strengths showed a 2.4-fold increase in delivered cell density compared to gravity alone. After cell delivery, HCECs formed a functional monolayer, with no difference in tight junction formation between MNP-loaded and control HCECs. These data suggest that nanoparticle-mediated magnetic cell delivery may increase the efficiency of cell delivery without compromising HCEC survival, identity or function. Future studies may assess the safety and efficacy of this therapeutic modality in vivo. From the clinical editor: The authors show in this article that magnetic force facilitates the delivery of human corneal endothelial cells loaded by superparamagnetic nanoparticles to cornea, without changing their morphology, identity or functional properties. This novel idea can potentially have vast impact in the treatment of corneal endothelial dystrophies by providing self-endothelial cells after ex-vivo expansion.

Improving knowledge and decreasing depressive symptoms in caregivers of children with asthma through the asthma academy: A randomized controlled trial.

Background: Caregivers of children with asthma demonstrate higher levels of anxious and depressive symptoms when compared to caregivers of healthy children. Objectives: The objectives of this study were to: 1) Evaluate feasibility and acceptability of two nurse-led, remotely offered interventions for caregivers of children with asthma; 2) Compare effectiveness of two interventions (a virtual education session and a virtual education session supplemented with a telehealth visit) in relation to caregiver outcomes, and 3) Assess the preliminary effect of the interventions on caregivers' knowledge of asthma, sleep, anxiety and depressive symptoms. Methods: A mixed methods approach was used inclusive of a qualitative, descriptive design and randomized controlled trial design. Caregivers were provided virtual education and telehealth visits and evaluated from pre-posttest. Results: The intervention was found to be feasible and acceptable. Both the virtual education session and telehealth visit were effective. The intervention had a significant effect on caregiver's asthma knowledge and depressive symptoms (p<.05), but did not affect caregiver's sleep or anxiety. Qualitative analysis of the virtual educational session revealed themes of 1) valuable learning experience, 2) more medication education needed, and 3) appreciated remote format. Qualitative analysis of the telehealth visits revealed themes of 1) educational, helpful, and worthwhile and 2) virtual offering was easy and convenient. Conclusions: Remotely conducted, nurse-led interventions such as virtual education sessions and telehealth visits are a feasible, acceptable, and effective way to improve caregiver outcomes.

Gamma-synuclein as a marker of retinal ganglion cells.

PURPOSE: Previous studies have described gamma-synuclein as a protein highly expressed in retinal ganglion cells (RGCs), and a loss of RGCs correlates with a downregulation of gamma-synuclein gene expression in glaucoma. Here we asked whether gamma-synuclein expression in the retina can be considered a specific marker of RGCs. METHODS: gamma-Synuclein expression was examined with immunohistochemistry in retinal sections from normal and glaucomatous human eyes. Primary cultures of RGCs from Sprague-Dawley rats purified by sequential immunopanning using a monoclonal antibody to Thy1-1, cultures of A7 immortalized optic nerve astrocytes from newborn rats, and the immortalized RGC-5 cell line were studied using immunofluorescence and quantitative RT-PCR. RESULTS: gamma-Synuclein was highly expressed in RGCs in the human retina and was localized in cytoplasm adjacent to the RGC nuclear marker, Brn-3a. Axons of RGCs were immunopositive for gamma-synuclein in the nerve fiber layer (NFL), the lamina cribrosa and the retrobulbar optic nerve. In the optic nerve of glaucoma patients, axon swellings were likewise immunopositive, whereas in the retina of patients with retinoblastoma, NFL staining appeared reduced. In primary rat RGCs and in immortalized RGC-5 cultures, gamma-synuclein was localized predominantly in the perinuclear area and in cell processes. Among rat retinal cells in culture, all Brn-3a positive cells were stained with a gamma-synuclein antibody; rare gamma-synuclein-positive cells were not stained by the Brn-3a antibody. CONCLUSIONS: gamma-Synuclein is selectively and abundantly expressed in human RGCs in vivo, primary rat RGCs in vitro, and immortalized RGC-5 cells. In pathology, gamma-synuclein abundance may vary between RGC somas and axons. Coincident Brn-3a and gamma-synuclein expression suggests that strong gamma-synuclein expression can be considered a marker of RGCs. Future translational approaches might include using a gamma-synuclein promoter for the specific delivery of siRNA or therapeutic proteins to RGCs.

Novel Identity and Functional Markers for Human Corneal Endothelial Cells.

PURPOSE: Human corneal endothelial cell (HCEC) density decreases with age, surgical complications, or disease, leading to vision impairment. Such endothelial dysfunction is an indication for corneal transplantation, although there is a worldwide shortage of transplant-grade tissue. To overcome the current poor donor availability, here we isolate, expand, and characterize HCECs in vitro as a step toward cell therapy. METHODS: Human corneal endothelial cells were isolated from cadaveric corneas and expanded in vitro. Cell identity was evaluated based on morphology and immunocytochemistry, and gene expression analysis and flow cytometry were used to identify novel HCEC-specific markers. The functional ability of HCEC to form barriers was assessed by transendothelial electrical resistance (TEER) assays. RESULTS: Cultured HCECs demonstrated canonical morphology for up to four passages and later underwent endothelial-to-mesenchymal transition (EnMT). Quality of donor tissue influenced cell measures in culture including proliferation rate. Cultured HCECs expressed identity markers, and microarray analysis revealed novel endothelial-specific markers that were validated by flow cytometry. Finally, canonical HCECs expressed higher levels of CD56, which correlated with higher TEER than fibroblastic HCECs. CONCLUSIONS: In vitro expansion of HCECs from cadaveric donor corneas yields functional cells identifiable by morphology and a panel of novel markers. Markers described correlated with function in culture, suggesting a basis for cell therapy for corneal endothelial dysfunction.

Clinical and Electrophysiologic Characteristics of a Large Kindred with X-Linked Retinitis Pigmentosa Associated with the RPGR Locus.

PURPOSE: To phenotypically and genotypically characterize a large Puerto Rican kindred with X-linked retinitis pigmentosa associated with a novel RP GTPase regulator (RPGR) genotype. METHODS: A total of 100 family members of a single kindred with X-linked RP were evaluated with ophthalmic examinations and blood DNA analysis. Visual fields, OCT, and full-field ERG were obtained on all affected males and carriers. RESULTS: Of the 100 family members examined, 13 were affected males and 18 were carriers. A deletion of 2 base pair of the RPGR gene in the ORF15 region at position c.2267-2268 (Lys756del2aaAG hemi) was identified with the affected and carriers. Best eye visual acuity was correlated with age (Spearman coefficient = 0.95) with hand-motion acuity by age 35 and light perception to no light perception by age 50-60. Visual fields were minimally plottable by age 40, and ERG responses reached non-detectable levels by late teens. Carriers had no or mild visual symptoms. All carriers had visual acuity of at least 20/50 or better in one eye, and the amount of retinal degeneration was variable with ERG responses ranging from severely impaired to normal. CONCLUSIONS: Profound visual loss occurred by the second decade of life with progression to near no light perception by age 60 in this kindred of X-linked RP associated with the RPGR genotype. Female carriers maintained visual acuity with age and were identifiable by clinical and ERG examination. The information from this study is important to determine the optimal age for intervention, as new RP treatments are being developed and tested.