The large GTPase dynamin 2 controls both endosomal fission and microtubule acetylation. Here we report that dynamin 2 alters microtubules and regulates the trafficking of human adenovirus type 37. Dynamin 2 knockdown by siRNA in infected cells resulted in accumulation of acetylated tubulin, repositioning of microtubule organizing centers (MTOCs) closer to cell nuclei, increased virus in the cytosol (with a compensatory decrease in endosomal virus), reduced proinflammatory cytokine induction, and increased binding of virus to the nucleoporin, Nup358. These events led to increased viral DNA nuclear entry and viral replication. Overexpression of dynamin 2 generated opposite effects. Therefore, dynamin 2 inhibits adenovirus replication and promotes innate immune responses by the infected cell. MTOC transposition in dynamin 2 knockdown promotes a closer association with nuclear pore complexes to facilitate viral DNA delivery. Dynamin 2 plays a key role in adenoviral trafficking and influences host responses to infection.

OBJECTIVE:  To evaluate to what extent indicators of placenta insufficiency are associated with low concentrations of insulin-like growth factor 1 (IGF-1) and IGF-1-binding protein-1 (IGFBP-1) in neonatal blood, and to what extent the concentrations of these growth factors are associated with concentrations of proteins with inflammatory, neurotrophic, or angiogenic properties. STUDY DESIGN:  Using multiplex immunoassays, we measured the concentrations of IGF-1 and IGFBP-1, as well as 25 other proteins in blood spots collected weekly from ≥ 880 infants born before the 28th week of gestation, and sought correlates of concentrations in the top and bottom quartiles for gestational age and day the specimen was collected. RESULTS:  Medically indicated delivery and severe fetal growth restriction (sFGR) were associated with low concentrations of IGF-1 on the first postnatal day and with high concentrations of IGFBP-1 on almost all days. Elevated concentrations of IGF-1 and IGFBP-1 were accompanied by elevated concentrations of many other proteins with inflammatory, neurotrophic, or angiogenic properties. CONCLUSION:  Disorders associated with impaired placenta implantation and sFGR appear to account for a relative paucity of IGF-1 on the first postnatal day. Elevated concentrations of IGF-1 and especially IGFBP-1 were associated with same-day elevated concentrations of inflammatory, neurotrophic, and angiogenic proteins.

PURPOSE: To evaluate the therapeutic effect of subconjunctival injection of human mesenchymal stromal cells (hMSCs) in the cornea of mice with graft versus host disease (GVHD). METHODS: GVHD was induced in mice after hematopoietic stem cell transplantation (HSCT) between MHC-mismatched mouse strains. Subconjunctival injection of hMSCs was applied at day 10 post-HSCT. Infiltration of CD3 cells in the cornea and epithelial alterations were analyzed by immunofluorescence. Tear was assessed using the PRT test and TearLab Osmolarity System. qPCR was used to evaluate changes in cytokines, Pax6 and Sprr1b expression. To evaluate the effect of irradiation, we analyzed the expression of these genes in TBI mice. RESULTS: Immune cell invasion occurs in mice with GVHD, as shown by the presence of CD3 cells in the cornea. Interestingly, eyes treated with hMSC did not present CD3 cells. Tear osmolarity was increased in GVHD eyes, but not in treated eyes. TNFa expression was highly increased in all corneas except in Control and treated eyes. Pax6 in corneal epithelium showed a similar pattern in GVHD and Control mice, and its gene expression was enhanced in GVHD corneas. In contrast, Pax6 was reduced in GVHD + MSC corneas. We also found an increase in SPRR1B staining in GVHD eyes that was lower in GVHD + MSC mice, demonstrating that corneal keratinization is less frequent after treatment with hMSC. CONCLUSIONS: The treatment with hMSCs by subconjunctival injection is effective in reducing corneal inflammation and squamous metaplasia in ocular GVHD (oGVHD). Local treatment with hMSCs is a promising strategy for oGVHD.

The adeno-associated virus (AAV) serves as a broadly used vector system for gene delivery. The process of AAV capsid assembly remains poorly understood. The viral cofactor assembly-activating protein (AAP) is required for maximum AAV production and has multiple roles in capsid assembly, namely, trafficking of the structural proteins (VP) to the nuclear site of assembly, promoting the stability of VP against multiple degradation pathways, and facilitating stable interactions between VP monomers. The N-terminal 60 amino acids of AAP (AAPN) are essential for these functions. Presumably, AAP must physically interact with VP to execute its multiple functions, but the molecular nature of the AAP-VP interaction is not well understood. Here, we query how structurally related AAVs functionally engage AAP from AAV serotype 2 (AAP2) toward virion assembly. These studies led to the identification of key residues on the lumenal capsid surface that are important for AAP-VP and for VP-VP interactions. Replacing a cluster of glutamic acid residues with a glutamine-rich motif on the conserved VP beta-barrel structure of variants incompatible with AAP2 creates a gain-of-function mutant compatible with AAP2. Conversely, mutating positively charged residues within the hydrophobic region of AAP2 and conserved core domains within AAPN creates a gain-of-function AAP2 mutant that rescues assembly of the incompatible variant. Our results suggest a model for capsid assembly where surface charge/neutrality dictates an interaction between AAPN and the lumenal VP surface to nucleate capsid assembly. Efforts to engineer the AAV capsid to gain desirable properties for gene therapy (e.g., tropism, reduced immunogenicity, and higher potency) require that capsid modifications do not affect particle assembly. The relationship between VP and the cofactor that facilitates its assembly, AAP, is central to both assembly preservation and vector production. Understanding the requirements for this compatibility can inform manufacturing strategies to maximize production and reduce costs. Additionally, library-based approaches that simultaneously examine a large number of capsid variants would benefit from a universally functional AAP, which could hedge against overlooking variants with potentially valuable phenotypes that were lost during vector library production due to incompatibility with the cognate AAP. Studying interactions between the structural and nonstructural components of AAV enhances our fundamental knowledge of capsid assembly mechanisms and the protein-protein interactions required for productive assembly of the icosahedral capsid.

OBJECTIVES: Ocular hypertension is a primary risk factor for glaucoma and results in retinal ganglion cell (RGC) degeneration. Current animal models of glaucoma lack severe RGC cell death as seen in glaucoma, making assessment of physiological mediators of cell death difficult. We developed a modified mouse model of ocular hypertension whereby long-lasting elevation of intraocular pressure (IOP) is achieved, resulting in significant reproducible damage to RGCs. RESULTS: In this model, microbeads are mixed with hyaluronic acid and injected into the anterior chamber of C57BL/6J mice. The hyaluronic acid allows for a gradual release of microbeads, resulting in sustained blockage of Schlemm's canal. IOP elevation was bimodal during the course of the model's progression. The first peak occurred 1 hours after beads injection, with an IOP value of 44.69 ± 6.00 mmHg, and the second peak occurred 6-12 days post-induction, with an IOP value of 34.91 ± 5.21 mmHg. RGC damage was most severe in the peripheral retina, with a loss of 64.1% compared to that of untreated eyes, while the midperiphery exhibited a 32.4% loss, 4 weeks following disease induction. CONCLUSIONS: These results suggest that sustained IOP elevation causes more RGC damage in the periphery than in the midperiphery of the retina. This model yields significant and reproducible RGC degeneration.

Importance: Health care prices may drive differences in health care costs across high-income nations. Adalimumab, ranibizumab, and aflibercept are high-cost medications in the United States and Australia. A comparison of their prices over time may elucidate how ophthalmic medication prices contribute to health care costs. Objective: To compare changes in the prices of adalimumab, ranibizumab, and aflibercept in the United States and Australia, the highest and lowest spenders on health care, respectively, among high-income nations. Design, Setting, and Participants: This retrospective price comparison study examined prices paid by government entities in the United States (Medicare) and Australia (Pharmaceuticals and Benefits Scheme). The analysis and data collection were conducted from March 28 to May 4, 2018, in accordance with guidelines set by the International Society for Pharmacoeconomics and Outcomes Research Task Force on Good Research Practices and prior published studies. No human participants or related data were included in this study. Exposures: The change in mean prices of adalimumab, ranibizumab, and aflibercept in the United States and Australia. Main Outcomes and Measures: Initial, final, and change in medication price annually from 2013 to 2017 in inflation-adjusted 2017 US dollars. Results: The mean prices (US dollar prices unadjusted for inflation) in 2013 and 2017 in the United States were $1114 ($1053) and $1818 ($1818), respectively, for adalimumab; $2102 ($1988) and $1904 ($1904), respectively, for ranibizumab; and $2074 ($1961) and $1956 ($1956), respectively, for aflibercept. The mean (Australian dollar prices unadjusted for inflation) 2013 and 2017 prices in Australia were $1854 (A $1797) and $1206 (A $1574), respectively, for adalimumab; $2157 (A $2090) and $972 (A $1268), respectively, for ranibizumab; and $2030 ($1967) and $996 ($1300), respectively, for aflibercept. The estimated annual change in price for adalimumab was +12.8% (95% CI, 9.1%-16.5%) in the United States compared with -11.1% (95% CI, -15.0% to -7.1%) in Australia, a difference of 23.9% per year (95% CI, 19.7%-28.0%; P < .001). The annual change in price for ranibizumab was -2.6% (95% CI, -3.9% to -1.3%) in the United States compared with -18.5% (95% CI, -29.3% to -7.8%) in Australia, a difference of 15.9% per year (95% CI, 7.6%-24.2%; P = .003). The annual change in price for aflibercept was -1.5% (95% CI, -2.2% to -0.7%) in the United States compared with -16.9% (95% CI, -25.1% to -8.6%) in Australia, a difference of 15.4% (95% CI, 9.1%-21.8%; P = .001). Conclusions and Relevance: Results of this study indicate that the prices of adalimumab, ranibizumab, and aflibercept significantly decreased during the past 5 years in Australia compared with the United States. These data do not indicate why these differences are noted or what actions might affect future pricing in either country.

: Boston keratoprosthesis (KPro) patients are prone to glaucoma even with well-controlled intraocular pressure (IOP). Recent experimental data have shown that soluble tumor necrosis factor alpha (TNF-) after ocular injury may contribute to progressive retinal damage and subsequent glaucoma. This study evaluates the blood plasma levels of soluble TNF-, TNF receptors 1 (TNFR1) and 2 (TNFR2), and leptin in patients with Boston type I KPro. : Venous blood samples were collected from KPro patients with glaucoma (KPro G, = 19), KPro patients without glaucoma (KPro NoG, = 12), primary angle closure glaucoma without KPro (PACG, = 13), and narrow angles without glaucoma or KPro (NA, = 21). TNF-, TNFR1, TNFR2, and leptin levels were quantified using the enzyme-linked immunosorbent assay. Erythrocyte sedimentation rate (ESR) was assessed using the Westergren test. Patients with underlying autoimmune conditions or diabetes were excluded from the study. : All groups had similar age, body mass index (BMI), IOP, and ESR ( ≥ 0.11). The mean time from KPro surgery to blood draw was 5.3 ± 3.7 years. Compared to NA patients (0.72 ± 0.3 pg/ml), KPro G and KPro NoG patients had higher blood plasma levels of TNF- (1.18 ± 0.58 pg/ml, = 0.006; 1.16 ± 0.50 pg/ml, = 0.04, respectively). Similarly, KPro G patients had higher blood plasma levels of TNFR2 (2768 ± 1368 pg/ml) than NA patients (2020 ± 435 pg/ml, = 0.048). In multivariate analysis, KPro status remained positively associated with TNF- levels ( = 0.36; 95% confidence intervals [CI]: 0.14-0.58; = 0.002) and TNFR2 levels ( = 458.3; 95% CI: 32.8-883.7; = 0.035) after adjusting for age, gender, BMI, glaucoma status, and ESR. TNFR1 and leptin levels were not significantly different in the study groups. : We detected elevated serum levels of TNF- and TNFR2 in KPro patients. Longitudinal studies are needed to establish TNF- and TNFR2 as serum biomarkers related to KPro surgery. : BCVA: best corrected visual acuity; BMI: body mass index; CDR: cup-to-disc ratio; EDTA: ethylenediaminetetraacetic acid; ELISA: enzyme-linked immunosorbent assay; ESR: erythrocyte sedimentation rate; HVF: Humphrey visual field; IOP: intraocular pressure; KPro G: keratoprosthesis with glaucoma; KPro NoG: keratoprosthesis without glaucoma; KPro: keratoprosthesis; MD: mean deviation; NA: narrow angle; non-KPro: without keratoprosthesis; PACG: primary angle closure glaucoma; RNFL: retinal nerve fiber layer; TNF-α: tumor necrosis factor alpha; TNFR1: tumor necrosis factor receptor 1; TNFR2: tumor necrosis factor receptor 2.

Anesthetics have profound effects on the brain and central nervous system. Vital signs, along with the electroencephalogram and electroencephalogram-based indices, are commonly used to assess the brain states of patients receiving general anesthesia and sedation. Important information about the patient's arousal state during general anesthesia can also be obtained through use of the neurologic examination. This article reviews the main components of the neurologic examination focusing primarily on the brainstem examination. It details the components of the brainstem examination that are most relevant for patient management during induction, maintenance, and emergence from general anesthesia. The examination is easy to apply and provides important complementary information about the patient's arousal level that cannot be discerned from vital signs and electroencephalogram measures.

The therapeutic potential of mesenchymal stem cells (MSCs) has been heralded by their multipotentiality and immunomodulatory capacity. MSCs migrate toward sites of tissue damage, where specific pro-inflammatory factors 'license' their immunosuppressive functions. Recent studies in animal models of ocular surface disease have demonstrated the potential of MSC-derived therapies to limit inflammation and promote tissue repair. Herein, we review the immunoregulatory mechanisms of MSCs, as well as strategies to harness their regenerative function at the cornea. We examine reports of the therapeutic application of MSCs in the setting of ocular surface inflammation; including corneal injury, transplantation, ocular surface autoimmunity and allergy.