Development of finely tuned liposome nanoplatform for macrophage depletion.

BACKGROUND: Immunotherapy with clodronate-encapsulated liposomes, which induce macrophage depletion, has been studied extensively. However, previously reported liposomal formulation-based drugs (Clodrosome® and m-Clodrosome®) are limited by their inconsistent size and therapeutic efficacy. Thus, we aimed to achieve consistent therapeutic effects by effectively depleting macrophages with uniform-sized liposomes. RESULTS: We developed four types of click chemistry-based liposome nanoplatforms that were uniformly sized and encapsulated with clodronate, for effective macrophage depletion, followed by conjugation with Man-N3 and radiolabeling. Functionalization with Man-N3 improves the specific targeting of M2 macrophages, and radioisotope labeling enables in vivo imaging of the liposome nanoplatforms. The functionalized liposome nanoplatforms are stable under physiological conditions. The difference in the biodistribution of the four liposome nanoplatforms in vivo were recorded using positron emission tomography imaging. Among the four platforms, the clodronate-encapsulated mannosylated liposome effectively depleted M2 macrophages in the normal liver and tumor microenvironment ex vivo compared to that by Clodrosome® and m-Clodrosome®. CONCLUSION: The newly-developed liposome nanoplatform, with finely tuned size control, high in vivo stability, and excellent ex vivo M2 macrophage targeting and depletion effects, is a promising macrophage-depleting agent.

Supramolecular Senolytics via Intracellular Oligomerization of Peptides in Response to Elevated Reactive Oxygen Species Levels in Aging Cells.

Senolytics, which eliminate senescent cells from tissues, represent an emerging therapeutic strategy for various age-related diseases. Most senolytics target antiapoptotic proteins, which are overexpressed in senescent cells, limiting specificity and inducing severe side effects. To overcome these limitations, we constructed self-assembling senolytics targeting senescent cells with an intracellular oligomerization system. Intracellular aryl-dithiol-containing peptide oligomerization occurred only inside the mitochondria of senescent cells due to selective localization of the peptides by RGD-mediated cellular uptake into integrin αvß3-overexpressed senescent cells and elevated levels of reactive oxygen species, which can be used as a chemical fuel for disulfide formation. This oligomerization results in an artificial protein-like nanoassembly with a stable α-helix secondary structure, which can disrupt the mitochondrial membrane via multivalent interactions because the mitochondrial membrane of senescent cells has weaker integrity than that of normal cells. These three specificities (integrin αvß3, high ROS, and weak mitochondrial membrane integrity) of senescent cells work in combination; therefore, this intramitochondrial oligomerization system can selectively induce apoptosis of senescent cells without side effects on normal cells. Significant reductions in key senescence markers and amelioration of retinal degeneration were observed after elimination of the senescent retinal pigment epithelium by this peptide senolytic in an age-related macular degeneration mouse model and in aged mice, and this effect was accompanied by improved visual function. This system provides a strategy for the treatment of age-related diseases using supramolecular senolytics.

Mannosylated-serum albumin nanoparticle imaging to monitor tumor-associated macrophages under anti-PD1 treatment.

BACKGROUND: Immune checkpoint inhibitors such as anti-programmed cell death protein 1 (PD1) block tumor growth by reinvigorating the immune system; however, determining their efficacy only by the changes in tumor size may prove inaccurate. As the immune cells including macrophages in the tumor microenvironment (TME) are associated with the response to anti-PD1 therapy, tumor-associated macrophages (TAMs) imaging using nanoparticles can noninvasively provide the immune enrichment status of TME. Herein, the mannosylated-serum albumin (MSA) nanoparticle was labeled with radioactive isotope 68Ga to target the mannose receptors on macrophages for noninvasive monitoring of the TME according to anti-PD1 therapy. RESULTS: B16F10-Luc and MC38-Luc tumor-bearing mice were treated with anti-PD1, and the response to anti-PD1 was determined by the tumor volume. According to the flow cytometry, the responders to anti-PD1 showed an increased proportion of TAMs, as well as lymphocytes, and the most enriched immune cell population in the TME was also TAMs. For noninvasive imaging of TAMs as a surrogate of immune cell augmentation in the TME via anti-PD1, we acquired [68Ga] Ga-MSA positron emission tomography. According to the imaging study, an increased number of TAMs in responders at the early phase of anti-PD1 treatment was observed in both B16F10-Luc and MC38-Luc tumor-bearing mice models. CONCLUSION: As representative immune cells in the TME, non-invasive imaging of TAMs using MSA nanoparticles can reflect the immune cell enrichment status in the TME closely associated with the response to anti-PD1. As non-invasive imaging using MSA nanoparticles, this approach shows a potential to monitor and evaluate anti-tumor response to immune checkpoint inhibitors.

QUANTIFIED ANASTOMOTIC AREAS OF NEOVASCULARIZATION AS FACTORS ASSOCIATED WITH FREQUENT RECURRENCE IN NEOVASCULAR AGE-RELATED MACULAR DEGENERATION.

PURPOSE: To investigate the quantitative characteristics of anastomoses of macular neovascularization (MNV) in neovascular age-related macular degeneration using optical coherence tomography angiography according to the frequency of intravitreal injections. METHODS: Eighty-six eyes of 86 patients treated for neovascular age-related macular degeneration were classified into two groups based on the number of intravitreal injections administered over 12 months: stable (<3) and unstable (≥3). Anastomotic areas were defined as areas surrounded by neighboring vessels in the MNV; their total number, mean area, maximal and minimal diameters (i.e., maximal and minimum Feret diameters), and ratio (Feret aspect ratio) were analyzed in the inner and outer areas of the MNV. RESULTS: Forty-four and 42 eyes were classified into the stable and unstable groups, respectively. The eyes in the unstable group had larger anastomotic areas with longer minimum Feret diameters and longer perimeters in the outer MNV. In the logistic regression analysis, instability was associated with a larger anastomotic area and a longer minimum Feret diameter in the outer MNV. Multivariate analysis revealed that a longer minimum Feret diameter in the outer MNV was the most significant factor ( P = 0.03). CONCLUSION: The quantitative characteristics of the anastomotic areas in the MNV might indicate the need for intravitreal injections in patients with neovascular age-related macular degeneration.

Accelerated aging phenotypes in the retinal pigment epithelium of Zmpste24-deficient mice.

Age-related macular degeneration (AMD) is a chronic and progressive disease characterized by degeneration of the retinal pigment epithelium (RPE) and retina that ultimately leads to loss of vision. The pathological mechanisms of AMD are not fully known. Cellular senescence, which is a state of cell cycle arrest induced by DNA-damage or aging, is hypothesized to critically affect the pathogenesis of AMD. In this study, we examined the relationship between cellular senescence and RPE/retinal degeneration in mouse models of natural aging and accelerated aging. We performed a bulk RNA sequencing of the RPE cells from adult (8 months old) and naturally-aged old (24 months old) mice and found that common signatures of senescence and AMD pathology - inflammation, apoptosis, and blood vessel formation - are upregulated in the RPE of old mice. Next, we investigated markers of senescence and the degree of RPE/retinal degeneration in Zmpste24-deficient (Zmpste24-/-) mice, which is a model for progeria and accelerated aging. We found that Zmpste24-/- mice display markedly greater level of senescence-related markers in RPE and significant RPE/retinal degeneration compared to wild-type mice, in a manner consistent with natural aging. Overall, these results provide support for the association between cellular senescence of RPE and the pathogenesis of AMD, and suggest the use of Zmpste24-/- mice as a novel senescent RPE model of AMD.

Evaluation of safety and pharmacokinetics of bismuth-containing quadruple therapy with either vonoprazan or lansoprazole for Helicobacter pylori eradication.

AIMS: Helicobacter pylori (Hp) eradication plays a key role in the treatment and prevention of peptic ulcer diseases. Increasing clarithromycin resistance in Hp necessitates more effective treatments for eradication, such as bismuth-containing quadruple therapy. We aimed to compare the safety and pharmacokinetics (PK) of bismuth between vonoprazan- and lansoprazole-containing quadruple therapy in Hp-positive subjects. METHODS: In this randomised, double-blind, parallel-group study, Hp-positive subjects were randomised to receive vonoprazan- or lansoprazole-containing quadruple therapy. Each subject received vonoprazan 20 mg or lansoprazole 30 mg combined with bismuth 220 mg, clarithromycin 500 mg and amoxicillin 1000 mg twice daily for 14 days. Blood sampling and urine collection for bismuth PK were conducted predose and up to 12 hours postdose at steady-state. The PK parameters of bismuth were derived using a noncompartmental method and compared between treatments. An exploratory breath test for Hp was conducted at screening and at the follow-up visit on day 42. Safety was assessed by adverse event monitoring, physical examinations, vital signs, 12-lead electrocardiograms and clinical laboratory tests. RESULTS: A total of 30 subjects were randomised and 26 subjects completed the study (12 in the vonoprazan group and 14 in the lansoprazole group). The systemic exposure of bismuth in the 2 treatments was comparable (~5% difference). All subjects turned negative for Hp at the follow-up visit. No significant difference in safety profiles was noted between the 2 treatments. CONCLUSION: The systemic exposure of bismuth was similar between vonoprazan- and lansoprazole-containing quadruple therapy. Vonoprazan-containing quadruple therapy was safe and well tolerated.

Randomized controlled trial of Regulation Focused Psychotherapy for children: A manualized psychodynamic treatment for externalizing behaviors.

OBJECTIVE: This article examines outcomes of the first randomized controlled trial of Regulation Focused Psychotherapy for Children (RFP-C), a manualized, short-term, psychodynamic intervention for decreasing symptoms of the oppositional defiant disorder (ODD) in school-aged children. METHOD: Participants (n = 43) were school-aged children who were randomly assigned to RFP-C or a waitlist control group. Symptoms of ODD and explicit emotion regulation capacities were assessed at baseline, end of waitlist, and end of treatment. Multilevel modeling was used to account for patient and therapist factors in outcomes. RESULTS: At the end of treatment, parents reported significant reductions in children's ODD symptoms on the primary outcome measure. There were no observed changes in explicit emotion regulation. Reliable change index scores indicated that 79.4% of children were recovered or improved after 10 weeks of treatment. There were no identifiable patient or therapist effects. Treatment adherence and completion was high. CONCLUSION: This study is the first randomized controlled trial of a manualized psychodynamic intervention for children with ODD. Participants demonstrated significant reductions in symptoms of ODD after 10 weeks of treatment. Further investigation is needed to compare RFP-C relative to active treatment, assess changes in implicit emotion, and to determine long-term maintenance of symptom improvement.Clinical trial registration information: Evaluation of Regulation Focused Psychotherapy for Children (RFP-C); https://clinicaltrials.gov/ct2/show/NCT03594253.

Recombinant sulfated CCR2 peptide trap reduces retinal degeneration in mice.

Chemokine receptors are generally sulfated at tyrosine residues of the N-terminal region. Tyrosine sulfation of the C-C chemokine receptor type 2 (CCR2) enhances its interaction with the chemokine ligand CCL2. Here, we generated a recombinant sulfated CCR2 peptide trap (mCCR2-S2) and investigated its effects on retinal degeneration in mice. Treatment with mCCR2-S2 reduced choroidal neovascularization (CNV) in a laser-induced CNV mouse model. In NaIO3-injected mice, treatment with mCCR2-S2 increased the outer nuclear layer thickness and rhodopsin expression in the retinas compared to that in mice treated with mCCR2-wild-type or glutathione S-transferase controls. Furthermore, glial fibrillary acidic protein (GFAP) expression and macrophage infiltration were decreased in mCCR2-S2-treated retinas. Recombinant mCCR2-S2 suppressed CNV development and retinal degeneration, possibly by regulating macrophage infiltration. Thus, the sulfated form of the CCR2 peptide trap may be a useful tool for treating patients with retinal degeneration, such as those with age-related macular degeneration and intraocular inflammatory disorders.

Mitochondrial Membrane Disrupting Molecules for Selective Killing of Senescent Cells.

Cellular senescence, a stable form of cell cycle arrest, facilitates protection from tumorigenesis and aids in tissue repair as they accumulate in the body at an early age. However, long-term retention of senescent cells causes inflammation, aging of the tissue, and progression of deadly diseases such as obesity, diabetes, and atherosclerosis. Various attempts have been made to achieve selective elimination of senescent cells from the body, yet little has been explored in designing the mitochondria-targeted senolytic agent. Many characteristics of senescence are associated with mitochondria. Here we have designed a library of alkyl-monoquaternary ammonium-triphenyl phosphine (TPP) and alkyl-diquaternary ammonium-TPP of varying alkyl chain lengths, which target the mitochondria; we also studied their senolytic properties. It was observed that the alkyl-diquaternary ammonium-TPP with the longest chain length induced apoptosis in senescent cells selectively via an increase of reactive oxygen species (ROS) and mitochondrial membrane disruption. This study demonstrates that mitochondria could be a potential target for designing new small molecules as senolytic agents for the treatment of a variety of dysfunctions associated with pathological aging.

AUTOMATED QUANTIFICATION OF VITREOUS HYPERREFLECTIVE FOCI AND VITREOUS HAZE USING OPTICAL COHERENCE TOMOGRAPHY IN PATIENTS WITH UVEITIS.

PURPOSE: Development of an automated method to quantify the count of vitreous hyperreflective foci (vHF) and intensity of vitreous haze in patients with uveitis by optical coherence tomography. METHODS: A method based on deep learning to automatically segment the vHF, vitreous, and retinal pigment epithelium (RPE) in optical coherence tomography was developed using 1,058 scans from 88 optical coherence tomography volumes of 33 patients with intermediate, posterior or panuveitis. Based on segmented images, the vHF count and the relative intensity of vitreous to RPE (VIT/RPE-relative intensity) were quantified. Dice coefficient and intraclass correlation coefficient were calculated between ground truth and the trained network. RESULTS: The segmented area of vHF, vitreous, and RPE by the deep learning-based model showed good agreement with the clinicians' results, yielding a Dice coefficient of 0.69, 0.99, and 0.88, respectively. The intraclass correlation coefficient of the vHF count and the VIT/RPE-relative intensity per scan was 0.99 and 1.00, respectively. In eyes of test set, changes in vHF and VIT/RPE-relative intensity during treatment did not show similar patterns. CONCLUSION: Automated segmentation of the vHF, vitreous, and RPE in optical coherence tomography images of patients with uveitis was accomplished by a deep learning approach. The vHF count and VIT/RPE-relative intensity could be quantified with high reliability.

POST-TREATMENT PREDICTION OF OPTICAL COHERENCE TOMOGRAPHY USING A CONDITIONAL GENERATIVE ADVERSARIAL NETWORK IN AGE-RELATED MACULAR DEGENERATION.

PURPOSE: To develop a deep learning model to generate posttreatment optical coherence tomography (OCT) images of neovascular age-related macular degeneration. METHODS: Two hundred ninety-eight patients with neovascular age-related macular degeneration were included. The conditional generative adversarial network was trained using 15,183 augmented paired OCT B-scan images obtained from 723 scans of 241 patients at baseline and 1 month after 3 loading doses of an anti-vascular endothelial growth factor treatment. The network was also trained using baseline fluorescein angiography (FA) or indocyanine green angiography (ICGA) images together with baseline OCT images. A test set of 150 images of 50 eyes was used to evaluate its ability to predict the presence of intraretinal fluid, subretinal fluid, PED, and subretinal hyperreflective material. Posttreatment OCT images were compared with images generated from baseline OCT with or without FA and indocyanine green angiography images. RESULTS: The predicted images inferred from baseline OCT images achieved an acceptable accuracy, specificity, and negative predictive value for four lesions (range: 77.0-91.9, 94.1-95.1, and 54.7-96.5%, respectively). The addition of both FA and indocyanine green angiography images improved the accuracy, specificity, and negative predictive value (range: 80.7-96.3, 97.3-99.0, and 59.0-98.3%, respectively). CONCLUSION: A conditional generative adversarial network is able to generate posttreatment OCT images from baseline OCT, FA, and indocyanine green angiography images.

Comparative pharmacokinetics of Theracurmin, a highly bioavailable curcumin, in healthy adult subjects.

OBJECTIVE: Theracurmin is a submicron dispersed formulation of curcumin, which was developed to increase the bioavailability of curcumin. This study aimed to compare the pharmacokinetics of curcumin administered as two Theracurmin powder products and unformulated curcumin powder. MATERIALS AND METHODS: This randomized, three-treatment, six-sequence, and three-period crossover study enrolled 24 healthy subjects. Blood sampling was done until 12 hours after the administration of Theracurmin and curcumin powder to assess pharmacokinetics using a non-compartmental method. The plasma concentration of curcumin was determined using high-performance liquid chromatography coupled with tandem mass spectrometry. RESULTS: The median time to reach the maximum concentration was 1.5 - 3 hours for Theracurmin and 8 hours for curcumin powder. The two Theracurmin products showed systemic exposure profiles that were comparable to each other. The exposure ratio of Theracurmin to curcumin powder was 18.4 - 20.5 for the maximum plasma concentration and 35.9 - 42.6 for the area under the concentration-time curve from dosing to the last measurable time. CONCLUSION: In conclusion, this study showed similar systemic exposure between the two Theracurmin products. The absorption of curcumin after the administration of Theracurmin was significantly enhanced compared with curcumin powder.

Ilimaquinone inhibits neovascular age-related macular degeneration through modulation of Wnt/ß-catenin and p53 pathways.

Neovascular age-related macular degeneration (nAMD) is a common cause of irreversible vision loss in the elderly. Anti-vascular endothelial growth factor has been effective in treating pathological ocular neovascularization, but it has limitations including the need for repeated intraocular injections for the maintenance of therapeutic effects in most patients and poor or non-response to this agent in some patients. in vitro cellular studies were conducted using retinal pigment epithelial cell lines (ARPE-19 and hTERT-RPE1), human umbilical vein endothelial cells (HUVECs), and human umbilical vein smooth muscle cells (HUVSMCs). in vivo efficacy of ilimaquinone (IQ) was tested in laser-induced choroidal neovascularization mouse and rabbit models. Tissue distribution study was performed in male C57BL6/J mice. IQ, 4,9-friedodrimane-type sesquiterpenoid isolated from the marine sponge, repressed the expression of angiogenic/inflammatory factors and restored the expression of E-cadherin in retinal pigment epithelial cells by inhibiting the Wnt/ß-catenin pathway. In addition, it selectively inhibited proliferation and tube formation of HUVECs by activating the p53 pathway. Topical and intraperitoneal administration of IQ significantly reduced choroidal neovascularization in rabbits and mice with laser-induced choroidal neovascularization. Notably, IQ by the oral route of exposure was highly permeable to the eyes and suppressed abnormal vascular leakage by downregulation of ß-catenin and stabilization of p53 in vivo. Our findings demonstrate that IQ functions through regulation of p53 and Wnt/ß-catenin pathways with conceivable advantages over existing cytokine-targeted anti-angiogenic therapies.

ASSOCIATION OF TREATMENT RESPONSE WITH QUANTITATIVE CHANGES IN CHOROIDAL NEOVASCULARIZATION AND CHOROIDAL VESSEL IN NEOVASCULAR AGE-RELATED MACULAR DEGENERATION.

PURPOSE: To evaluate the association between treatment response and quantitative morphological changes in choroidal neovascularization and outer choroidal vessels using optical coherence tomography angiography (OCTA) and en face OCT in neovascular age-related macular degeneration (nAMD). METHODS: We retrospectively analyzed 75 eyes of typical nAMD patients and 53 polypoidal choroidal vasculopathy eyes of 124 patients with OCTA performed at least 6 months after initial antivascular endothelial growth factor treatment. Quantitative parameters, including vessel area, vessel diameter, branch vessel length, fractal dimension, and lacunarity were analyzed based on en face images of the choroidal neovascularization and choroidal vessel in Haller's layer. Parameters associated with loss of logarithm of the minimum angle of resolution visual acuity with the basis of 0.3 and the treatment interval (good vs. poor responder based on 12 weeks) were analyzed. Analyses were conducted for "before OCTA" (initial visit to OCTA) and "after OCTA" (OCTA to 6 months post-OCTA). RESULTS: In typical nAMD, visual acuity loss before OCTA was associated with a higher SD of choroidal neovascularization diameter and lower choroidal fractal dimension. Visual acuity loss after OCTA in typical nAMD was associated with higher lacunarity of the choroid. Poor responders before OCTA were not associated with any factor. Poor responders after OCTA were associated with a lower SD of outer choroidal vessel diameter in typical nAMD. In polypoidal choroidal vasculopathy, no factor was associated with clinical outcomes in either period. CONCLUSION: Quantitative analyses of choroidal neovascularization on OCTA and choroidal vessels on en face OCT provide information about treatment response, including changes in visual acuity and treatment interval, in nAMD.

Diabetic macular edema with pachychoroid features.

BACKGROUND: To investigate the clinical features of diabetic macular edema (DME) in eyes with pachychoroid phenotypes using multimodal retinal imaging. METHODS: We retrospectively reviewed 210 eyes from 210 DME patients and analyzed the clinical and imaging parameters, including visual acuity, central macular thickness (CMT), subfoveal choroidal thickness (SFCT) and neural retina layer thickness (NRT). The DME eyes were divided into two groups: group 1 (80 eyes with submacular detachment [SMD]) and group 2 (130 eyes without SMD). The clinical and imaging parameters of 285 eyes from 285 diabetic patients without DME were collected as a control group. RESULTS: DME eyes with pachychoroid phenotypes were more frequent in group 1 than in group 2 (53 eyes [66.25%] and 53 eyes [40.77%], respectively, P < 0.001). Pachychoroid phenotypes were identified in 108 (37.90%) of the control eyes. CMT and NRT were greater in group 1 than in group 2. In group 1, 37 eyes had SMD combined with focal edema, and 43 eyes had SMD combined with diffuse-type edema. No significant difference in pachychoroid phenotypes was found between the focal and diffuse types (26 [70.27%] and 27 [62.79%], respectively, P = 0.481). In group 2, 70 eyes had focal-type edema, and 60 eyes had diffuse-type edema. No significant difference in the frequency of pachychoroid phenotypes was found (32 [45.71%] and 21 [35.00%], respectively, P = 0.215). Interestingly, among the 70 eyes with focal edema in group 2, 13 (40.6%) and 5 (13.2%) eyes with and without pachychoroid phenotypes showed no definite microaneurysms, respectively. CONCLUSION: SMD and focal edema without definite microaneurysms may be clinical manifestations of DME with pachychoroid phenotypes and possibly related to choroidal circulation disturbance in DME.

Disruption of the polyubiquitin gene Ubb causes retinal degeneration in mice.

We have previously demonstrated that a reduction in ubiquitin (Ub) levels via disruption of the polyubiquitin gene Ubb results in reactive gliosis and hypothalamic neurodegeneration in mice. However, it is not known whether other neural tissues, apart from the brain, can also be affected by Ubb disruption. We examined the retina, which, being derived from the diencephalon, has the same developmental origin as the hypothalamus. We found that expression levels of Ubb were much higher than those of the other polyubiquitin gene Ubc in the retina. In retinal tissues from Ubb knockout (KO) mice, we found that Ubc expression was upregulated to compensate for the loss of Ubb; however, the Ub pool remained disrupted, with reduced levels of free Ub. To directly demonstrate whether the disrupted Ub pools affect neural integrity in retinal tissues, we investigated retinal layers in control and Ubb KO mice. Using optical coherence tomography and histological analysis, we demonstrated that the thickness of the outer nuclear layer of the retina was decreased in Ubb KO mice compared to control mice, suggesting that retinal degeneration was induced by Ub deficiency. Furthermore, the mRNA and protein levels of rhodopsin decreased and those of glial fibrillary acidic protein increased in Ubb KO mouse retinas. Therefore, the maintenance of Ub pools in the retina appears to be crucial for the survival of photoreceptor cells and the prevention of excessive glial cell activation.

Ursodeoxycholic acid exerts hepatoprotective effects by regulating amino acid, flavonoid, and fatty acid metabolic pathways.

INTRODUCTION: Ursodeoxycholic acid (UDCA) is an intestinal bacterial metabolite with hepatoprotective effects. However, molecular mechanisms underlying its effects remain unclear. OBJECTIVES: The aim of this study was to investigate the mechanisms underlying the therapeutic effects of UDCA by using global metabolomics analyses in healthy subjects. METHODS: Healthy Korean men were administered UDCA at dosage of 400, 800, or 1200 mg daily for 2 weeks. Serum samples were collected and used for liver function tests and to determine miR-122 expression levels. Urinary and plasma global metabolomics analyses were conducted using a liquid chromatography system coupled with quadrupole-time-of-flight mass spectrometry (LC/QTOFMS) and gas chromatography-TOFMS (GC/TOFMS). Unsupervised multivariate analysis (principal component analysis) was performed to identify discriminative markers before and after treatment. RESULTS: Alanine transaminase score and serum miR-122 levels decreased significantly after 2 weeks of treatment. Through LC- and GC-based metabolomic profiling, we identified 40 differential metabolites in plasma and urine samples. CONCLUSIONS: Regulation of liver function scores and metabolic alternations highlight the potential hepatoprotective action of UDCA, which were primarily associated with amino acid, flavonoid, and fatty acid metabolism in healthy men.

QUANTIFICATION OF RETINAL VESSEL TORTUOSITY IN DIABETIC RETINOPATHY USING OPTICAL COHERENCE TOMOGRAPHY ANGIOGRAPHY.

PURPOSE: To investigate the association of vessel tortuosity with severity of diabetic retinopathy (DR) using optical coherence tomography angiography. METHODS: We retrospectively analyzed 30 healthy eyes and 121 eyes of diabetic subjects with no DR, mild nonproliferative DR (NPDR), moderate to severe NPDR and proliferative DR (PDR). Binarized images were used to quantify the vessel tortuosity, vessel density, foveal avascular zone (FAZ) area, and FAZ acircularity. The vessels were divided vertically as superficial retinal layer and deep retinal layer, and horizontally as circular areas with 3 mm and 1.5 mm diameters. Analysis of variance was performed for multiple comparisons. Correlation analysis evaluated the association between the quantified parameters. RESULTS: Compared with healthy eyes, vessel tortuosity increased as DR severity was more in NPDR, but decreased in PDR (P = 0.033). The decrease in vessel density and the increase in both FAZ area and FAZ acircularity were consistent, while DR approached PDR. Among all parameters, statistically significant difference between no DR and mild NPDR was observed only in vessel tortuosity, especially within the 1.5 mm area of superficial retinal layer (P = 0.011). Correlations of vessel tortuosity with FAZ area and acircularity were confined to the 3 mm and 1.5 mm areas of superficial retinal layer (r = -0.185, P = 0.023 for FAZ area; r = 0.268, P = 0.001 for FAZ acircularity), while vessel density strongly correlated with FAZ parameters in the superficial retinal layer and deep retinal layer. CONCLUSION: Vessel tortuosity increased as the stage of NPDR was more severe, but decreased in PDR. The vessel tortuosity determined using optical coherence tomography angiography might be a useful parameter indicating the progression to PDR, circumventing the risk from invasive conventional angiography.

Quantitative proteomic analysis of aqueous humor from patients with drusen and reticular pseudodrusen in age-related macular degeneration.

BACKGROUND: To identify novel biomarkers related to the pathogenesis of dry age-related macular degeneration (AMD), we adopted a human retinal pigment epithelial (RPE) cell culture model that mimics some features of dry AMD including the accumulation of intra- and sub-RPE deposits. Then, we investigated the aqueous humor (AH) proteome using a data-independent acquisition method (sequential window acquisition of all theoretical fragment ion mass spectrometry) for dry AMD patients and controls. METHODS: After uniformly pigmented polarized monolayers of human fetal primary RPE (hfRPE) cells were established, the cells were exposed to 4-hydroxy-2-nonenal (4-HNE), followed by Western blotting, immunofluorescence analysis and ELISA of cells or conditioned media for several proteins of interest. Data-dependent acquisition for identification of the AH proteome and SWATH-based mass spectrometry were performed for 11 dry AMD patients according to their phenotypes (including soft drusen and reticular pseudodrusen [RPD]) and 2 controls (3 groups). RESULTS: Increased intra- and sub-RPE deposits were observed in 4-HNE-treated hfRPE cells compared with control cultures based on APOA1, cathepsin D, and clusterin immunoreactivity. Additionally, the differential abundance of proteins in apical and basal chambers with or without 4-HNE treatment confirmed the polarized secretion of proteins from hfRPE cells. A total of 119 proteins were quantified in dry AMD patients and controls by SWATH-MS. Sixty-five proteins exhibited significantly altered abundance among the three groups. A two-dimensional principal component analysis plot was generated to identify typical proteins related to the pathogenesis of dry AMD. Among the identified proteins, eight proteins, including APOA1, CFHR2, and CLUS, were previously considered major components or regulators of drusen. Three proteins (SERPINA4, LUM, and KERA proteins) have not been previously described as components of drusen or as being related to dry AMD. Interestingly, the LUM and KERA proteins, which are related to extracellular matrix organization, were upregulated in both RPD and soft drusen. CONCLUSIONS: Differential protein expression in the AH between patients with drusen and RPD was quantified using SWATH-MS in the present study. Detailed proteomic analyses of dry AMD patients might provide insights into the in vivo biology of drusen and RPD.

Polycystic ovary morphology is associated with insulin resistance in women with polycystic ovary syndrome.

BACKGROUND/OBJECTIVES: Polycystic ovary syndrome (PCOS) is a heterogeneous disorder characterized by chronic anovulation, hyperandrogenism, polycystic ovary morphology (PCOM) and metabolic disturbances including insulin resistance and type 2 diabetes mellitus. Although insulin resistance could be associated with PCOM, recent studies have shown controversial results. The aim of this study was to determine the relationship between PCOM and insulin resistance. SUBJECTS/METHODS: This was a cross-sectional clinical study. A total of 679 women with PCOS who were diagnosed using the National Institute of Child Health and Human Disease (NICHD) criteria and 272 control women were analysed. We measured fasting glucose and insulin levels, 75 g oral glucose tolerance test-derived glucose and insulin levels, testosterone levels, ovarian volume and follicle number. RESULTS: Polycystic ovary morphology was described in 543 women (80.0%) with PCOS. Women with PCOS had significantly higher 2 hours postload glucose, fasting and 2 hours postload insulin levels, ovarian volume, ovarian follicle numbers and lower insulin sensitivity compared with those of the controls (all P<.01). In women with PCOS, ovarian volume and ovarian follicle number were negatively associated with the quantitative insulin sensitivity check index after adjusting for age, body mass index and total testosterone; however, this association was not observed in the controls. In the logistic regression analysis, increased ovarian follicle number was associated with decreased insulin sensitivity in women with PCOS. CONCLUSIONS: In PCOS, enlarged ovarian volume and follicle excess were associated with insulin resistance, and the number of ovarian follicles could be a predictor of insulin resistance.