Novel extracellular role of REIC/Dkk-3 protein in PD-L1 regulation in cancer cells.

The adenovirus-REIC/Dkk-3 expression vector (Ad-REIC) has been the focus of numerous clinical studies due to its potential for the quenching of cancers. The cancer-suppressing mechanisms of the REIC/DKK-3 gene depend on multiple pathways that exert both direct and indirect effects on cancers. The direct effect is triggered by REIC/Dkk-3-mediated ER stress that causes cancer-selective apoptosis, and the indirect effect can be classified in two ways: (i) induction, by Ad-REIC-mis-infected cancer-associated fibroblasts, of the production of IL-7, an important activator of T cells and NK cells, and (ii) promotion, by the secretory REIC/Dkk-3 protein, of dendritic cell polarization from monocytes. These unique features allow Ad-REIC to exert effective and selective cancer-preventative effects in the manner of an anticancer vaccine. However, the question of how the REIC/Dkk-3 protein leverages anticancer immunity has remained to be answered. We herein report a novel function of the extracellular REIC/Dkk-3-namely, regulation of an immune checkpoint via modulation of PD-L1 on the cancer-cell surface. First, we identified novel interactions of REIC/Dkk-3 with the membrane proteins C5aR, CXCR2, CXCR6, and CMTM6. These proteins all functioned to stabilize PD-L1 on the cell surface. Due to the dominant expression of CMTM6 among the proteins in cancer cells, we next focused on CMTM6 and observed that REIC/Dkk-3 competed with CMTM6 for PD-L1, thereby liberating PD-L1 from its complexation with CMTM6. The released PD-L1 immediately underwent endocytosis-mediated degradation. These results will enhance our understanding of not only the physiological nature of the extracellular REIC/Dkk-3 protein but also the Ad-REIC-mediated anticancer effects. KEY MESSAGES: • REIC/Dkk-3 protein effectively suppresses breast cancer progression through an acceleration of PD-L1 degradation. • PD-L1 stability on the cancer cell membrane is kept high by binding with mainly CMTM6. • Competitive binding of REIC/Dkk-3 protein with CMTM6 liberates PD-L1, leading to PD-L1 degradation.

Changes in Inner and Outer Retinal Layer Thicknesses after Vitrectomy for Idiopathic Macular Hole: Implications for Visual Prognosis.

PURPOSE: To investigate sequential post-operative thickness changes in inner and outer retinal layers in eyes with an idiopathic macular hole (MH). METHODS: Retrospective case series. Twenty-four eyes of 23 patients who had received pars plana vitrectomy (PPV) for the closure of MH were included in the study. Spectral domain optical coherence tomography C-scan was used to automatically measure the mean thickness of the inner and outer retinal layers pre-operatively and up to 6 months following surgery. The photoreceptor outer segment (PROS) length was measured manually and was used to assess its relationship with best-corrected visual acuity (BCVA). RESULTS: Compared with the pre-operative thickness, the inner layers significantly thinned during follow-up (P = 0.02), particularly in the parafoveal (P = 0.01), but not perifoveal, area. The post-operative inner layer thinning ranged from the ganglion cell layer to the inner plexiform layer (P = 0.002), whereas the nerve fiber layer was unaltered. Outer layer thickness was significantly greater post-operatively (P = 0.002), and especially the PROS lengthened not only in the fovea but also in the parafovea (P < 0.001). Six months after surgery, BCVA was significantly correlated exclusively with the elongated foveal PROS (R = 0.42, P = 0.03), but not with any of the other thickness parameters examined. CONCLUSIONS: Following PPV for MH, retinal inner layers other than the nerve fiber layer thinned, suggestive of subclinical thickening in the inner layers where no cyst was evident pre-operatively. In contrast, retinal outer layer thickness significantly increased, potentially as a result of PROS elongation linking tightly with favorable visual prognosis in MH eyes.

Alteration of N-Glycan Profiles in Diabetic Retinopathy.

PURPOSE: To investigate the alteration of vitreal N-glycans in patients with proliferative diabetic retinopathy (PDR). METHODS: Plasma and vitreous samples were collected from 17 patients (10 females and 7 males) with PDR (PDR group) and 17 nondiabetic patients (8 females and 9 males) with epiretinal membrane (ERM) and idiopathic macular hole (MH) (non-diabetes mellitus [DM] group). Profiles of N-glycans were analyzed by a glycoblotting-based high-throughput protocol that we recently developed. Human retinal microvascular endothelial cells (HRMECs) were cultivated with culture media containing either low glucose (5 mM) or high glucose (25 mM), and expression levels of sialyltransferases were analyzed by real-time PCR and ELISA. RESULTS: Amount of N-glycans in the vitreous fluid of the PDR group was significantly higher than that of the non-DM group (495.5 ± 37.4 vs. 142.7 ± 30.8 pmol/100 µg protein, P < 0.005), whereas there was no significant difference in the plasma samples between the PDR and the non-DM group. In addition, profile analysis showed that N-glycans with sialic acids increased in the vitreous of the PDR group (328.4 ± 25.8 pmol/100 µg protein) compared to the non-DM group (92.1 ± 21.2 pmol/100 µg protein, P < 0.0005). Expression levels of sialyltransferases ST3GAL1 and ST3GAL4 were upregulated in the HRMECs after high-glucose stimulation. Consistent with the real-time PCR data, high-glucose stimulation elevated the protein levels of ST3GAL1 (117.4 ± 14.9 pg/mg, P < 0.01) and ST3GAL4 (6.1 ± 0.9 pg/mg, P < 0.05) in the HRMECs compared with the cells cultured with low-glucose culture media (ST3GAL1, 64.4 ± 5.8 pg/mg; ST3GAL4, 3.8 ± 0.3 pg/mg). CONCLUSIONS: Our data demonstrate distinct changes in the N-glycan profile and an increase in sialylated N-glycans in eyes with PDR.

A comparison of N-glycan profiles in human plasma and vitreous fluid.

PURPOSE: To investigate the concentration and composition of N-glycans in plasma and vitreous samples obtained from patients with non-proliferative vitreoretinal diseases. METHODS: Plasma and vitreous samples were collected from 11 patients with idiopathic macular hole (MH) and 9 patients with epiretinal membrane (ERM). The samples were pretreated for enzymatic cleaving, and subsequently glycans released from proteins were captured on BlotGlyco H beads. Sialic acids were methyl-esterified. Processed glycans were tagged with aminooxy-functionalized peptide reagent (aoWR) and released from the beads, followed by detection by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The concentration and composition of N-glycans in the samples were assessed. RESULTS: Concentration of N-glycans in vitreous samples (132 ± 29 pmol/100 µg protein) was significantly lower compared with those in plasma samples (714 ± 29 pmol/100 µg protein, p < 0.001). Predominant N-glycan in both plasma (39.7 ± 1.1 %) and vitreous fluid (37.2 ± 3.1 %) was identical, and the composition was presumed as [(Hex)2(HexNAc)2(NeuAc)2+ (Man)3(GlcNAc)2]. By contrast, the second-ranked N-glycan in vitreous samples (15.6 ± 1.5 %) was the seventh in plasma (2.3 ± 0.2 %). CONCLUSIONS: The current data provide useful information on N-glycan profile in the vitreous fluid, which is distinct from that in the plasma.

Pars plana vitrectomy for macular schisis associated with peripapillary intrachoroidal cavitation.

A case of retinoschisis associated with peripapillary intrachoroidal cavitation (ICC) was successfully treated with pars plana vitrectomy. A 70-year-old woman developed macular elevation and peripapillary ICC in her right eye. Optical coherence tomography revealed retinoschisis with retinal detachment in the macula. A microhole was observed within the myopic conus. A narrow passage was observed linking the ICC with the schisis cavity, complicated by a peripapillary epiretinal membrane tissue adjacent to the passage. The patient underwent vitrectomy with removal of the membrane tissue. Complete regression of the retinoschisis was observed, with closure of the connection channel between the ICC and schisis cavity. These findings suggest that the communication channel between the vitreous and schisis cavities was formed due to the traction exerted by the membrane tissue on the ICC that had generated a connecting path to the schisis cavity.

Correlation between decreased choroidal blood flow velocity and the pathogenesis of acute zonal occult outer retinopathy.

BACKGROUND: To evaluate changes of choroidal circulation quantitatively using laser speckle flowgraphy in patients with acute zonal occult outer retinopathy. DESIGN: Retrospective observational case series. PARTICIPANTS: Sixteen eyes of 11 acute zonal occult outer retinopathy patients: seven non-treated eyes with good visual acuity and nine systemic corticosteroid-treated eyes with progressive visual acuity loss. Six eyes with thyroid-associated ophthalmopathy receiving systemic corticosteroid therapy served as controls. METHODS: The mean blur rate, an index of quantitative relative blood flow velocity, in the affected area was measured by laser speckle flowgraphy. The changes of mean blur rate, best-corrected visual acuity and the average threshold at the affected area on Humphrey perimetry during 24-week follow up were analysed. MAIN OUTCOME MEASURES: Mean blur rate, best-corrected visual acuity, and the average threshold. RESULTS: In non-treated eyes, the average mean blur rate significantly increased at 24 weeks, with a significant increase of the average threshold. In corticosteroid-treated eyes, the mean blur rates at 1, 4, 12 and 24 weeks were significantly higher than the pretreatment value, with significant improvement of best-corrected visual acuity and the average threshold. The increase in mean blur rate at 4 weeks in corticosteroid-treated acute zonal occult outer retinopathy eyes was significantly higher than that in corticosteroid-treated control eyes. CONCLUSIONS: In eyes with acute zonal occult outer retinopathy, the mean blur rate at the affected area significantly increases along with improvement of visual functions. These results suggest that impaired choroidal circulation is involved in the pathogenesis of acute zonal occult outer retinopathy.

Retinal outer layer thickness increases after vitrectomy for epiretinal membrane, and visual improvement positively correlates with photoreceptor outer segment length.

PURPOSE: To investigate postoperative thickness changes in the retinal layers in eyes with epiretinal membrane (ERM). Correlations between these changes and visual outcomes were also examined. METHODS: Retrospective review of 25 eyes (24 patients) that had undergone pars plana vitrectomy for ERM and had a postoperative follow-up period ≥6 months. Optical coherence tomography (6 × 6 mm macular thickness map) was used to measure mean thickness of the inner and outer retinal layers 1 week and 1, 3, and 6 months following surgery. Photoreceptor outer segment (PROS) length was evaluated manually, and used to assess the association between best-corrected visual acuity (BCVA) and retinal layer thickness at the fovea. RESULTS: At 1 week and 1, 3, and 6 months, retinal layer thickness was 388, 377, 362, and 352 µm for the whole layer; 133, 115, 107, and 101 µm for the inner layer; 138, 145, 147, and 148 µm for the outer layer; and 28, 35, 36, and 40 µm for the PROS length, respectively. In comparison to 1-week data, the inner layers were significantly thinner at 1 month and later, as was the thickness of the entire retina. Outer layer thickness and PROS length were also significantly thicker at these time points. Six months following surgery, BCVA was significantly correlated with an elongated PROS length (R = 0.49, P = 0.01). CONCLUSION: Retinal outer layer thickness significantly increased following pars plana vitrectomy for ERM. Visual improvement was positively correlated with PROS length recovery.

Macular choroidal blood flow velocity decreases with regression of acute central serous chorioretinopathy.

AIM: To quantitatively evaluate the time course of macular choroidal blood flow velocity in acute central serous chorioretinopathy (CSC). METHODS: This retrospective observational case series included 21 eyes of 20 patients (17 men, 3 women; mean age, 53.0 years) with treatment-naïve acute CSC. Laser speckle flowgraphy was performed to calculate macular mean blur rate (MBR), an indicator of relative blood flow velocity at the first visit, 3 and 6 months thereafter. Changes in average MBR values were compared with visual improvement at 6 months. RESULTS: Subretinal fluid completely resolved in all eyes within 6 months, while best-corrected visual acuity (BCVA) significantly improved at 6 months compared to the initial BCVA. During the follow-up period, the average MBR significantly decreased to 92.8% and 82.3% at 3 and 6 months, respectively, against baseline (100%). Importantly, there was a negative correlation between the BCVA recovery and the MBR decrease, showing the possible association of MBR increase with poor visual prognosis. Multiple regression analysis demonstrated no significant correlation between MBR and ocular perfusion pressure. CONCLUSIONS: These results indicate that macular choroidal blood flow velocity decreases concurrently with regression of CSC, suggesting a validity of choroidal blood flow elevation in the pathogenesis of acute CSC.

Photodynamic therapy combined with intravitreal bevacizumab and sub-tenon triamcinolone acetonide injections for age-related macular degeneration.

PURPOSE: To report the results of triple therapy with photodynamic therapy (PDT) (PDT combined with intravitreal injection of bevacizumab (IVB) and sub-tenon injection of triamcinolone acetonide (STTA)) for the treatment of age-related macular degeneration (AMD) in Japanese patients. METHODS: This retrospective case series included 38 eyes of 38 patients with exudative AMD treated with PDT combined with IVB (1.25 mg) and STTA (40 mg). Retreatment was performed in the same manner with intervals of at least 3 months. All patients had been treatment naïve, with a follow-up period of 12 months. Best-corrected visual acuity (BCVA), macular retinal thickness (MRT) on optical coherence tomography, and the number of treatments were analyzed. RESULTS: The mean logarithm of the minimum angle of resolution BCVA in patients treated with PDT triple therapy was 0.86 ± 0.55 at baseline and 0.62 ± 0.55 at 12 months (p < 0.001). The mean MRT was 554.0 ± 202.6 µm at baseline and 205.1 ± 78.6 µm at 12 months (p < 0.001). During the 1-year follow-up, the average number of PDT triple therapy (treatments per patient) was 1.1. No complications, for example increase in intraocular pressure, cataract, or endophthalmitis, were observed. CONCLUSIONS: In AMD patients, PDT triple therapy significantly improved visual acuity with a minimum number of treatments and a low risk of complications during the 1-year follow-up.

Clinical Features of Central Retinal Vein Occlusion With Inflammatory Etiology.

PURPOSE: The purpose of this study was to characterize central retinal vein occlusion (CRVO) with inflammatory etiology. DESIGN: A retrospective study. METHODS: We retrospectively reviewed 51 eyes of 48 patients with CRVO between 2004 and 2009 and investigated whether they had inflammatory findings related to retinal vasculitis, that is, severe peripheral retinal vascular changes based on fluorescein angiography and anterior vitreous cells. The extent of vascular leakage was classified as "mild," "moderate," and "severe" in angiographic images. RESULTS: Six eyes of 5 patients (11.8%) had both inflammatory criteria. All patients were male and with a diagnosis of ischemic CRVO. No causative etiology was detected in any patient. The mean age was 46.4 (SD, 23.3) years. The age range was not limited to young generation. These patients received systemic corticosteroid therapy with a mean follow-up period of 41.8 months. The mean logMAR visual acuity [1.29 (SD, 0.77)] and central retinal thickness [1290.5 (SD, 399.0) µm)] at the initial visit were significantly (P = 0.004 and P = 0.001) improved at the final visit [0.94 (SD, 0.69) and 266.7 (SD, 135.4) µm, respectively]. CONCLUSIONS: Central retinal vein occlusion with idiopathic inflammatory conditions appears to have clinical characteristics of broad age range and good response to systemic corticosteroid therapy.