NRF2/ARE mediated antioxidant response to glaucoma: role of glia and retinal ganglion cells.

Glaucoma, the second leading cause of irreversible blindness worldwide, is associated with age and sensitivity to intraocular pressure (IOP). We have shown that elevated IOP causes an early increase in levels of reactive oxygen species (ROS) in the microbead occlusion mouse model. We also detected an endogenous antioxidant response mediated by Nuclear factor erythroid 2-Related Factor 2 (NRF2), a transcription factor that binds to the antioxidant response element (ARE) and increases transcription of antioxidant genes. Our previous studies show that inhibiting this pathway results in earlier and greater glaucoma pathology. In this study, we sought to determine if this endogenous antioxidant response is driven by the retinal ganglion cells (RGCs) or glial cells. We used Nrf2fl/fl mice and cell-type specific adeno-associated viruses (AAVs) expressing Cre to alter Nrf2 levels in either the RGCs or glial cells. Then, we quantified the endogenous antioxidant response, visual function and optic nerve histology after IOP elevation. We found that knock-down of Nrf2 in either cell type blunts the antioxidant response and results in earlier pathology and vision loss. Further, we show that delivery of Nrf2 to the RGCs is sufficient to provide neuroprotection. In summary, both the RGCs and glial cells contribute to the antioxidant response, but treatment of the RGCs alone with increased Nrf2 is sufficient to delay onset of vision loss and axon degeneration in this induced model of glaucoma.

Intraocular Sustained Release of EPO-R76E Mitigates Glaucoma Pathogenesis by Activating the NRF2/ARE Pathway.

Erythropoietin (EPO) is neuroprotective in multiple models of neurodegenerative diseases, including glaucoma. EPO-R76E retains the neuroprotective effects of EPO but diminishes the effects on hematocrit. Treatment with EPO-R76E in a glaucoma model increases expression of antioxidant proteins and is neuroprotective. A major pathway that controls the expression of antioxidant proteins is the NRF2/ARE pathway. This pathway is activated endogenously after elevation of intraocular pressure (IOP) and contributes to the slow onset of pathology in glaucoma. In this study, we explored if sustained release of EPO-R76E in the eye would activate the NRF2/ARE pathway and if this pathway was key to its neuroprotective activity. Treatment with PLGA.EPO-E76E prevented increases in retinal superoxide levels in vivo, and caused phosphorylation of NRF2 and upregulation of antioxidants. Further, EPO-R76E activates NRF2 via phosphorylation by the MAPK pathway rather than the PI3K/Akt pathway, used by the endogenous antioxidant response to elevated IOP.

Teaching bedside ultrasound to medical students.

OBJECTIVE: Point-of-care ultrasound (POCUS) assists in the rapid diagnosis of conditions in the Emergency Department (ED). POCUS has been introduced to international medical curricula; however, there is no described implementation of clinically focused POCUS education in Australian medical schools. We wanted to investigate whether a formal curriculum can be effective and feasible in an Australian medical school. METHODS: Pre-post intervention study of a focused curriculum based on the Extended Focused Assessment with Sonography in Trauma (E-FAST) examination, consisting of online and practical teaching, was implemented for Year-4 and -5 medical students. An online questionnaire was used to measure knowledge, image interpretation and confidence prior to the intervention. After the intervention and ED placement, the questionnaire was repeated and students were assessed performing the E-FAST examination on a healthy volunteer. RESULTS: Twenty-seven students participated in both the pre-intervention and post-intervention questionnaires. There was a significant improvement in confidence in performing the E-FAST after the intervention [p < 0.001]. There was also a significant improvement in ultrasound knowledge and image interpretation skills. For the formative assessment, the mean score was 31.8 out of 33 and 22 of 27 students (82%) passed the assessment. There is no described implementation of clinically focused point-of-care ultrasound education in Australian medical schools CONCLUSIONS: We have demonstrated that a focused curriculum can improve POCUS knowledge and skills. The curriculum was feasible and well received. With global trends to include POCUS in medical education, Australian institutions should consider upskilling their medical graduates.

Protective immunity against a lethal respiratory Yersinia pestis challenge induced by V antigen or the F1 capsular antigen incorporated into adenovirus capsid.

The aerosol form of the bacterium Yersinia pestis causes pneumonic plague, a rapidly fatal disease that is a biothreat if deliberately released. At present, no plague vaccines are available for use in the United States, but subunit vaccines based on the Y. pestis V antigen and F1 capsular protein show promise when administered with adjuvants. In the context that adenovirus (Ad) gene transfer vectors have a strong adjuvant potential related to the ability to directly infect dendritic cells, we hypothesized that modification of the Ad5 capsid to display either the Y. pestis V antigen or the F1 capsular antigen on the virion surface would elicit high V antigen- or F1-specific antibody titers, permit boosting with the same Ad serotype, and provide better protection against a lethal Y. pestis challenge than immunization with equivalent amounts of V or F1 recombinant protein plus conventional adjuvant. We constructed AdYFP-pIX/V and AdLacZ-pIX/F1, E1(-), E3(-) serotype 5 Ad gene transfer vectors containing a fusion of the sequence for either the Y. pestis V antigen or the F1 capsular antigen to the carboxy-terminal sequence of pIX, a capsid protein that can accommodate the entire V antigen (37 kDa) or F1 protein (15 kDa) without disturbing Ad function. Immunization with AdYFP-pIX/V followed by a single repeat administration of the same vector at the same dose resulted in significantly better protection of immunized animals compared with immunization with a molar equivalent amount of purified recombinant V antigen plus Alhydrogel adjuvant. Similarly, immunization with AdLacZ-pIX/F1 in a prime-boost regimen resulted in significantly enhanced protection of immunized animals compared with immunization with a molar-equivalent amount of purified recombinant F1 protein plus adjuvant. These observations demonstrate that Ad vaccine vectors containing pathogen-specific antigens fused to the pIX capsid protein have strong adjuvant properties and stimulate more robust protective immune responses than equivalent recombinant protein-based subunit vaccines administered with conventional adjuvant, suggesting that F1-and/or V-modified capsid Ad-based recombinant vaccines should be considered for development as anti-plague vaccines.

Adenovirus-mediated delivery of an anti-V antigen monoclonal antibody protects mice against a lethal Yersinia pestis challenge.

Pneumonic plague, caused by inhalation of Yersinia pestis, represents a major bioterrorism threat for which no vaccine is available. Based on the knowledge that genetic delivery of monoclonal antibodies (MAbs) with adenovirus (Ad) gene transfer vectors results in rapid, high-level antibody expression, we evaluated the hypothesis that Ad-mediated delivery of a neutralizing antibody directed against the Y. pestis V antigen would protect mice against a Y. pestis challenge. MAbs specific for the Y. pestis V antigen were generated, and the most effective in protecting mice against a lethal intranasal Y. pestis challenge was chosen for further study. The coding sequences for the heavy and light chains were isolated from the corresponding hybridoma and inserted into a replication-defective serotype 5 human Ad gene transfer vector (AdalphaV). Western analysis of AdalphaV-infected cell supernatants demonstrated completely assembled antibodies reactive with V antigen. Following AdalphaV administration to mice, high levels of anti-V antigen antibody titers were detectable as early as 1 day postadministration, peaked by day 3, and remained detectable through a 12-week time course. When animals that received AdalphaV were challenged with Y. pestis at day 4 post-AdalphaV administration, 80% of the animals were protected, while 0% of control animals survived (P < 0.01). Ad-mediated delivery of a V antigen-neutralizing antibody is an effective therapy against plague in experimental animals and could be developed as a rapidly acting antiplague therapeutic.

An evaluation of silicone-hydrogel lenses worn on a daily wear basis.

PURPOSE: To examine the clinical performance of two brands of silicone-hydrogel lenses when worn on a daily wear basis. METHODS: Fifty-six subjects with no ocular disease were enrolled at multiple sites in Australasia. Contact lenses made from galyfilcon A or lotrafilcon A were randomly assigned to each eye of the subject and the lenses were worn on a daily wear basis for a period of two weeks. Subjects did not know the identity of the lenses they wore. Clinical data and patient responses to a questionnaire were gathered at an initial visit and after two weeks of wear. RESULTS: For both lenses, the degree of limbal hyperaemia and bulbar conjunctival hyperaemia decreased significantly over the two-week wearing period. The eyes wearing galyfilcon A lenses showed an increase in conjunctival staining compared to the baseline measures. On average, galyfilcon A lenses decentred more and moved less than the lotrafilcon A lenses. The lotrafilcon A lenses showed a greater loss of wettability, as judged by practitioner grading, than the galyfilcon A lenses over the two-week period. The subjective responses showed strong preference for the galyfilcon A lens across 26 of 27 questions relating to comfort, vision, handling, preference and other subjective outcomes. DISCUSSION: The results show that different silicone-hydrogel lenses have different performance characteristics on the eye, when worn on a daily wear basis. Striving for high oxygen transmissibility at the expense of other properties may lead to a range of undesirable performance characteristics.