Myeloid Protease-Activated Receptor-2 Contributes to Influenza A Virus Pathology in Mice.

Background: Innate immune responses to influenza A virus (IAV) infection are initiated in part by toll-like receptor 3 (TLR3). TLR3-dependent signaling induces an antiviral immune response and an NFκB-dependent inflammatory response. Protease-activated receptor 2 (PAR2) inhibits the antiviral response and enhances the inflammatory response. PAR2 deficiency protected mice during IAV infection. However, the PAR2 expressing cell-types contributing to IAV pathology in mice and the mechanism by which PAR2 contributes to IAV infection is unknown. Methods: IAV infection was analyzed in global (Par2-/- ), myeloid (Par2fl/fl;LysMCre+) and lung epithelial cell (EpC) Par2 deficient (Par2fl/fl ;SPCCre+) mice and their respective controls (Par2+/+ and Par2fl/fl). In addition, the effect of PAR2 activation on polyinosinic-polycytidylic acid (poly I:C) activation of TLR3 was analyzed in bone marrow-derived macrophages (BMDM). Lastly, we determined the effect of PAR2 inhibition in wild-type (WT) mice. Results: After IAV infection, Par2-/- and mice with myeloid Par2 deficiency exhibited increased survival compared to infected controls. The improved survival was associated with reduced proinflammatory mediators and reduced cellular infiltration in bronchoalveolar lavage fluid (BALF) of Par2-/- and Par2fl/fl;LysMCre+ 3 days post infection (dpi) compared to infected control mice. Interestingly, Par2fl/fl;SPCCre+ mice showed no survival benefit compared to Par2fl/fl . In vitro studies showed that Par2-/- BMDM produced less IL6 and IL12p40 than Par2+/+ BMDM after poly I:C stimulation. In addition, activation of PAR2 on Par2+/+ BMDM increased poly I:C induction of IL6 and IL12p40 compared to poly I:C stimulation alone. Importantly, PAR2 inhibition prior to IAV infection protect WT mice. Conclusion: Global Par2 or myeloid cell but not lung EpC Par2 deficiency was associated with reduced BALF inflammatory markers and reduced IAV-induced mortality. Our study suggests that PAR2 may be a therapeutic target to reduce IAV pathology.

HANDHELD SPECTRAL DOMAIN OPTICAL COHERENCE TOMOGRAPHY FINDINGS OF X-LINKED RETINOSCHISIS IN EARLY CHILDHOOD.

BACKGROUND/PURPOSE: Using handheld spectral domain optical coherence tomography (SDOCT) imaging to investigate in vivo microanatomic retinal changes and their progression over time in young children with juvenile X-linked retinoschisis (XLRS). METHODS: This retrospective analysis was of handheld SD OCT images obtained under a prospective research protocol in children who had established XLRS diagnosis based on genetic testing or clinical history. Three OCT graders performed standardized qualitative and quantitative assessment of retinal volume scans, which were divided into foveal, parafoveal, and extrafoveal regions. Visual acuity data were obtained when possible. RESULTS: Spectral domain OCT images were available of both eyes in 8 pediatric patients with ages 7 months to 10 years. The schisis cavities involved inner nuclear layer in over 90% (15/16) of eyes in all 3 regions. Retinal nerve fiber and ganglion cell layer involvement was present only in the extrafoveal region in 63% (10/16) eyes and outer nuclear and plexiform layer in few others. In 7 children followed over 2 months to 15 months, the location of schisis remained consistent. Central foveal thickness decreased from the baseline to final available visit in 4/6 eyes. Ellipsoid zone disruption seemed to accompany lower visual acuity in 1/4 eyes. CONCLUSION: Early in life, the SD OCT findings in XLRS demonstrate differences in schisis location in fovea-parafoveal versus extrafoveal region, possible association between poor visual acuity and degree of ellipsoid zone disruption and decrease in central foveal thickness over time in this group. Furthermore, they illustrates that the pattern of XLRS in adults is already present in very young children, and unlike in older children and adults, those presenting with earlier disease may have a more aggressive course. Further studies in this early age group may provide more insights into treatment and prevention of progressive visual impairment in children with XLRS.

Real-Time Volumetric Imaging of Vitreoretinal Surgery with a Prototype Microscope-Integrated Swept-Source OCT Device.

PURPOSE: To evaluate the images produced in an initial surgical series of intraoperative near-real-time volumetric swept-source (SS) OCT imaging. DESIGN: Prospective translational study. PARTICIPANTS: Forty-one consecutive adult patients undergoing vitreoretinal surgery between July 22, 2014, and July 1, 2015, at the Duke University Eye Center who agreed to participate. METHODS: A novel microscope-integrated SS-OCT prototype captured volumetric renderings of imaging of macular surgery in near-real-time and showed them to the surgeon via a heads-up display through the microscope oculars. Then the images were analyzed formally after surgery. MAIN OUTCOME MEASURES: Image quality, successful capture of surgical instruments, maneuvers and associated retinal deformation volumetrically over time, and qualitative image analysis. RESULTS: Volumetric SS-OCT images were graded as acceptable in 92% of patients. Volumetric imaging of scraping and peeling procedures was achieved in 75% and 78% of patients in whom it was performed, respectively. Imaging provided the surgeon with near-real-time volumetric visualization of the position of the instrument relative to the retinal surface, flap initiation, flap removal, and retinal deformation during instrumentation via a heads-up display. CONCLUSIONS: This volumetric, microscope-integrated SS-OCT prototype seems to provide high-detail, near-real-time volumetric imaging of delicate maneuvers during macular surgery.

Optical Coherence Tomography Reflective Drusen Substructures Predict Progression to Geographic Atrophy in Age-related Macular Degeneration.

PURPOSE: Structural and compositional heterogeneity within drusen comprising lipids, carbohydrates, and proteins have been previously described. We sought to detect and define phenotypic patterns of drusen heterogeneity in the form of optical coherence tomography-reflective drusen substructures (ODS) and examine their associations with age-related macular degeneration (AMD)-related features and AMD progression. DESIGN: Retrospective analysis in a prospective study. PARTICIPANTS: Patients with intermediate AMD (n = 349) enrolled in the multicenter Age-Related Eye Disease Study 2 (AREDS2) ancillary spectral-domain optical coherence tomography (SD OCT) study. METHODS: Baseline SD OCT scans of 1 eye per patient were analyzed for the presence of ODS. Cross-sectional and longitudinal associations of ODS presence with AMD-related features visible on SD OCT and color photographs, including drusen volume, geographic atrophy (GA), and preatrophic features, were evaluated for the entire macular region. Similar associations were also made locally within a 0.5-mm-diameter region around individual ODS and corresponding control region without ODS in the same eye. MAIN OUTCOME MEASURES: Preatrophy SD OCT changes and GA, central GA, and choroidal neovascularization (CNV) from color photographs. RESULTS: Four phenotypic subtypes of ODS were defined: low reflective cores, high reflective cores, conical debris, and split drusen. Among the 349 participants, there were 307 eligible eyes and 74 (24%) had at least 1 ODS. The ODS at baseline were associated with (1) greater macular drusen volume at baseline (P < 0.001), (2) development of preatrophic changes at year 2 (P = 0.001-0.01), and (3) development of macular GA (P = 0.005) and preatrophic changes at year 3 (P = 0.002-0.008), but not development of CNV. The ODS at baseline in a local region were associated with (1) presence of preatrophy changes at baseline (P = 0.02-0.03) and (2) development of preatrophy changes at years 2 and 3 within the region (P = 0.008-0.05). CONCLUSIONS: Optical coherence tomography-reflective drusen substructures are optical coherence tomography-based biomarkers of progression to GA, but not to CNV, in eyes with intermediate AMD. Optical coherence tomography-reflective drusen substructures may be a clinical entity helpful in monitoring AMD progression and informing mechanisms in GA pathogenesis.

Association of the Rad9-Rad1-Hus1 checkpoint clamp with MYH DNA glycosylase and DNA.

Cell cycle checkpoints provide surveillance mechanisms to activate the DNA damage response, thus preserving genomic integrity. The heterotrimeric Rad9-Rad1-Hus1 (9-1-1) clamp is a DNA damage response sensor and can be loaded onto DNA. 9-1-1 is involved in base excision repair (BER) by interacting with nearly every enzyme in BER. Here, we show that individual 9-1-1 components play distinct roles in BER directed by MYH DNA glycosylase. Analyses of Hus1 deletion mutants revealed that the interdomain connecting loop (residues 134-155) is a key determinant of MYH binding. Both the N-(residues 1-146) and C-terminal (residues 147-280) halves of Hus1, which share structural similarity, can interact with and stimulate MYH. The Hus1(K136A) mutant retains physical interaction with MYH but cannot stimulate MYH glycosylase activity. The N-terminal domain, but not the C-terminal half of Hus1 can also bind DNA with moderate affinity. Intact Rad9 expressed in bacteria binds to and stimulates MYH weakly. However, Rad9(1-266) (C-terminal truncated Rad9) can stimulate MYH activity and bind DNA with high affinity, close to that displayed by heterotrimeric 9(1-266)-1-1 complexes. Conversely, Rad1 has minimal roles in stimulating MYH activity or binding to DNA. Finally, we show that preferential recruitment of 9(1-266)-1-1 to 5'-recessed DNA substrates is an intrinsic property of this complex and is dependent on complex formation. Together, our findings provide a mechanistic rationale for unique contributions by individual 9-1-1 subunits to MYH-directed BER based on subunit asymmetry in protein-protein interactions and DNA binding events.

Coordination of MYH DNA glycosylase and APE1 endonuclease activities via physical interactions.

MutY homologue (MYH) is a DNA glycosylase which excises adenine paired with the oxidative lesion 7,8-dihydro-8-oxoguanine (8-oxoG, or G(o)) during base excision repair (BER). Base excision by MYH results in an apurinic/apyrimidinic (AP) site in the DNA where the DNA sugar-phosphate backbone remains intact. A key feature of MYH activity is its physical interaction and coordination with AP endonuclease I (APE1), which subsequently nicks DNA 5' to the AP site. Because AP sites are mutagenic and cytotoxic, they must be processed by APE1 immediately after the action of MYH glycosylase. Our recent reports show that the interdomain connector (IDC) of human MYH (hMYH) maintains interactions with hAPE1 and the human checkpoint clamp Rad9-Rad1-Hus1 (9-1-1) complex. In this study, we used NMR chemical shift perturbation experiments to determine hMYH-binding site on hAPE1. Chemical shift perturbations indicate that the hMYH IDC peptide binds to the DNA-binding site of hAPE1 and an additional site which is distal to the APE1 DNA-binding interface. In these two binding sites, N212 and Q137 of hAPE1 are key mediators of the MYH/APE1 interaction. Intriguingly, despite the fact that hHus1 and hAPE1 both interact with the MYH IDC, hHus1 does not compete with hAPE1 for binding to hMYH. Rather, hHus1 stabilizes the hMYH/hAPE1 complex both in vitro and in cells. This is consistent with a common theme in BER, namely that the assembly of protein-DNA complexes enhances repair by efficiently coordinating multiple enzymatic steps while simultaneously minimizing the release of harmful repair intermediates.