mTORC1 and mTORC2 are differentially engaged in the development of laser-induced CNV.

BACKGROUND: The mechanistic target of rapamycin (mTOR) pathway is a potential target to inhibit pathologic processes in choroidal neovascularization. However, the exact role of mTOR signaling in the development of CNV remains obscure. In this study, we assessed the role of mTORC1 and mTORC2 as well as the effect of rapamycin (sirolimus) on choroidal neovascularization (CNV) in a laser-induced mouse model. METHODS: In experiment A, we observed the natural course of CNV development and the dynamics of mTOR-related proteins during the 12 days after the laser injury. The expression of mTOR-related proteins was evaluated using Western blot (WB). Cryosections of CNV-induced mice were immunostained for the visualization of the vascular and extravascular components of the CNV. Experiment B was performed to confirm the critical period of mTOR signaling in the development of laser-induced CNV, we administered rapamycin before and/or during the active period of mTOR complexes. WB and immunofluorescence staining was performed to evaluate the mode of action and the effect of mTOR inhibition on CNV development. RESULTS: In experiment A, we detected high levels of p-mTOR S2448 and p-mTOR S2481 from the 5th to 12th day of laser injury. Immunofluorescence imaging of cryosections of mice sacrificed on day 7 revealed greater co-immunoreactivity of p-mTOR S2448 positive cells with CD11b and F4/80, while p-mTOR S2481 positive cells showed colocalization with CD31, α-SMA, and cytokeratin. In experiment B, rapamycin injection during the active period of mTOR signaling demonstrated near-complete inhibition of CNV lesion as well as significant induction of autophagy. CONCLUSION: Our study suggests the mTOR as a critical player during CNV development in laser-induced mouse model through differentially acting with the mTORC1 and mTORC2. mTORC1 activity was high predominantly in inflammatory cells in CNV lesion, while mTORC2 activity was higher in vascular components and the RPE.

Adeno-Associated Viral Vector 2 and 9 Transduction Is Enhanced in Streptozotocin-Induced Diabetic Mouse Retina.

Adeno-associated viruses (AAVs) are currently the most popular vector platform technology for ocular gene therapy. While transduction efficiency and tropism of intravitreally administered AAV has been fairly well established in various retinal conditions, its transduction pattern in diabetic retinas has not previously been characterized. Here, we describe the transduction efficiencies of four different AAV serotypes, AAV2, 5, 8, and 9, in streptozotocin (STZ)-induced diabetic mouse retinas after intravitreal injections, which differed according to the duration of diabetic induction. STZ was intraperitoneally injected into C57/B6 diabetic mice subjected to unilateral intravitreal injection of AAV2, AAV5, AAV8, and AAV9 packaged with EGFP. Significantly enhanced AAV2 and AAV9 transduction was observed in 2-month-old diabetic mouse retinas compared to the 2-week-old diabetic mouse retinas and nondiabetic, vector uninjected or injected retinas. Intravitreal injection of AAV5 or AAV8 serotype in 2-month- and 2-week-old diabetic mouse retinas did not show any significant vector transduction enhancement compared to the nondiabetic control retinas. The tropism of AAV2 and AAV9 in diabetic mouse retinas differed. AAV2 was transduced into various retinal cells, including Müller cells, microglia, retinal ganglion cells (RGCs), bipolar cells, horizontal cells, and amacrine cells, whereas AAV9 was effectively transduced only into RGC and horizontal cells. The expression levels of receptors and co-receptors for AAV2 and AAV9 were significantly increased in 2-month-old diabetic mouse retinas. The results of our study demonstrated that AAV2 and AAV9 may be the vector of choice in treating diabetic retinopathy (DR) with gene therapy, and DR-related retinal changes may improve AAV vector transduction efficiency.

The retinal pigment epithelial response after retinal laser photocoagulation in diabetic mice.

To investigate the characteristics of regenerated retinal pigment epithelial (RPE) cells after retinal laser photocoagulation in diabetic mice. C57BL/6J mice were used to induce diabetes using intraperitoneal injection of streptozotocin. The proliferation of RPE cells after laser photocoagulation was determined using the 5-ethynyl-2'-deoxyuridine (EdU) assay in both diabetic and wild-type mice. The morphological changes of RPE cells were evaluated by using Voronoi diagram from immunostaining for ß-catenin. Characteristics of regenerated cells were evaluated by quantifying the mRNA and protein levels of RPE and epithelial-mesenchymal transition (EMT) markers. There were significantly less EdU-positive cells in laser-treated areas in diabetic mice than wild-type mice. Hexagonality was extensively lost in diabetic mice. Many EdU-positive cells were co-localized with Otx2-positive cells in the center of the laser-treated areas in wild-type mice, but only EdU-positive cells were widely distributed in diabetic mice. Quantitative analysis of mRNA and protein levels showed that the expression levels of RPE markers, Pax6, Mitf, and Otx2, were significantly decreased in RPE of diabetic mice compared with that of wild-type mice, whereas the expression levels of EMT markers, vimentin and fibronectin, were significantly increased. The proliferation and hexagonality of regenerating RPE cells were impaired after laser photocoagulation, and the regenerated RPE cells lost their original properties in diabetic mice. Further clinical research is needed to elucidate the RPE response after laser photocoagulation in diabetic patients.

Essential Role of mTOR Signaling in Human Retinal Pigment Epithelial Cell Regeneration After Laser Photocoagulation.

This study assessed the role of mechanistic target of rapamycin (mTOR) pathway in the human adult retinal pigment epithelial (ARPE) cell response after laser photocoagulation (LP). The effect of mTOR inhibition on ARPE-19 cell was investigated by rapamycin treatment after LP. Cell viability and proliferation were explored using MTT and EdU assays, respectively. The expression of mTOR-related proteins and epithelial-mesenchymal transition (EMT) markers was verified by Western blot. Rapamycin retarded the LP area recovery in a dose-dependent manner by the 120 h, while LP+DMSO vehicle-treated cells completely restored the lesion zone (P ≤ 0.01). ARPE-19 cell viability is significantly lower in LP + rapamycin 80 and 160 ng/ml treated cultures compared to LP control at 120 h (P ≤ 0.001). LP control group demonstrated significantly more proliferative cells compared to untreated cells at the 72 and 120 h, whereas EdU-positive cell numbers in cultures treated with rapamycin at concentrations of 80 and 160 ng/ml were similar to baseline values (P ≤ 0.01). mTOR pathway activation is essential for regulation of the RPE cell migration and proliferation after LP. mTOR inhibition with rapamycin effectively blocks the migration and proliferation of the RPE cells. Our results demonstrate that mTOR has an important role in ARPE-19 cell as a regulator of cell behavior under stress conditions, suggesting that mTOR could be a promising therapeutic target for numerous retinal diseases.

The role of Wnt/ß-catenin signaling in the restoration of induced pluripotent stem cell-derived retinal pigment epithelium after laser photocoagulation.

To investigate the role of Wnt/ß-catenin signaling pathway in the restoration of induced pluripotent stem cell-derived retinal pigment epithelium (hiPSC-RPE) after laser photocoagulation. After differentiation of RPE cells from hiPSCs, laser photocoagulation was performed. Activation of Wnt/ß-catenin signaling at days 1 and 5 after laser photocoagulation was evaluated by expression of ß-catenin. Cell proliferation and alteration in cell-to-cell contact at day 5 after laser photocoagulation with or without Dickkopf-1 (Dkk-1) treatment were studied using ethynyl-2'-deoxyuridine (EdU) assay and zonula occludens-1 (ZO-1) expression analysis, respectively. The mRNA levels of Wnt genes at day 5 after laser photocoagulation were evaluated by quantitative real-time polymerase chain reaction (qRT-PCR). Activation of Wnt/ß-catenin signaling at days 1 and 5 after laser photocoagulation was confirmed by ß-catenin accumulation in the cytoplasm and nucleus of hiPSC-RPE. Many EdU-positive cells also expressed ß-catenin, and the number of EdU-positive cells was decreased at day 5 after laser photocoagulation after Dkk-1 treatment, indicating that Wnt/ß-catenin signaling mediated hiPSC-RPE proliferation. ZO-1 expression was not decreased with Dkk-1 treatment at day 5 after laser photocoagulation, indicating that Wnt/ß-catenin signaling mediated hiPSC-RPE restoration. At day 5, after laser photocoagulation, mRNA levels of Wnt2b, Wnt3, Wnt5a, Wnt7a, and Wnt10b were increased. Wnt/ß-catenin signaling has a crucial role in restoration of hiPSC-RPE proliferation after laser photocoagulation. Manipulation of Wnt/ß-catenin signaling while elucidating the underlying mechanisms of RPE restoration might have a therapeutic potential in retinal degenerative diseases.

Transduction Patterns of Adeno-associated Viral Vectors in a Laser-Induced Choroidal Neovascularization Mouse Model.

Adeno-associated virus (AAV) vector is a promising platform technology for ocular gene therapy. Recently clinical successes to treat choroidal neovascularization (CNV) in wet type age-related macular degeneration have been reported. However, because pathologic conditions of the retina may alter the tropism of viral vectors, it is necessary to evaluate the transduction efficiency of different serotypes of AAV vectors in the retinas with CNVs. Here, we show the patterns and efficacy of transduction of AAV2, -5, and -8 vectors in a laser-induced CNV mouse model. C57BL/6J mice were subjected to unilateral laser photocoagulation on the right eye to induce CNV 5 days prior to intravitreal injection of AAV2, -5, and -8 capsids expressing EGFP. Transduction was increased around CNV lesions for all AAV capsid types, and AAV2 resulted in the highest transduction efficiency. In the absence of CNV, the AAV2 vector transduced ganglion and inner nuclear layer (INL) cells, and AAV5 and AAV8 transduced only a small proportion of cells in the retinal ganglion cell layer. CNV increased AAV2 vector expression throughout the retina and in and around CNVs; the transduced cells included retinal ganglion cells, Müller cells, cells from the INL and outer nuclear layer (ONL), photoreceptors, and retinal pigment epithelium (RPE) cells. Inflammatory cells and endothelial cells in CNVs were also transduced by AAV2. AAV5 and AAV8 were transduced in retinal ganglion, Müller, INL, ONL, and RPE cells in a localized pattern, and only endothelial cells at the surface of CNV lesions showed EGFP expression. Taken together, CNV formation resulted in enhanced transduction of AAV2, -5, and -8, and AAV2 exhibited the highest transduction efficiency in cells in CNV lesions.

Fit Characteristics of N95 Filtering Facepiece Respirators and the Accuracy of the User Seal Check among Koreans.

Adequate facepiece fit of N95 filtering facepiece respirators (FFRs) is crucial for optimal protection against airborne pathogens. The quantitative fit test (QNFT) pass rates of the 4 N95 FFR models commonly used in Korea were below 50%. Male sex was identified as a single independent predictive factor for QNFT pass. Infect Control Hosp Epidemiol 2018;39:104-107.

Heat-Killed Saccharomyces cerevisiae, A Dectin-1 Agonist, Selectively Induces IgG4 Production by Human B Cells.

Dectin-1 is a major receptor that recognizes fungal cell wall ß-glucan. We previously reported that heat-killed Saccharomyces cerevisiae (HKSC), a Dectin-1 agonist, selectively induces IgG1 class switching in mouse B cells. Dectin-1 is also expressed on human B cells; however, Dectin-1 function in human B cells remains unknown. This study aimed to investigate the direct effect of in vitro stimulation using HKSC on Ig class switching in human B cells. HKSC selectively induced the expression of germline γ4 transcripts (GLTγ4) by human B cell line 2E2, and HKSC significantly augmented GLTγ4 promoter activity. Moreover, HKSC selectively enhanced GLTγ4 expression and IgG4 production by anti-CD40-activated human tonsillar resting B cells. Thus, these results suggest that Dectin-1 maybe involved in selective IgG4 class switching by human B cells.

Aberrant expression of interleukin-10 and activation-induced cytidine deaminase in B cells from patients with Behçet's disease.

Despite extensive studies, the pathogenesis of Behçet's disease (BD) remains unclear. In particular, the roles of B cells in patients with BD have not been elucidated. Activation-induced cytidine deaminase (AID) is a critical enzyme for immunoglobulin (Ig) heavy chain class switching and somatic hypermutation in B cells and the abnormal expression of AID in various immune conditions has previously been studied. B10 cells, an interleukin (IL)-10-secreting subset of regulatory B cells, function to downregulate inflammation and autoimmunity. Thus, in the present study, the relevance of B cells in patients with BD was investigated. The plasma levels of IL-10 and IgA and the proportions of cluster of differentiation (CD)43+ B cells, excluding naïve B cells, were measured in 16 patients with BD and 16 age- and sex-matched healthy controls (HCs). Additionally, the mRNA levels of IL-10 and AID were assessed in B cells from fresh peripheral blood samples of the BD patients and HCs. The plasma level of IL-10 in patients with BD did not differ significantly from that in HCs. Similarly, there was no significant difference in the plasma level of IgA, although a slight increase was observed in patients with BD compared with that in HCs. There were no differences in CD43+CD19+ B cell numbers between patients with BD and HCs. However, IL-10 mRNA levels were significantly reduced (P<0.05), while AID mRNA levels were significantly increased (P<0.01) in the B cells of patients with BD compared with those in HCs. These results provide insight into the role of B cells in patients with BD.

Adeno-Associated Viral Vector-Mediated mTOR Inhibition by Short Hairpin RNA Suppresses Laser-Induced Choroidal Neovascularization.

Choroidal neovascularization (CNV) is the defining characteristic feature of the wet subtype of age-related macular degeneration (AMD) and may result in irreversible blindness. Based on anti-vascular endothelial growth factor (anti-VEGF), the current therapeutic approaches to CNV are fraught with difficulties, and mammalian target of rapamycin (mTOR) has recently been proposed as a possible therapeutic target, although few studies have been conducted. Here, we show that a recombinant adeno-associated virus-delivered mTOR-inhibiting short hairpin RNA (rAAV-mTOR shRNA), which blocks the activity of both mTOR complex 1 and 2, represents a promising therapeutic approach for the treatment of CNV. Eight-week-old male C57/B6 mice were treated with the short hairpin RNA (shRNA) after generating CNV lesions in the eyes via laser photocoagulation. The recombinant adeno-associated virus (rAAV) delivery vehicle was able to effectively transduce cells in the inner retina, and significantly fewer inflammatory cells and less extensive CNV were observed in the animals treated with rAAV-mTOR shRNA when compared with control- and rAAV-scrambled shRNA-treated groups. Presumably related to the reduction of CNV, increased autophagy was detected in CNV lesions treated with rAAV-mTOR shRNA, whereas significantly fewer apoptotic cells detected in the outer nuclear layer around the CNV indicate that mTOR inhibition may also have neuroprotective effects. Taken together, these results demonstrate the therapeutic potential of mTOR inhibition, resulting from rAAV-mTOR shRNA activity, in the treatment of AMD-related CNV.

Dectin-1 agonist selectively induces IgG1 class switching by LPS-activated mouse B cells.

Heat-killed Saccharomyces cerevisiae (HKSC) is an agonist for Dectin-1, a major fungal cell wall ß-glucan receptor. We previously reported that HKSC selectively enhances IgG1 production by LPS-activated mouse B cells. To determine if this IgG1 selectivity is caused by selective IgG1 class switching, we performed RT-PCRs for measuring germline transcripts (GLTs), flow cytometric analyses for detecting Ig-expressing cells, and ELISPOT assays for measuring the number of Ig-secreting cells in HKSC/LPS-stimulated mouse B cell cultures. HKSC selectively enhanced expression of GLTγ1, the number of IgG1-expressing cells, and the number of IgG1-secreting B cells in the presence of LPS stimulation. In addition, HKSC induced the expression of CD69, an activation marker for B lymphocytes, and the expression of surface Dectin-1. Two Dectin-1 antagonists, laminarin and a neutralizing Dectin-1 antibody, selectively diminished HKSC-reinforced IgG1 production by LPS-stimulated B cells. Furthermore, depleted zymosan (dzn), a Dectin-1 agonist with increased selectivity, also selectively enhanced GLTγ1 transcription. The Dectin-1 antagonists blocked dzn-induced IgG1 production by LPS-activated B cells. Collectively, these results suggest that Dectin-1 agonists selectively induce IgG1 class switching by direct stimulation of Dectin-1 on LPS-activated B cells resulting in selective production of IgG1.

Ginsenoside Rg1 and 20(S)-Rg3 Induce IgA Production by Mouse B Cells.

Ginsenosides are the major components of ginseng, which is known to modulate blood pressure, metabolism, and immune function, and has been used to treat various diseases. It has been reported that ginseng and several ginsenosides have immunoregulatory effects on the innate and T cell-mediated immune response. However, their effects on the humoral immune response have not been fully explored. The present study examined the direct effects of red ginseng extract (RGE) and ginsenosides on mouse B cell proliferation and on antibody production and the expression of germline transcripts (GLT) by mouse B cells in vitro. RGE slightly reduced B cell proliferation, but increased IgA production by LPS-stimulated B cells. Furthermore, ginsenoside Rg1 and 20(S)-Rg3 selectively induced IgA production and expression of GLTα transcripts by LPS-stimulated B cells. Collectively, these results suggest that ginsenoside Rg1 and 20(S)-Rg3 can drive the differentiation of B cells into IgA-producing cells through the selective induction of GLTα expression.

Characterization and cDNA cloning of a defensin-like peptide, harmoniasin, from Harmonia axyridis.

We compared the mRNA expression profile of the Harmonia axyridis larvae that were either untreated or treated with LPS. The extracted mRNAs were subjected to ACP RTPCR analysis using a combination of arbitrary primers and oligo (dT) primer. Among the 47 DEGs differentially expressed, we identified a cDNA showing homology with defensin-like antibacterial peptide. The cDNA showed a putative 32-residue signal sequence and a 50-residue mature peptide named harmoniasin. We also investigated the antibacterial activity of the harmoniasin analog, which exhibited potent antibacterial activities against Gramnegative and -positive bacteria strains and it also evidenced no hemolytic activity.