A single-cell transcriptomic atlas of the human ciliary body.

The ciliary body critically contributes to the ocular physiology with multiple responsibilities in the production of aqueous humor, vision accommodation and intraocular immunity. Comparatively little work, however, has revealed the single-cell molecular taxonomy of the human ciliary body required for studying these functionalities. In this study, we report a comprehensive atlas of the cellular and molecular components of human ciliary body as well as their interactions using single-cell RNA sequencing (scRNAseq). Cluster analysis of the transcriptome of 14,563 individual ciliary cells from the eyes of 3 human donors identified 14 distinct cell types, including the ciliary epithelium, smooth muscle, vascular endothelial cell, immune cell and other stromal cell populations. Cell-type discriminative gene markers were also revealed. Unique gene expression patterns essential for ciliary epithelium-mediated aqueous humor inflow and ciliary smooth muscle contractility were identified. Importantly, we discovered the transitional states that probably contribute to the transition of ciliary macrophage into retina microglia and verified no lymphatics in the ciliary body. Moreover, the utilization of CellPhoneDB allowed us to systemically infer cell-cell interactions among diverse ciliary cells including those that potentially participate in the pathogenesis of glaucoma and uveitis. Altogether, these new findings provide insights into the regulation of intraocular pressure, accommodation reflex and immune homeostasis under physiological and pathological conditions.

Th17 Activation and Th17/Treg Imbalance in Prolonged Anterior Intraocular Inflammation after Ocular Alkali Burn.

Ocular alkali burn (OAB) is a sight-threatening disease with refractory ocular inflammation causing various blinding complications. Th17 lymphocytes account for the pathogeneses of the autoimmune disease and chronic inflammation, but their role in prolonged anterior intraocular inflammation after OAB is still unknown. A rat OAB model was established for this purpose. Anterior intraocular inflammation was observed in both the acute and late phases of OAB, and histological examination confirmed the presence of inflammatory cell infiltration and fibrin exudation in the anterior segment. Luminex xMAP technology and qPCR were used to evaluate the intraocular levels of cytokines. The levels of IL-1ß, IL-6, and TNF-α were significantly elevated during the acute phase. The expression of IL-17A gradually increased from day 7 onwards and remained at a relatively high level. Immunofluorescence was performed to identify Th17 cells. CD4 and IL-17A double positive cells were detected in the anterior chamber from days 7 to 28. Flow cytometry showed that the frequency of Th17 cells increased in both lymph nodes and spleen, while the frequency of Treg cells remained unchanged, resulting in an elevated Th17/Treg ratio. The present study suggests that Th17 activation and Th17/Treg imbalance account for prolonged anterior intraocular inflammation after OAB.

METTL3 attenuates proliferative vitreoretinopathy and epithelial-mesenchymal transition of retinal pigment epithelial cells via wnt/ß-catenin pathway.

Proliferative vitreoretinopathy (PVR) is a refractory vitreoretinal fibrosis disease, and epithelial-mesenchymal transition (EMT) of retinal pigment epithelial (RPE) cells is the key pathological mechanism of PVR. However, few studies focused on the role of METTL3, the dominating methyltransferase for m6A RNA modification in PVR pathogenesis. Immunofluorescence staining and qRT-PCR were used to determine the expression of METTL3 in human tissues. Lentiviral transfection was used to stably overexpress and knockdown METTL3 in ARPE-19 cells. MTT assay was employed to study the effects of METTL3 on cell proliferation. The impact of METTL3 on the EMT of ARPE-19 cells was assessed by migratory assay, morphological observation and expression of EMT markers. Intravitreal injection of cells overexpressing METTL3 was used to assess the impact of METTL3 on the establishment of the PVR model. We found that METTL3 expression was less in human PVR membranes than in the normal RPE layers. In ARPE-19 cells, total m6A abundance and the METTL3 expression were down-regulated after EMT. Additionally, METTL3 overexpression inhibited cell proliferation through inducing cell cycle arrest at G0/G1 phase. Furthermore, METTL3 overexpression weakened the capacity of TGFß1 to trigger EMT by regulating wnt/ß -catenin pathway. Oppositely, knockdown of METTL3 facilitated proliferation and EMT of ARPE-19 cells. In vivo, intravitreal injection of METTL3-overexpressing cells delayed the development of PVR compared with injection of control cells. In summary, this study suggested that METTL3 is involved in the PVR process, and METTL3 overexpression inhibits the EMT of ARPE-19 cells in vitro and suppresses the PVR process in vivo.

Effect of vitrectomy with intrasurgical regulation of intraocular pressure in a rabbit model of central retinal artery occlusion.

Central retinal artery occlusion (CRAO) is an ophthalmic emergency that causes severe and permanent visual impairment. The effects of conventional treatments on recanalizing retinal arteries and improving visual outcome are equivocal. This study was designed to determine the possible benefits of pars plana vitrectomy (PPV) with intrasurgical regulation of intraocular pressure using intraocular vascular counterpulsation (IVT). CRAO was induced by 532-nm argon green laser activation of auricular intravenous injected rose bengal, a photosensitive dye, in the central retinal arteries (CRA) of eighty-four New Zealand white albino rabbits. CRAO rabbits were randomly assigned to photocoagulation, vitrectomy and counterpulsation groups. Depending on the time intervals between surgery and CRAO induction, vitrectomy and counterpulsation groups were further divided into 2 h (2h), 6 h (6h) and 24 h (24h) subgroups. The proportion of eyes with complete recanalization was significantly higher in the 2h counterpulsation subgroup after three days (P = 0.032) and in all counterpulsation subgroups after one week (P = 0.020). After one month, the 2h and 6h counterpulsation subgroups showed greater oscillatory potential (OPs) responses (F = 3.519, P = 0.049). The 2h counterpulsation subgroup also exhibited greater b-wave amplitude in photopic 3.0 Flicker(F = 4.530, P = 0.044). Histologic evaluation revealed less destruction in the inner retina for the 2h and 6h counterpulsation subgroups. Expression of HSP70 was higher in the 2h and 6h counterpulsation subgroups (F = 48.915,P < 0.001). Levels of HSP90 were lower in all counterpulsation subgroups (F = 30.065,P < 0.001). Levels of TNF-α were lower in the 2h counterpulsation subgroup (F = 14.762,P < 0.001). These results indicate that PPV with IVT was effective to recanalize retinal arteries after CRAO. Early intervention provided better morphologic and functional prognosis for inner retina. The protective effect was related with higher retinal levels of HSP70 and lower levels of HSP90 and TNF-α.

Shifts in renin-angiotensin system components, angiogenesis, and oxidative stress-related protein expression in the lamina cribrosa region of streptozotocin-induced diabetic mice.

PURPOSE: This study aimed to analyse shifts in renin-angiotensin system (RAS) components, angiogenesis, and oxidative stress-related protein expression in the lamina cribrosa (LC) region in streptozotocin (STZ)-induced diabetic mice. METHODS: Six months after diabetes induction, the retinal vessels of male C57BL/6 J mice were observed by colour photography, fundus fluorescein angiography (FFA), and immunofluorescent staining following incubation with CD31. Immunofluorescence for glial fibrillary acidic protein (GFAP), alpha-smooth muscle actin (α-SMA),and NG2 was also performed. Angiotensin-converting enzyme 1 (ACE1), angiotensin II type I receptor (AT1R), renin, hypoxia-inducible factor 1-alpha (HIF-1α), vascular endothelial growth factor (VEGF), vascular endothelial growth factor receptor 2 (VEGFR2), and haeme oxygenase 1 (HO-1) expression levels were confirmed by immunohistochemical and western blotting analyses. RESULTS: Compared with control mice, diabetic mice had significantly higher blood glucose concentrations (p < 0.001) and significantly lower body weights (p < 0.001). Colour photography and FFA did not reveal any vessel abnormalities in the diabetic mice; however, immunostaining of whole-mount retinas revealed an increased number of retinal vessels. Furthermore, histopathological staining showed significant reduction in the whole retinal thickness. GFAP expression was slightly higher, whereas fewer NG2+ pericytes were observed in diabetic mice than in control mice. ACE1, AT1R, renin, HIF-1α, VEGF, VEGFR2, and HO-1 expression were up-regulated in the LC of the STZ-induced diabetic mice. CONCLUSIONS: Collectively, ACE 1, AT1R, HIF-1α, VEGF, VEGFR2, and HO-1 activation in the LC region in diabetic mice may be involved in diabetes via the RAS and induction of angiogenesis and oxidative stress.

Retinal vessel oxygen saturation and vessel diameter in retinitis pigmentosa at various ages.

PURPOSE: This study was conducted to determine whether oxygen saturation and retinal blood vessel diameter are affected by retinitis pigmentosa (RP) at various ages. METHODS: Relative oxygen saturation was measured in retinal blood vessels in 68 RP patients and 136 normal subjects using the Oxymap T1 retinal oximeter. Subjects were divided into two age groups: Group A (20-40 years) and Group B (> 40 years). One randomly selected eye of each subject was used for statistical analysis. Student's t tests were used to analyze the mean saturation and diameter of retinal arterioles and venules and arteriovenous differences between RP and normal subjects in the two age groups. A Spearman test was used to analyze the correlation of mean saturation of retinal arterioles (AS) and arteriovenous differences (AVS) with visual acuity, disease duration, and electroretinogram (ERG) b-wave amplitude in patients with RP. RESULTS: AS was significantly higher in patients with RP (105.5 ± 9.4 %) than in normal subjects (94.5 ± 4.4 %, p = 0.000) in Group A, while in Group B, AS was significantly lower in RP patients (86.8 ± 10.3 %) than in healthy subjects (96.0 ± 4.8 %, p = 0.000). Vessel diameter was smaller in RP patients than in normal subjects. AS and AVS showed a negative correlation with disease duration and a tendency toward positive correlation with ERG b-wave in patients with RP. CONCLUSIONS: The shifting characteristics of retinal vessel oxygen saturation suggest that the pathological mechanism of retinal oxygen metabolic disorder differs by age in patients with RP.

Cytarabine chemotherapy induces meibomian gland dysfunction.

PURPOSE: Cytarabine (Ara-C) chemotherapy causes symptoms resembling meibomian gland dysfunction (MGD), suggesting potential associations between Ara-C and MGD. In this study, the pathological effects of Ara-C on MGD were investigated in a rodent model. METHODS: Mice received Ara-C with or without rosiglitazone (PPARγ agonist) for 7 consecutive days. Slit-lamp biomicroscope was used for ocular examinations. Immunofluorescence detected acinar cell proliferation, differentiation, and ductal keratinization in the meibomian gland (MG). Lipid accumulation was evaluated by Oil Red O and LipidTox staining. Lipogenic status, FoxO1/FoxO3a cellular localization, and oxidative stress were visualized via immunohistochemistry. Western blotting assessed relative protein expression and AKT/FoxO1/FoxO3a pathway phosphorylation. RESULTS: Ara-C (50 mg/kg) did not affect mouse survival but induced damage to ocular surface microenvironment, including corneal epithelial defects, MG orifice plugging and acinar dropout, and lacrimal gland (LG) dysfunction. Ara-C intervention inhibited proliferation and caused progenitor loss in the MG, as evidenced by reduced PCNA + labeling and P63+/Lrig1+ basal cell numbers. The MG ducts of Ara-C-treated mice exhibited marked dilatation, lipid deposition, and hyperkeratinization (K1/K10 overexpression). Ara-C disrupted MG lipid metabolism by downregulating PPARγ and its downstream lipogenic targets AWAT2/SOAT1/ELOVL4 and upregulating HMGCR. Dephosphorylation of AKT and the subsequent nuclear translocation of FoxO1/FoxO3a contributed to Ara-C-induced PPARγ downregulation. Ara-C triggered oxidative stress with increases in 4-HNE and 8-OHdG and Keap1/Nrf2/HO-1/SOD1 axis dysregulation. Rosiglitazone treatment ameliorated MGD-associated pathological manifestations, LG function, MG lipid metabolism, and oxidative stress in Ara-C-exposed mice. CONCLUSIONS: Systemic Ara-C chemotherapy exerted topical cytotoxic effects on the ocular surface, and PPARγ restoration by rosiglitazone mitigated Ara-C-induced MGD alterations.

Extraction from different parts of Citrus maxima flowers using ultrasound as an aid and study of their composition and function.

Ultrasonic assisted extraction is frequently referred to as a green environmental protection method. The flower of Citrus maxima (FCM) has been used as a health tea drink in China, although the tea drink lacks clear compound composition identification and functional research. In order to fully use Citrus fruit by-products and further explore the functional features of FCM, this paper isolated, identified, and assessed the chemical compounds in the petals, stems, styles, receptacles, stamens, and buds of FCM extract. There are 88 compounds were recovered, including 23 compounds in the bud, 21 compounds in the petal, 19 compounds in the stem, 11 compounds in the receptacle, 20 compounds in the stamen, and 13 compounds in the style. Antioxidant experiments revealed that the FCM's various compounds had observable impacts in scavenging free radicals (38.44%-58.35%). The aforementioned study demonstrates that the pomelo by-products were developed into useful components using ultrasonic aided extraction technique. FCM has flavor-rich compounds that make it suited for use as an antioxidant tea beverage and offers practical suggestions for preparing healthy products.

Intravitreal slow-release dexamethasone alleviates traumatic proliferative vitreoretinopathy by inhibiting persistent inflammation and Müller cell gliosis in rabbits.

AIM: To evaluate the effects of intravitreal slow-release dexamethasone on traumatic proliferative vitreoretinopathy (PVR) and Müller cell gliosis and preliminarily explored the possible inflammatory mechanism in a rabbit model induced by penetrating ocular trauma. METHODS: Traumatic PVR was induced in the right eyes of pigmented rabbits by performing an 8-mm circumferential scleral incision placed 2.5 mm behind the limbus, followed by treatment with a slow-release dexamethasone implant (Ozurdex) or sham injection. Left eyes were used as normal controls. The intraocular pressure (IOP) was monitored using an iCare tonometer. PVR severity was evaluated via anatomical and histopathological examinations every week for 6wk; specific inflammatory cytokine and proliferative marker levels were measured by quantitative real-time polymerase chain reaction, Western blot, protein chip analysis, or immunofluorescence staining. RESULTS: During the observation period, PVR severity gradually increased. Intense Müller cell gliosis was observed in the peripheral retina near the wound and in the whole retina of PVR group. Ozurdex significantly alleviated PVR development and Müller cell gliosis. Post-traumatic inflammation fluctuated and was persistent. The interleukin-1ß (IL-1ß) mRNA level was significantly upregulated, peaking on day 3 and increasing again on day 21 after injury. The expression of nod-like receptor family pyrin domain containing 3 (NLRP3) showed a similar trend that began earlier than that of IL-1ß expression. Ozurdex suppressed the expression of IL-1ß, NLRP3, and phosphorylated nuclear factor-kappa B (NF-κB). The average IOP after treatment was within normal limits. CONCLUSION: The present study demonstrates chronic and fluctuating inflammation in a traumatic PVR rabbit model over 6wk. Ozurdex treatment significantly inhibites inflammatory cytokines expression and Müller cell gliosis, and thus alleviates PVR severity. This study highlights the important role of IL-1ß, and Ozurdex inhibites inflammation presumably via the NF-κB/NLRP3/IL-1ß inflammatory axis. In summary, Ozurdex provides a potential therapeutic option for traumatic PVR.

Optic nerve head astrocytes contribute to vascular associated effects.

Purpose: This study was conducted in order to test the expression of vasoactive substances within rat lamina cribrosa (LC) and optic nerve head (ONH) astrocytes, so as to investigate the role and potential mechanism of ONH astrocytes in vascular associated effects. Methods: LC tissue sections and primary cultured ONH astrocytes were obtained from adult Sprague-Dawley (SD) rats. Immunofluorescent staining was then used to detect the expression of vasoactive substances. Hyperoxia exposure was carried out both in vivo and in vitro, after which nitric oxide (NO) levels in LC tissue and cell supernatant were detected. The variations of protein and gene expression associated with vasoactive substances were subsequently tested. ONH astrocytes and vascular smooth muscle cells (VSMCs) were then incubated in a direct co-culture manner. Morphological parameters of VSMCs were finally analyzed in order to evaluate cell contraction. Results: Endothelin-1 (ET-1), nitric oxide synthase (NOS) and renin-angiotensin system (RAS) were detected in both LC tissue and ONH astrocytes. Retinal vessel diameter was found obviously decreased following hyperoxia exposure. Moreover, hyperoxia inhibited NO production both in vivo and in vitro. ET-1 and RAS elements were observed to be upregulated, whereas NOS was downregulated. In ONH astrocytes and VSMCs co-culture system, the length-to-width ratio of VSMCs was shown to significantly increase on days 3 and 7 in hyperoxia compared with normoxia. Conclusions: There is an abundance of expression of vasoactive substances within LC tissue and ONH astrocytes. The contractile response of VSMCs in the co-culture system provided direct evidence for the involvement of ONH astrocytes in vascular associated effects, which may signify a potentially novel direction for future research.

Safety assessment of polymeric micelles as an ophthalmic drug delivery system for intravitreal administration of dasatinib.

BACKGROUND AND AIMS: Ocular safety/biocompatibility is an essential element of ophthalmic drug delivery. We previously applied poly(ethylene glycol)-block-poly(ɛ-caprolactone) (PEG-b-PCL) micelles to deliver dasatinib for the management of proliferative vitreoretinopathy (PVR) in vitro. Herein, we seek to ascertain the ocular safety/compatibility of blank and dasatinib loaded PEG-b-PCL micelles, which will set the stage for the future in vivo efficacy evaluations and/or clinical translation for PVR or other eye diseases. METHODS: To access the safety of blank and dasatinib loaded micelles, in vitro cell based assays (LDH cell membrane damage test, SRB cytotoxicity, TEER and permeability of RPE tight junctions), in vivo slit lamp biomicroscopy and optical coherence tomography, Ex vivo histology (H&E staining, GFAP immunofluorescence staining and TUNEL assay) were undertaken. RESULTS: Both blank and dasatinib loaded micelles showed remarkable safety profiles at cellular levels. They also caused negligible ocular toxicity/abnormalities up to 28 days post-intravitreal injection in mice. The micelles did not insult the cornea, as demonstrated by slit-lamp biomicroscopy. Ex vivo histology and in vivo optical coherence tomography revealed a normal retinal structure with minimal apoptosis and stresses. CONCLUSION: Taken together, both blank and dasatinib loaded micelles appear to be safe and their applications in drug delivery for eye diseases should be explored.

HLA-A, -B, and -DRB1 gene polymorphism in the Miao ethnic group of Hunan, China.

OBJECTIVE: To investigate HLA-A, -B, and -DRB1 gene polymorphism in the Miao ethnic group in Hunan, China. METHODS: PCR-sequence specific oligonucleotide probes (SSO) reverse flow chip method was used to type HLA-A, -B, and -DRB1 genes of 154 unrelated healthy Miao ethnic individuals in Hunan. The allele and haplotype frequencies were calculated and compared with other populations in China. RESULTS: A total of 10 HLA-A, 25 HLA-B, and 13 HLA-DRB1 alleles were observed in the population. The higher frequency alleles included A 11(38.96%), A 02(27.27%), A 24(18.83%), B 40(60)(17.53%), B 46(13.96%), B 15(75)(11.69%), DRB1 09(15.26%), DRB1 12(15.26%), DRB1 15(15.26%), and DRB1 04(12.66%). The most frequent haplotypes were A11-B60(9.60%), A2-B46(9.27%), A11-B75(9.22%), B75-DR12(6.31%), A11-DR12(9.62%), A11-DR4(9.07%), A11-DR15(6.69%), A11-B75-DR12(5.43%), A11-B60-DR4(4.24%), and A2-B46-DR9(3.71%). Compared with other populations in China, HLA gene polymorphism of Hunan Miao population was close to that of southern population. CONCLUSION: HLA loci are highly polymorphic in the Miao population of Hunan, and their distribution has the character of South China population.

Artesunate induces mitochondria-mediated apoptosis of human retinoblastoma cells by upregulating Kruppel-like factor 6.

Retinoblastoma (RB) is the most common primary intraocular malignancy in children. Intravitreal chemotherapy achieves favorable clinical outcomes in controlling RB vitreous seeds, which are a common reason for treatment failure. Thus, a novel, effective and safe intravitreal chemotherapeutic drug is urgently required. The malaria drug artesunate (ART) recently demonstrated remarkable anticancer effects with mild side effects. The purpose of this study is to investigate the anti-RB efficacy, the underlying mechanism and the intraocular safety of ART. Herein, we verified that ART inhibits RB cell viability and induces cell apoptosis in a dose- and time-dependent manner. Microarray analysis revealed that Kruppel-like factor 6 (KLF6) was upregulated after ART treatment, and this was further confirmed by real-time PCR and western blot assays. Silencing of KLF6 expression significantly reversed ART-induced RB cell growth inhibition and apoptosis. Furthermore, ART activated mitochondria-mediated apoptosis of RB cells, while silencing KLF6 expression significantly inhibited this effect. In murine xenotransplantation models of RB, we further confirmed that ART inhibits RB tumor growth, induces tumor cell apoptosis and upregulates KLF6 expression. In addition, KLF6 silencing attenuates ART-mediated inhibition of tumor growth in vivo. Furthermore, we proved that intravitreal injection of ART in Sprague-Dawley (SD) rats is safe, with no obvious retinal function damage or structural disorders observed by electrophysiology (ERG), fundal photographs, fundus fluorescein angiography (FFA) or optical coherence tomography (OCT) examinations. Collectively, our study revealed that ART induces mitochondrial apoptosis of RB cells via upregulating KLF6, and our results may extend the application of ART to the clinic as an effective and safe intravitreal chemotherapeutic drug to treat RB, especially RB with vitreous seeds.

A Pilot Clinical Study of Intravitreal Injection of Artesunate for Ocular Neovascularization.

Purpose: To evaluate the efficacy and primary safety of treatment with artesunate in reducing ocular neovascularization in humans. Methods: Five patients with corneal, iris, and retinal neovascularization and no light perception were treated with intravitreal injections of artesunate 80 µg. Visual acuity, anterior segment photography, fundus fluorescein angiography, and optical coherence tomography were used to evaluate efficacy, while intraocular pressure (IOP) and lens opacity degree were employed to evaluate safety. The primary endpoint was attenuation of neovascularization as determined at 24 weeks, with the last posttreatment follow-up at 52 weeks. Results: Corneal and iris neovascularization, which were secondary to fundus ischemic diseases and retinal neovascularization in all 5 patients, were attenuated after 1 or 2 injections by the 52-week follow-up. Retinal neovascularization was also attenuated, and papilledema was alleviated. The average IOP fell from 25.5 mmHg to 17.66 mmHg. Conclusions: This pilot study determined that intravitreal artesunate injection is efficacious for reducing corneal, iris, and retinal neovascularization. These results indicate that this drug may be a novel alternative to the currently popular antivascular endothelial growth factor drugs used to suppress ocular neovascularization and improve visual function.

Intravitreal anti-VEGF agents, oral glucocorticoids, and laser photocoagulation combination therapy for macular edema secondary to retinal vein occlusion: preliminary report.

AIM: To evaluate the efficacy and safety of combined anti-vascular endothelial growth factor (VEGF) agents, oral glucocorticoid, and laser photocoagulation therapy for macular edema (ME) secondary to retinal vein occlusion (RVO). METHODS: This study included 16 eyes of 16 patients with RVO-associated ME. Patients were initially treated with oral prednisone and an intravitreal anti-VEGF agent. Two weeks later, patients underwent standard laser photocoagulation. Best-corrected visual acuity (BCVA), central retinal thickness (CRT), and retinal vessel oxygenation were examined over 12mo. RESULTS: Patients received 1.43±0.81 anti-VEGF injections. Mean baseline and 12-month logMAR BCVA were 0.96±0.51 (20/178) and 0.31±0.88 (20/40), respectively, in eyes with central retinal vein occlusion (CRVO) (P<0.00), and 1.02±0.45 (20/209) and 0.60±0.49 (20/80), respectively, in eyes with branch retinal vein occlusion (BRVO) (P<0.00). At 12mo, CRT had significantly decreased in eyes with CRVO (P<0.00) and BRVO (P<0.00). Venous oxygen saturation had significantly increased in eyes with CRVO (P<0.00) and BRVO (P<0.00). No examined parameters were significantly different between the 2 RVO groups. No serious adverse effects occurred. CONCLUSION: Anti-VEGF, glucocorticoid, and photocoagulation combination therapy improves visual outcome, prolongs therapeutic effect, and reduces the number of intravitreal injections in eyes with RVO-associated ME.

The FOP Connection Registry: Design of an international patient-sponsored registry for Fibrodysplasia Ossificans Progressiva.

The Fibrodysplasia Ossificans Progressiva (FOP) Connection Registry is an international, voluntary, observational study that directly captures demographic and disease information initially from patients with FOP (the patient portal) and in the near future from treating physicians (the physician portal) via a secure web-based tool. It was launched by the International FOP Association (IFOPA) with a guiding vision to develop and manage one unified, global, and coordinated Registry allowing the assembly of the most comprehensive data on FOP. This will ultimately facilitate greater access and sharing of patient data and enable better and faster development of therapies and tracking their long-term treatment effectiveness and safety. This report outlines the FOP Connection Registry's design and procedures for data collection and reporting, as well as the long-term sustainability of Registry. Patient-reported, aggregate data are summarized for the first 196 enrolled patients, representing participation from 42 countries and approximately 25% of the world's known FOP population. Fifty-seven percent of the current Registry participants are female with a mean age of 23.8years (median=21years, range=1, 76years). Among the Registry participants who provided their FOP type, 51% reported FOP Classic (R206H), 41% reported FOP Type Unknown, and 8% reported FOP Variant. Patients reported 5.4years (median=3.0years, range=0, 45.8years) as the mean age at which they noticed their first FOP symptoms and a mean age at final FOP diagnosis of 7.5years (median=5.0years, range=0.1, 48.4years). Information on the patients' diagnostic journeys in arriving at a correct diagnosis of FOP is also presented. These early patient-reported data suggest that the IFOPA's vision of one, unified, global, and coordinated approach to the FOP Connection Registry is well underway to being realized. In addition, the positive response from the FOP patient community to the initial launch of the Registry's patient portal has created a solid foundation upon which to build the largest international registry for monitoring the clinical progression of FOP among patients.

Chemical genetic transcriptional fingerprinting for selectivity profiling of kinase inhibitors.

The importance of protein kinases as a major class of drug targets across multiple diseases has generated a critical need for technologies that enable the identification of potent and selective kinase inhibitors. Bruton's tyrosine kinase (Btk) is a compelling drug target in multiple therapeutic areas, including systemic lupus erythematosus, asthma, rheumatoid arthritis, and B cell malignancies. We have combined potent, selective kinase inhibition through chemical genetics with gene expression profiling to identify a "fingerprint" of transcriptional changes associated with selective Btk kinase inhibition. The Btk transcriptional fingerprint shows remarkable relevance for Btk's biological roles and was used for functional selectivity profiling of two kinase inhibitor compounds. The fingerprint was able to rank the compounds by relative selectivity for Btk, and revealed broader off-target effects than observed in a broad panel of biochemical kinase cross screens. In addition to being useful for functional selectivity profiling, the fingerprint genes are themselves potential preclinical and clinical biomarkers for developing Btk-directed therapies.

Macular Retinal Vessel Oxygen Saturation Elevation in Chinese Central Serous Chorioretinopathy.

PURPOSE: To evaluate the retinal vessel oxygen saturation in central serous chorioretinopathy (CSC) cases among the Chinese. METHODS: Relative oxygen saturation of retinal blood vessels was measured in 33 Chinese patients with single-eye CSC using the Oxymap T1 retinal oximeter. The contralateral eyes were the control. The mean saturation of the retinal arteriole (AS) and venule (VS), arteriovenous difference (AVS), and arteriole and venule diameters (AD, VD) was analyzed in the optic disc area and macular region. RESULTS: In the optic disc area, the inferotemporal quadrant (TI) AS (93.2 ± 10.2%) and inferonasal quadrant (NI) VS (61.3 ± 7.3%) were higher in the affected eyes than in the contralateral eyes (88.7 ± 7.7% and 56.9 ± 6.5%) and AVS in NI (36.7 ± 10.4%) decreased compared to the contralateral eyes (41.5 ± 11.2%). The VD in TI was expanded (19.9 ± 2.5 pixels versus 18.1 ± 3.4 pixels). Around the macular region, AS was 93.6 ± 7.6%, higher than in the contralateral eyes (89.5 ± 6.3%). No other significant changes were found. CONCLUSIONS: AS increased in the TI, and VS decreased in the NI in the eyes with CSC. In addition, AS also increased around the macular region, suggesting that these are contributors to CSC pathophysiology.

The Expansion of Heterotopic Bone in Fibrodysplasia Ossificans Progressiva Is Activin A-Dependent.

Fibrodysplasia ossificans progressiva (FOP) is a rare autosomal dominant disorder that is characterized by episodic yet cumulative heterotopic ossification (HO) in skeletal muscles, tendons, and ligaments over a patient's lifetime. FOP is caused by missense mutations in the type I bone morphogenetic protein (BMP) receptor ACVR1. We have determined that the formation of heterotopic bone in FOP requires activation of mutant ACVR1 by Activin A, in part by showing that prophylactic inhibition of Activin A blocks HO in a mouse model of FOP. Here we piece together a natural history of developing HO lesions in mouse FOP, and determine where in the continuum of HO Activin A is required, using imaging (T2-MRI, µCT, 18 F-NaF PET/CT, histology) coupled with pharmacologic inhibition of Activin A at different times during the progression of HO. First, we show that expansion of HO lesions comes about through growth and fusion of independent HO events. These events tend to arise within a neighborhood of existing lesions, indicating that already formed HO likely triggers the formation of new events. The process of heterotopic bone expansion appears to be dependent on Activin A because inhibition of this ligand suppresses the growth of nascent HO lesions and stops the emergence of new HO events. Therefore, our results reveal that Activin A is required at least up to the point when nascent HO lesions mineralize and further demonstrate the therapeutic utility of Activin A inhibition in FOP. These results provide evidence for a model where HO is triggered by inflammation but becomes "self-propagating" by a process that requires Activin A. © 2017 The Authors. Journal of Bone and Mineral Research Published by Wiley Periodicals Inc.

Small Molecular-Sized Artesunate Attenuates Ocular Neovascularization via VEGFR2, PKCα, and PDGFR Targets.

Ocular neovascularization (NV) is the primary cause of blindness in many ocular diseases. Large molecular weight anti- vascular endothelial growth factor (VEGF) protein drugs, such as Avastin and Lucentis, have saved the vision of millions. However, approximately 20-30% of patients respond poorly to anti-VEGF treatment. We found that artesunate (ART), a small molecular derivative of artemisinin, had a significant inhibitory effect on ocular NV by downregulating the expression of VEGFR2, PKCα, and PDGFR. ART significantly inhibited retinal NV in rabbits and macular edema in monkeys with greater anterior chamber penetrability and more durable efficacy than Avastin. Our pilot study showed that intravitreal injection of 80 µg ART significantly inhibited iris and corneal NV in a severe retinal detachment case. Our results suggest that ART might be a potential persistent small-molecule drug to manage ocular NV via multi-targets.