A surfactant-based dressing can reduce the appearance of Pseudomonas aeruginosa pigments and uncover the dermal extracellular matrix in an ex vivo porcine skin wound model.

From previous studies, we have shown that viable colony forming units of bacteria and bacterial biofilms are reduced after sequential treatment with a surfactant-based dressing. Here, we sought to test the impact on visible bacterial pigments and the ultrastructural impact following the sequential treatment of the same surfactant-based dressing. Mature Pseudomonas aeruginosa biofilms were grown on ex vivo porcine skin explants, and an imaging-based analysis was used to compare the skin with and without a concentrated surfactant. In explants naturally tinted by bacterial chromophores, wiping alone had no effect, while the use of a surfactant-based dressing reduced coloration. Similarly, daily wiping led to increased immunohistochemical staining for P. aeruginosa antigens, but not in the surfactant group. Confocal immunofluorescent imaging revealed limited bacterial penetration and coating of the dermis and loose pieces of sloughing material. Ultrastructural analysis confirmed that the biofilms were masking the extracellular matrix (ECM), but the surfactant could remove them, re-exposing the ECM. The masking of the ECM may provide another non-inflammatory explanation for delayed healing, as the ECM is no longer accessible for wound cell locomotion. The use of a poloxamer-based surfactant appears to be an effective way to remove bacterial chromophores and the biofilm coating the ECM fibres.

Coxsackievirus B3 infects and disrupts human induced-pluripotent stem cell derived brain-like endothelial cells.

Coxsackievirus B3 (CVB3) is a significant human pathogen that is commonly found worldwide. CVB3 among other enteroviruses, are the leading causes of aseptic meningo-encephalitis which can be fatal especially in young children. How the virus gains access to the brain is poorly-understood, and the host-virus interactions that occur at the blood-brain barrier (BBB) is even less-characterized. The BBB is a highly specialized biological barrier consisting primarily of brain endothelial cells which possess unique barrier properties and facilitate the passage of nutrients into the brain while restricting access to toxins and pathogens including viruses. To determine the effects of CVB3 infection on the BBB, we utilized a model of human induced-pluripotent stem cell-derived brain-like endothelial cells (iBECs) to ascertain if CVB3 infection may alter barrier cell function and overall survival. In this study, we determined that these iBECs indeed are susceptible to CVB3 infection and release high titers of extracellular virus. We also determined that infected iBECs maintain high transendothelial electrical resistance (TEER) during early infection despite possessing high viral load. TEER progressively declines at later stages of infection. Interestingly, despite the high viral burden and TEER disruptions at later timepoints, infected iBEC monolayers remain intact, indicating a low degree of late-stage virally-mediated cell death, which may contribute to prolonged viral shedding. We had previously reported that CVB3 infections rely on the activation of transient receptor vanilloid potential 1 (TRPV1) and found that inhibiting TRPV1 activity with SB-366791 significantly limited CVB3 infection of HeLa cervical cancer cells. Similarly in this study, we observed that treating iBECs with SB-366791 significantly reduced CVB3 infection, which suggests that not only can this drug potentially limit viral entry into the brain, but also demonstrates that this infection model could be a valuable platform for testing antiviral treatments of neurotropic viruses. In all, our findings elucidate the unique effects of CVB3 infection on the BBB and shed light on potential mechanisms by which the virus can initiate infections in the brain.

TBK1 and GABARAP family members suppress Coxsackievirus B infection by limiting viral production and promoting autophagic degradation of viral extracellular vesicles.

Host-pathogen dynamics are constantly at play during enteroviral infection. Coxsackievirus B (CVB) is a common juvenile enterovirus that infects multiple organs and drives inflammatory diseases including acute pancreatitis and myocarditis. Much like other enteroviruses, CVB is capable of manipulating host machinery to hijack and subvert autophagy for its benefit. We have previously reported that CVB triggers the release of infectious extracellular vesicles (EVs) which originate from autophagosomes. These EVs facilitate efficient dissemination of infectious virus. Here, we report that TBK1 (Tank-binding kinase 1) suppresses release of CVB-induced EVs. TBK1 is a multimeric kinase that directly activates autophagy adaptors for efficient cargo recruitment and induces type-1 interferons during viral-mediated STING recruitment. Positioning itself at the nexus of pathogen elimination, we hypothesized that loss of TBK1 could exacerbate CVB infection due to its specific role in autophagosome trafficking. Here we report that infection with CVB during genetic TBK1 knockdown significantly increases viral load and potentiates the bulk release of viral EVs. Similarly, suppressing TBK1 with small interfering RNA (siRNA) caused a marked increase in intracellular virus and EV release, while treatment in vivo with the TBK1-inhibitor Amlexanox exacerbated viral pancreatitis and EV spread. We further demonstrated that viral EV release is mediated by the autophagy modifier proteins GABARAPL1 and GABARAPL2 which facilitate autophagic flux. We observe that CVB infection stimulates autophagy and increases the release of GABARAPL1/2-positive EVs. We conclude that TBK1 plays additional antiviral roles by inducing autophagic flux during CVB infection independent of interferon signaling, and the loss of TBK1 better allows CVB-laden autophagosomes to circumvent lysosomal degradation, increasing the release of virus-laden EVs. This discovery sheds new light on the mechanisms involved in viral spread and EV propagation during acute enteroviral infection and highlights novel intracellular trafficking protein targets for antiviral therapy.

Assessment of Topical Therapies for Improving the Optical Clarity Following Stromal Wounding in a Novel Ex Vivo Canine Cornea Model.

Purpose: To evaluate the effect of topical suberanilohydroxamic acid (SAHA) and 5-methyl-1-phenyl-2[1H]-pyridone (pirfenidone) on the degree of corneal haze in the stromal wounded ex vivo canine cornea. Methods: Twenty-four corneoscleral rims from normal dogs were uniformly wounded with an excimer laser and placed into culture medium with an air-liquid interface. The control group (n = 8) contained placebo-treated corneas. Treatment group 1 (n = 8) received SAHA topically every 6 hours. Treatment group 2 (n = 8) received pirfenidone topically every 6 hours. Each cornea was fluorescein stained and macrophotographed every 6 hours to assess epithelialization rate. All corneas were also macrophotographed weekly to assess optical clarity (haze). Images were analyzed for differences in pixel intensity between wounded (haze) and unwounded (nonhaze) regions, and haze surface area for each cornea was calculated. Results: The mean epithelialization time was 47.25 hours in the control group, 45.00 hours in the SAHA group, and 43.50 hours in the pirfenidone group, revealing no significant difference (P = 0.368). The median difference in pixel intensity between haze and nonhaze areas was 21.5 in the control group, 8.0 in the SAHA group, and 8.0 in the pirfenidone group, which is significant (P < 0.01). The median haze surface area was 12.96 mm2 in the control group, 5.70 mm2 in the SAHA group, and 5.92 mm2 in the pirfenidone group, which is significant (P < 0.01). Conclusions: Stromal-wounded ex vivo canine corneas exhibited greater optical clarity when treated with SAHA and pirfenidone than when placebo treated at 21 days. There was no significant difference in epithelialization rate between groups. Corneal contour was correlated with geographic haze distribution.

Pathological consequences of long-term mitochondrial oxidative stress in the mouse retinal pigment epithelium.

Oxidative stress in the retinal pigment epithelium (RPE) is hypothesized to be a major contributor to the development of age-related macular degeneration (AMD). Mitochondrial manganese superoxide dismutase (MnSOD) is a critical antioxidant protein that scavenges the highly reactive superoxide radical. We speculated that specific reduction of MnSOD in the RPE will increase the level of reactive oxygen species in the retina/RPE/choroid complex leading to pathogenesis similar to geographic atrophy. To test this hypothesis, an Sod2-specific hammerhead ribozyme (Rz), delivered by AAV2/1 and driven by the human VMD2 promoter was injected subretinally into C57BL/6J mice. Dark-adapted full field electroretinogram (ERG) detected a decrease in the response to light. We investigated the age-dependent phenotypic and morphological changes of the outer retina using digital fundus imaging and SD-OCT measurement of ONL thickness. Fundus microscopy revealed pigmentary abnormalities in the retina and these corresponded to sub-retinal and sub-RPE deposits seen in SD-OCT B-scans. Light and electron microscopy documented the localization of apical deposits and thickening of the RPE. In RPE flat-mounts we observed abnormally displaced nuclei and regions of apparent fibrosis in the central retina of the oldest mice. This region was surrounded by enlarged and irregular RPE cells that have been observed in eyes donated by AMD patients and in other mouse models of AMD.

The 5HT1a receptor agonist 8-Oh DPAT induces protection from lipofuscin accumulation and oxidative stress in the retinal pigment epithelium.

Age-related macular degeneration (AMD), a major cause of blindness in the elderly, is associated with oxidative stress, lipofuscin accumulation and retinal degeneration. The aim of this study was to determine if a 5-HT(1A) receptor agonist can reduce lipofuscin accumulation, reduce oxidative damage and prevent retinal cell loss both in vitro and in vivo. Autophagy-derived and photoreceptor outer segment (POS)-derived lipofuscin formation was assessed using FACS analysis and confocal microscopy in cultured retinal pigment epithelial (RPE) cells in the presence or absence of the 5-HT(1A) receptor agonist, 8-OH DPAT. 8-OH DPAT treatment resulted in a dose-dependent reduction in both autophagy- and POS-derived lipofuscin compared to control. Reduction in autophagy-induced lipofuscin was sustained for 4 weeks following removal of the drug. The ability of 8-OH DPAT to reduce oxidative damage following exposure to 200 µM H(2)O(2) was assessed. 8-OH DPAT reduced superoxide generation and increased mitochondrial superoxide dismutase (MnSOD) levels and the ratio of reduced glutathione to the oxidized form of glutathione in H(2)O(2)-treated cells compared to controls and protected against H(2)O(2)-initiated lipid peroxidation, nitrotyrosine levels and mitochondrial damage. SOD2 knockdown mice, which have an AMD-like phenotype, received daily subcutaneous injections of either saline, 0.5 or 5.0 mg/kg 8-OH DPAT and were evaluated at monthly intervals. Systemic administration of 8-OH DPAT improved the electroretinogram response in SOD2 knockdown eyes of mice compared to knockdown eyes receiving vehicle control. There was a significant increase in the ONL thickness in mice treated with 8-OH DPAT at 4 months past the time of MnSOD knockdown compared to untreated controls together with a 60% reduction in RPE lipofuscin. The data indicate that 5-HT(1A) agonists can reduce lipofuscin accumulation and protect the retina from oxidative damage and mitochondrial dysfunction. 5-HT(1A) receptor agonists may have potential as therapeutic agents in the treatment of retinal degenerative disease.

Delivery of antioxidant enzyme genes to protect against ischemia/reperfusion-induced injury to retinal microvasculature.

PURPOSE: Retinal ischemia/reperfusion (I/R) injury results in the generation of reactive oxygen species (ROS). The aim of this study was to investigate whether delivery of the manganese superoxide dismutase gene (SOD2) or the catalase gene (CAT) could rescue the retinal vascular damage induced by I/R in mice. METHODS: I/R injury to the retina was induced in mice by elevating intraocular pressure for 2 hours, and reperfusion was established immediately afterward. One eye of each mouse was pretreated with plasmids encoding manganese superoxide dismutase or catalase complexed with cationic liposomes and delivered by intravitreous injection 48 hours before initiation of the procedure. Superoxide ion, hydrogen peroxide, and 4-hydroxynonenal (4-HNE) protein modifications were measured by fluorescence staining, immunohistochemistry, and Western blot analysis 1 day after the I/R injury. At 7 days after injury, retinal vascular cell apoptosis and acellular capillaries were quantitated. RESULTS: Superoxide ion, hydrogen peroxide, and 4-HNE protein modifications increased at 24 hours after I/R injury. Administration of plasmids encoding SOD2 or CAT significantly reduced levels of superoxide ion, hydrogen peroxide, and 4-HNE. Retinal vascular cell apoptosis and acellular capillary numbers increased greatly by 7 days after the injury. Delivery of SOD2 or CAT inhibited the I/R-induced apoptosis of retinal vascular cell and retinal capillary degeneration. CONCLUSIONS: Delivery of antioxidant genes inhibited I/R-induced retinal capillary degeneration, apoptosis of vascular cells, and ROS production, suggesting that antioxidant gene therapy might be a treatment for I/R-related disease.