CLINICAL CHARACTERIZATION OF AQUEOUS AND VITREOUS RETINOL-BINDING PROTEIN 3 CONCENTRATIONS IN RELATION TO DIABETIC RETINOPATHY SEVERITY, RETINAL STRUCTURES, AND SYSTEMIC COMPLICATIONS.

PURPOSE: To evaluate Retinol-Binding Protein 3 (RBP3) from photoreceptors in aqueous and its association with vitreous concentrations, diabetic retinopathy (DR) severity, retinal layer thickness, and clinical characteristics in people with diabetes. METHODS: RBP3 concentration was measured by custom-developed enzyme-linked immunosorbent assay in aqueous and correlated with vitreous concentrations in patients from the 50-Year Medalist study and Beetham Eye Institute at Joslin Diabetes Center. RESULTS: Aqueous RBP3 concentration (N = 131) was elevated in eyes with no to mild DR (mean ± SD 0.7 nM ± 0.2) and decreased in eyes with moderate to severe DR (0.65 nM ± 0.3) and proliferative DR (0.5 nM ± 0.2, P < 0.001) compared to eyes without diabetes. Aqueous and vitreous RBP3 concentrations correlated with each other (r = 0.34, P = 0.001) and between fellow eyes (P < 0.0001). History of retinal surgery did not affect aqueous RBP3 concentrations, but cataract surgery affected both vitreous and aqueous levels. Elevated aqueous RBP3 concentration associated with increased thickness of the outer nuclear layer (P = 0.004) and correlated with hemoglobin A1c, whereas vitreous RBP3 concentrations correlated with diabetic systemic complications. CONCLUSION: These findings suggest that aqueous RBP3 concentration may be an important endogenous clinical retinal protective factor, a biomarker for DR severity, and a promising VEGF-independent clinical intervention target in DR.

Enhanced immunosuppressive capability of mesenchymal stem cell-derived small extracellular vesicles with high expression of CD73 in experimental autoimmune uveitis.

BACKGROUND: Autoimmune uveitis is an inflammatory disease triggered by an aberrant immune response. Mesenchymal stem cell-derived small extracellular vesicles (MSC-sEVs) are emerging as potential therapeutic agents for this condition. CD73, an ectoenzyme present on MSC-sEVs, is involved in mitigating inflammation by converting extracellular adenosine monophosphate into adenosine. We hypothesize that the inhibitory effect of MSC-sEVs on experimental autoimmune uveitis (EAU) could be partially attributed to the surface expression of CD73. METHODS: To investigate novel therapeutic approaches for autoimmune uveitis, we performed lentiviral transduction to overexpress CD73 on the surface of MSC-sEVs, yielding CD73-enriched MSC-sEVs (sEVs-CD73). Mice with interphotoreceptor retinoid-binding protein (IRBP)-induced EAU were grouped randomly and treated with 50 µg MSC-sEVs, vector infected MSC-sEVs, sEVs-CD73 or PBS via single tail vein injection. We evaluated the clinical and histological features of the induced mice and analyzed the proportion and functional capabilities of T helper cells. Furthermore, T-cells were co-cultured with various MSC-sEVs in vitro, and we quantified the resulting inflammatory response to assess the potential therapeutic benefits of sEVs-CD73. RESULTS: Compared to MSC-sEVs, sEVs-CD73 significantly alleviates EAU, leading to reduced inflammation and diminished tissue damage. Treatment with sEVs-CD73 results in a decreased proportion of Th1 cells in the spleen, draining lymph nodes, and eyes, accompanied by an increased proportion of regulatory T-cells (Treg cells). In vitro assays further reveal that sEVs-CD73 inhibits T-cell proliferation, suppresses Th1 cells differentiation, and enhances Treg cells proportion. CONCLUSION: Over-expression of CD73 on MSC-sEVs enhances their immunosuppressive effects in EAU, indicating that sEVs-CD73 has the potential as an efficient immunotherapeutic agent for autoimmune uveitis.

Reappraisal of serum retinol-binding protein as a surrogate marker for retinol and discovery of a novel retinol estimation formula.

BACKGROUND & AIMS: Serum retinol (ROH) is commonly used for population level assessment of vitamin A status. High-performance liquid chromatography (HPLC) is considered most accurate method for measuring ROH. However, with the technical difficulty of using HPLC for routine assays, serum retinol-binding protein (RBP) measured by immunological assays is expected to be a surrogate marker for ROH, with reports of a close correlation between serum RBP and ROH. Nevertheless, RBP is not commonly tested to assess vitamin A status with concerns over RBP alterations under various physiopathological conditions. Thus, we reappraised the extent to which RBP could be used as a surrogate marker in representative disorders that alter serum RBP levels. As a related marker, diagnostic utility of transthyretin (TTR) was also evaluated. METHODS: To evaluate the reliability of ROH and RBP assays, specimen stability was assessed in terms of (1) storage at 25, 4, -20, and -80 °C for 1-28 days, (2) five-cycle freeze-thawing, and (3) fluorescent light exposure for 1-14 days. Sources of variation (sex, age, body mass index [BMI], and drinking habits) and reference intervals for ROH, RBP, and TTR were determined in 617 well-defined healthy individuals. To investigate the influence of disorders that affect serum RBP, patients with five diagnostic groups were enrolled: 26 with chronic kidney disease (CKD); 13 with various malignancies in advanced stages (AdM), 12 with acute bacterial infections (ABI), 6 with liver cirrhosis (LC), and 26 with simple obesity (BMI ≥ 27 kg/m2). RESULTS: The stability of RBP and ROH in serum was confirmed under all conditions. In healthy individuals, serum ROH, RBP, and TTR were appreciably high in males with a slight increase in proportion to age and BMI. The major-axis regression line between RBP (x) and ROH (y) in healthy individuals was y = x, with a correlation coefficient of 0.986. In the LC, AdM, and ABI groups, similar strong correlations were observed; however, the regression lines were shifted slightly rightward from the healthy group line, indicating a positive bias in estimating ROH. Interestingly, the same analyses between TTR and ROH revealed similar strong linear relationships in all groups; however, the regression line of each group showed a leftward (opposite) shift from the healthy group line. Based on these observations, we developed a novel regression model composed of RBP and TTR, which gave much improved accuracy in estimating ROH, even under these pathological conditions. CONCLUSIONS: The perfect RBP-ROH correlation in healthy individuals indicates the utility of RPB as a surrogate marker for ROH. Nevertheless, under RBP-altered conditions, a slight overestimation of ROH is inevitable. However, when the TTR was tested together, the bias can be corrected almost perfectly using the novel ROH estimation formula comprising RBP and TTR.

RBP3-Retinopathy-Inherited High Myopia and Retinal Dystrophy: Genetic Characterization, Natural History, and Deep Phenotyping.

PURPOSE: To examine the genetic and clinical features and the natural history of RBP3-associated retinopathy. DESIGN: Multi-center international, retrospective, case series of adults and children, with moleculraly confirmed RBP3-asociated retinopathy. METHODS: The genetic, clinical, and retinal imaging findings, including optical coherence tomography (OCT) and fundus autofluorescence (FAF), were investigated both cross-sectionally and longitudinally. The results of international standard full-field electroretinography (ERG) and pattern electroretinography (PERG) were reviewed. RESULTS: We ascertained 12 patients (5 female and 7 male) from 10 families (4 patients previously reported). Ten novel disease-causing RBP3 variants were identified. Ten patients were homozygous. The mean age (±SD, range) of the group was 21.4 years (±19.1, 2.9-60.5 years) at baseline evaluation. All 12 patients were highly myopic, with a mean spherical equivalent of -16.0D (range, -7.0D to -33.0D). Visual acuity was not significantly different between eyes, and no significant anisometropia was observed. Mean best-corrected visual acuity (BCVA) was 0.48 logMAR (SD, ±0.29; range, 0.2-1.35 logMAR); at baseline. Eleven patients had longitudinal BCVA assessment, with a mean BCVA of 0.46 logMAR after a mean follow-up of 12.6 years. All patients were symptomatic with reduced VA and myopia by the age of 7 years old. All patients had myopic fundi and features in keeping with high myopia on OCT, including choroidal thinning. The 4 youngest patients had no fundus pigmentary changes, with the rest of the patients presenting with a variable degree of mid-peripheral pigmentation and macular changes. FAF showed variable phenotypes, ranging from areas of increased signal to advanced atrophy in older patients. OCT showed cystoid macular edema at presentation in 3 patients, which persisted during follow-up in 2 patients and resolved to atrophy in the third patient. The ERGs were abnormal in 9 of 9 cases, revealing variable relative involvement of rod and cone photoreceptors with additional milder dysfunction post-phototransduction in some. All but 1 patient had PERG evidence of macular dysfunction, which was severe in most cases. CONCLUSIONS: This study details the clinical and functional phenotype of RBP3-retinopathy in the largest cohort reported to date. RBP3-retinopathy is a disease characterized by early onset, slow progression over decades, and high myopia. The phenotypic spectrum and natural history as described herein has prognostic and counseling implications. RBP3-related disease should be considered in children with high myopia and retinal dystrophy.

Unraveling the role of resistin, retinol-binding protein 4 and adiponectin produced by epicardial adipose tissue in cardiac structure and function: evidence of a paracrine effect.

PURPOSE: Adipokines produced by adipose tissue have been found to be involved in the pathophysiology of metabolic and cardiovascular diseases. We aimed to investigate the relationships of resistin, retinol-binding protein 4 (RBP4) and adiponectin produced by epicardial adipose tissue with coronary artery disease (CAD) and cardiac structure and function. METHODS: Forty-one non-diabetic males scheduled for cardiothoracic surgery were examined. Anthropometric measurements, echocardiography, coronary angiography, and blood analysis were performed preoperatively. We measured the serum levels of resistin, RBP4, and adiponectin and their mRNA expression in thoracic subcutaneous adipose tissue and two epicardial adipose tissue samples, one close to left anterior descending artery (LAD) (resistin-LAD, RBP4-LAD, adiponectin-LAD), and another close to the right coronary artery (RCA) (resistin-RCA, RBP4-RCA, adiponectin-RCA). RESULTS: Left ventricular (LV) ejection fraction correlated negatively with adiponectin-LAD (rho = - 0.390, p = 0.025). The ratio of early to late diastolic transmitral flow velocity, as an index of LV diastolic function, correlated negatively with resistin-LAD (rho = - 0.529, p = 0.024) and RBP4-LAD (rho = - 0.458, p = 0.049). There was no difference in epicardial adipose tissue mRNA expression of resistin, RBP4, and adiponectin between individuals with CAD and those without CAD. When we compared the individuals with CAD in the LAD with those without CAD in the LAD, there was no difference in resistin-LAD, RBP4-LAD, and adiponectin-LAD. There was no difference in resistin-RCA, RBP4-RCA, and adiponectin-RCA between the individuals with CAD in the RCA and those without CAD in the RCA. CONCLUSION: Elevation of epicardial adipose tissue mRNA expression of adiponectin was associated with LV systolic dysfunction, while that of both resistin and RBP4 was linked to LV diastolic dysfunction.

Free energy simulations to study mutational effect of a conserved residue, Trp24, on stability of human serum retinol-binding protein.

Human serum retinol-binding protein (RBP) is a plasma transport protein for vitamin A. RBP is a prime subclass of lipocalins, which bind nonpolar ligands within a ß-barrel. To understand the role of Trp 24, one of the highly conserved residues in RBP, free energy simulations have been carried out to understand the effects of the mutations from Trp at position 24 to Leu, Phe, and Tyr in the apo-RBP on its thermal stability. We examine various unfolded systems to study the dependence of the free energy differences on the denatured structure. Our calculated free energy difference values for the three mutations are in excellent agreement with the experimental values when the initial coordinates of the seven-residue peptide segments truncated from the crystal structure are used for the denatured systems. Our free energy change differences for the Trp→Leu, Trp→Phe, and Trp→Tyr mutations are 2.50 ± 0.69, 2.58 ± 0.50, and 2.49 ± 0.48 kcal/mol, respectively, when the native-like seven-residue peptides are used as models for the denatured systems. The main contributions to the free energy change differences for the Trp24→Leu and Trp24→Phe mutations are mainly from van der Waals and covalent interactions, respectively. Electrostatic, van der Waals and covalent terms equally contribute to the free energy change difference for the Trp24→Tyr mutation. The free energy simulation helps understand the detailed microscopic mechanism of the stability of the RBP mutants relative to the wild type and the role of the highly conserved residue, Trp24, of the human RBP.Communicated by Ramaswamy H. Sarma.

The prognostic value of retinol binding protein in geriatric hip fractures after surgeries: A propensity score matching and 1-year follow-up study.

BACKGROUND: We aimed to explore the predictive value of retinol binding protein (RBP) for outcomes of hip fractures. METHODS: Patients with hip fractures who underwent hip surgeries between December 2017 and February 2021 and met the inclusion criteria were analyzed. Propensity score matching was used to reduce the bias of co-factors and ROC curves based on matched populations were created to determine the optimal cutoff point of RBP. The outcomes between patients with low levels of RBP and high levels of RBP were compared. RESULTS: Four hundred eighty patients were enrolled in this study and 69 patients died within one year. After a 1:1 PSM, patients with more than 1-year survival had significantly higher RBP (p = 0.013) than those who died within one year, as well as patients divided by 6-months survival (p = 0.012). Logistics analysis showed that low RBP may be an independent risk factor for 3-month survival, 6-month survival, 1-year survival, and 3-month free walking ability. CONCLUSION: RBP may be associated with the survival and 3-month walking abilities of patients with hip fractures.

Hyperinsulinemia impairs the metabolic switch to ketone body utilization in proximal renal tubular epithelial cells under energy crisis via the inhibition of the SIRT3/SMCT1 pathway.

Objective: To investigate the effects and mechanism of hyperinsulinemia on the metabolic switch to ß-hydroxybutyrate (BHB) absorption and utilization under a starvation or hypoxic environment in proximal tubular epithelial cells. Methods: A high-fat diet-induced hyperinsulinemia model in ZDF rats was used to test the expression of key enzymes/proteins of ketone body metabolism in the kidney. Notably, 12-week-old renal tubule SMCT1 specific knockout mice (SMCT1 flox/floxCre+) and control mice (SMCT1 flox/floxCre-) were used to confirm the roles of SMCT1 in kidney protection under starvation. The changes of key enzymes/proteins of energy metabolism, mitochondrial function, and albumin endocytosis in HK2 cells under low glucose/hypoxic environments with or without 50 ng/mL insulin were studied. Silent information regulation 2 homolog 3 (SIRT3) was overexpressed to evaluate the effect of hyperinsulinemia on the metabolic switch to BHB absorption and utilization through the SIRT3/SMCT1 pathway in HK2 cells. Results: In ZDF rats, the expression of HMGCS2 increased, the SMCT1 expression decreased, while SCOT remained unchanged. In renal tubule SMCT1 gene-specific knockout mice, starvation for 48 h induced an increase in the levels of urine retinol-binding protein, N-acetyl-ß-glucosaminidase, and transferrin, which reflected tubular damages. In HK2 cells under an environment of starvation and hypoxia, the levels of key enzymes related to fatty acid oxidation and ketone body metabolism were increased, whereas glucose glycolysis did not change. The addition of 2 mmol/l BHB improved ATP production, mitochondrial biosynthesis, and endocytic albumin function, while cell apoptosis was reduced in HK2 cells. The addition of 50 ng/ml insulin resulted in the decreased expression of SMCT1 along with an impaired mitochondrial function, decreased ATP production, and increased apoptosis. The overexpression of SIRT3 or SMCT1 reversed these alterations induced by a high level of insulin both in low-glucose and hypoxic environments. Conclusions: The increased absorption and utilization of BHB is part of the metabolic flexibility of renal tubular epithelial cells under starvation and hypoxic environments, which exhibits a protective effect on renal tubular epithelial cells by improving the mitochondrial function and cell survival. Moreover, hyperinsulinemia inhibits the absorption of BHB through the inhibition of the SIRT3/SMCT1 pathway.

Genetic Ablation of Nrf2 Exacerbates Neuroinflammation in Ocular Autoimmunity.

Experimental autoimmune uveoretinitis (EAU) is an animal model of non-infectious uveitis and is developed by immunization with retinal antigen, interphotoreceptor retinoid-binding protein (IRBP). Nuclear factor erythroid 2- (NF-E2-) related factor 2 (Nrf2) is responsible for regulating antioxidant and inflammatory responses. In this study, we investigated the role of Nrf2 on the development of EAU. Clinical and pathological examination demonstrated that retinal inflammation was exacerbated in Nrf2 knockout (Nrf2 KO) mice compared to wild type (WT) mice, and the expression of inflammatory cytokines (IFN-γ, IL-6, and IL-17) in the retina was significantly elevated in Nrf2 KO mice. GFAP positive cells (astrocytes) and Iba-1 positive cells (microglia cells) in the retina were more numerous in Nrf2 KO mice compared to WT mice. Furthermore, we examined the suppressive effect of the Nrf2 activator CDDO-Im (2-cyano-3,12 dioxooleana-1,9 dien-28-oyl imidazoline) on the development of EAU. The treatment with CDDO-Im significantly reduced the clinical and pathological score of EAU compared to those of vehicle-treated mice. These findings suggest that Nrf2 plays a regulatory role in the pathogenesis of autoimmune uveoretinitis and the activation of the Nrf2 system may have therapeutic potential for protecting vision from autoimmune neuroinflammation.

Urinary RBP as an Independent Predictor of Renal Outcome in Diabetic Nephropathy.

Background: Renal tubular impairment is prevalent in diabetic nephropathy (DN) and the histological severity predicted renal outcome. Biomarkers of tubular injury also increased in the urine of DN patients. The retrospective study aimed to assess the prognostic value of clinically widely applied urinary tubular injury markers, retinol-binding protein (RBP), ß2-microglobulin (ß2-MG) and N-acetyl-ß-D-glucosaminidase (NAG) in DN. Method: A total of 305 patients with biopsy-proven DN were enrolled. The baseline urine total protein and components including albumin, IgG, RBP, ß2-MG and NAG were retrieved from medical records. The primary outcome was end stage renal disease (ESRD). Cox proportional hazard analysis and restricted cubic splines were performed to evaluate the association of parameters with ESRD. Nomograms were constructed and concordance index (C-index) was used to measure the prediction ability. Result: The levels of urinary RBP, ß2-MG and NAG were positively correlated with the severity of interstitial fibrosis and tubular atrophy (IFTA). Positive correlations were also observed among ß2-MG, NAG and mesangial expansion. Urinary RBP was not correlated with any glomerular lesions. Urinary RBP, ß2-MG and NAG were risk factors for ESRD in hazard analysis with adjustment for age, gender and body mass index (BMI). The hazard ratios increased with the increment of baseline levels. In the multivariate Cox model including serum creatinine (SCr), total urinary protein, urinary albumin, urinary IgG and the tubular injury biomarkers, urinary RBP (with every g/mol.Cr increase: HR 1.06, 95% CI 1.03-1.10, p =0.001) remained as an independent risk factor for ESRD in DN patients. Patients were divided by the medium value of urinary RBP into the low RBP and high RBP groups. Survival analysis showed that significantly more patients in the high RBP progressed to ESRD compared to those in the low RBP group (p =0.02) when urinary total protein was less than 3.5 g/g. The C-index of the nomogram incorporating age, gender, BMI, SCr and total urine protein was 0.757. The value increased to 0.777 after adding urinary RBP to the model. Conclusions: Urinary RBP excretion was only correlated with the severity of IFTA and independently predicted ESRD in DN patients.

Elevated Retinol Binding Protein 3 Concentrations Are Associated With Decreased Vitreous Inflammatory Cytokines, VEGF, and Progression of Diabetic Retinopathy.

OBJECTIVE: To correlate inflammatory cytokines and vascular endothelial growth factor (VEGF) in vitreous and plasma with vitreous retinol binding protein 3 (RBP3), diabetic retinopathy (DR) severity, and DR worsening in a population with type 1 and type 2 diabetes. RESEARCH DESIGN AND METHODS: RBP3, VEGF, and inflammatory cytokines were measured in plasma and vitreous samples (n = 205) from subjects of the Joslin Medalist Study and Beetham Eye Institute. RESULTS: Higher vitreous RBP3 concentrations were associated with less severe DR (P < 0.0001) and a reduced risk of developing proliferative DR (PDR) (P < 0.0001). Higher RBP3 correlated with increased photoreceptor segment thickness and lower vitreous interleukin-12 (IL-12), tumor necrosis factor-α (TNF-α), and TNF-ß (P < 0.05). PDR was associated with lower vitreous interferon-γ and IL-10 and higher VEGF, IL-6, and IL-15 (P < 0.05), but was not associated with their plasma concentrations. CONCLUSIONS: Higher vitreous RBP3 concentrations are associated with less severe DR and slower rates of progression to PDR, supporting its potential as a biomarker and therapeutic agent for preventing DR worsening, possibly by lowering retinal VEGF and inflammatory cytokines.

The innate immune receptor Nlrp12 suppresses autoimmunity to the retina.

BACKGROUND: Nod-like receptors (NLRs) are critical to innate immune activation and induction of adaptive T cell responses. Yet, their role in autoinflammatory diseases of the central nervous system (CNS) remains incompletely defined. The NLR, Nlrp12, has been reported to both inhibit and promote neuroinflammation in an animal model of multiple sclerosis (experimental autoimmune encephalomyelitis, EAE), where its T cell-specific role has been investigated. Uveitis resulting from autoimmunity of the neuroretina, an extension of the CNS, involves a breach in immune privilege and entry of T cells into the eye. Here, we examined the contribution of Nlrp12 in a T cell-mediated model of uveitis, experimental autoimmune uveitis (EAU). METHODS: Mice were immunized with interphotoreceptor retinoid-binding protein peptide 1-20 (IRBP1-20) emulsified in Complete Freund's adjuvant, CFA. Uveitis was evaluated by clinical and histopathological scoring, and comparisons were made in WT vs. Nlrp12-/- mice, lymphopenic Rag1-/- mice reconstituted with WT vs. Nlrp12-/- CD4+ T cells, or among bone marrow (BM) chimeric mice. Antigen-specific Th-effector responses were evaluated by ELISA and intracellular cytokine staining. Cellular composition of uveitic eyes from WT or Nlrp12-/- mice was compared using flow cytometry. Expression of Nlrp12 and of cytokines/chemokines within the neuroretina was evaluated by immunoblotting and quantitative PCR. RESULTS: Nlrp12-/- mice developed exacerbated uveitis characterized by extensive vasculitis, chorioretinal infiltrates and photoreceptor damage. Nlrp12 was dispensable for T cell priming and differentiation of peripheral Th1 or Th17 cells, and uveitis in immunodeficient mice reconstituted with either Nlrp12-/- or WT T cells was similar. Collectively, this ruled out T cells as the source of Nlrp12-mediated protection to EAU. Uveitic Nlrp12-/- eyes had more pronounced myeloid cell accumulation than uveitic WT eyes. Transplantation of Nlrp12-/- BM resulted in increased susceptibility to EAU regardless of host genotype, but interestingly, a non-hematopoietic origin for Nlrp12 function was also observed. Indeed, Nlrp12 was found to be constitutively expressed in the neuroretina, where it suppressed chemokine/cytokine induction. CONCLUSIONS: Our data identify a combinatorial role for Nlrp12 in dampening autoimmunity of the neuroretina. These findings could provide a pathway for development of therapies for uveitis and potentially other autoinflammatory/autoimmune diseases of the CNS.

Positive Expression of Retinol-Binding Protein 4 Is Related to the Malignant Clinical Features Leading to Poor Prognosis of Glioblastoma.

Backgrounds: Retinol-binding protein 4 (RBP4) is a monomeric-binding protein belonging to the lipocalin protein family, which has been reported to be dysregulated in several malignancies such as breast cancer and lung cancer. However, the expression and function of RBP4 in glioblastoma (GBM) are completely unknown. Materials and Methods: TCGA datasets were used for analyzing the mRNA level of RBP4 in GBM and its clinical relevance. A retrospective GBM cohort (n = 73) was enrolled from our hospital to test the protein expression profile of RBP4 in GBM tissues as well as its correlation with patients' prognoses. Two human GBM cell lines, LN229 and U251, were collected to conduct overexpression and knockdown experiments targeting RBP4. The tumor-related effects of RBP4 in GBM were finally evaluated by proliferation and invasion assays. Results: Both the higher mRNA level and protein level of RBP4 in GBM tissues were significantly correlated with poorer patients' overall survival. Multivariate analysis identified RBP4 as a novel independent prognostic predictor in GBM patients. Overexpression of RBP4 resulted in enhanced GBM proliferation capacity, which was consistent with clinical findings on the positive correlation between RBP4 level and tumor size. Meanwhile, overexpressing RBP4 promoted GBM cell migration and invasion, while silencing RBP4 led to the opposite results. Conclusions: RBP4 overexpression in tumor tissues is correlated with poorer prognosis of GBM patients, which functions by promoting GBM proliferation and invasion, thus, may serve as an invaluable predictive biomarker and therapeutic target.

Inhibition of Hepatic Stellate Cell Activation Suppresses Tumorigenicity of Hepatocellular Carcinoma in Mice.

Transdifferentiation (or activation) of hepatic stellate cells (HSCs) to myofibroblasts is a key event in liver fibrosis. Activated HSCs in the tumor microenvironment reportedly promote tumor progression. This study analyzed the effect of an inhibitor of HSC activation, retinol-binding protein-albumin domain III fusion protein (R-III), on protumorigenic functions of HSCs. Although conditioned medium collected from activated HSCs enhanced the migration, invasion, and proliferation of the hepatocellular carcinoma cell line Hepa-1c1c7, this effect was not observed in Hepa-1c1c7 cells treated with conditioned medium from R-III-exposed HSCs. In a subcutaneous tumor model, larger tumors with increased vascular density were formed in mice transplanted with Hepa-1c1c7+HSC than in mice transplanted with Hepa-1c1c7 cells alone. Intriguingly, when Hepa-1c1c7+HSC-transplanted mice were injected intravenously with R-III, a reduction in vascular density and extended tumor necrosis were observed. In an orthotopic tumor model, co-transplantation of HSCs enhanced tumor growth, angiogenesis, and regional metastasis accompanied by increased peritumoral lymphatic vessel density, which was abolished by R-III. In vitro study showed that R-III treatment affected the synthesis of pro-angiogenic and anti-angiogenic factors in activated HSCs, which might be the potential mechanism underlying the R-III effect. These findings suggest that the inhibition of HSC activation abrogates HSC-induced tumor angiogenesis and growth, which represents an attractive therapeutic strategy.

Macrophage-like iPS-derived Suppressor Cells Reduce Th1-mediated Immune Response to a Retinal Antigen.

PURPOSE: To investigate the immunotherapeutic effects of macrophage-like induced pluripotent stem (iPS) cell-derived suppressor cells (SCs) in ocular immune response and experimental autoimmune uveoretinitis (EAU). METHODS: The genes of Oct3/4, Sox2, Klf4, and c-Myc were transferred to B cells enriched from the spleen cells of C57BL/6 mice by using retrovirus vectors. Transferred B cells were cultured for 17 days to obtain colonies of iPS cells. Through additional steps, iPS-SCs were induced. An antigen-specific T cell proliferation assay was performed with CD4+ T cells collected from draining lymph nodes of the mice immunized with human interphotoreceptor retinoid-binding protein (hIRBP) peptide and co-cultured with iPS-SCs. Cytokine concentrations in the culture supernatant were examined. Mice were immunized with hIRBP peptide to induce EAU. The iPS-SCs were administered into the mice one day before the induction of EAU. RESULTS: The iPS-SCs decreased hIRBP-specific T cell proliferation depending on the number of cells. Productions of tumor necrosis factor-α and interferon-γ were significantly decreased; however, transforming growth factor-ß1, nitric oxide, interleukin (IL)-13, IL-17A, and IL-17 F levels were elevated in the supernatant when the collected T cells were co-cultured with iPS-SCs. The iPS-SCs had immunosuppressant effects even without cell-to-cell contact, and their effects were non-specific to the antigen preloaded on iPS-SCs. EAU was significantly milder in the mice administered iPS-SCs prior to immunization. CONCLUSIONS: Macrophage-like iPS-SCs reduced Th1 immune response to a retinal antigen and Th1-mediated EAU in mice. These results showed the possibility of the application of iPS technology to the treatment of noninfectious ocular inflammation, endogenous uveitis, in the future.

Induction of antigen-specific Treg cells in treating autoimmune uveitis via bystander suppressive pathways without compromising anti-tumor immunity.

BACKGROUND: Induction of autoantigen-specific Treg cells that suppress tissue-specific autoimmunity without compromising beneficial immune responses is the holy-grail for immunotherapy to autoimmune diseases. METHODS: In a model of experimental autoimmune uveitis (EAU) that mimics human uveitis, ocular inflammation was induced by immunization with retinal antigen interphotoreceptor retinoid-binding protein (IRBP). Mice were given intraperitoneal injection of αCD4 antibody (Ab) after the onset of disease, followed by administration of IRBP. EAU was evaluated clinically and functionally. Splenocytes, CD4+CD25- and CD4+CD25+ T cells were sorted and cultured with IRBP or αCD3 Ab. T cell proliferation and cytokine production were assessed. FINDINGS: The experimental approach resulted in remission of ocular inflammation and rescue of visual function in mice with established EAU. Mechanistically, the therapeutic effect was mediated by induction of antigen-specific Treg cells that inhibited IRBP-driven Th17 response in TGF-ß and IL-10 dependent fashion. Importantly, the Ab-mediated immune tolerance could be achieved in EAU mice by administration of retinal autoantigens, arrestin but not limited to IRBP only, in an antigen-nonspecific bystander manner. Further, these EAU-suppressed tolerized mice did not compromise their anti-tumor T immunity in melanoma model. INTERPRETATION: We successfully addressed a specific immunotherapy of EAU by in vivo induction of autoantigen-specific Treg cells without compromising host overall T cell immunity, which should have potential implication for patients with autoimmune uveitis. FUNDING: This study was supported by the Natural Science Foundation of Guangdong Province and the Fundamental Research Fund of the State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center.

Dose-response evaluation of urinary cadmium and kidney injury biomarkers in Chinese residents and dietary limit standards.

BACKGROUND: Cadmium (Cd) is a common heavy metal that mainly causes renal damage. There is a lack of research on the large-scale and systematic evaluation of the association between urinary Cd (U-Cd) and various effect biomarkers among Chinese residents. METHODS: Based on the establishment process of dietary Cd limit standards by European Food Safety Authority (EFSA), the dose-response relationships between U-Cd and four biomarkers, ß2-microglobulin (ß2-MG), N-acetyl-ß-glucosidase (NAG), microalbumin (mALB), and retinol binding Protein (RBP) were explored, respectively. Toxicokinetic model was used to derive the dietary Cd exposure limit for Chinese residents after critical U-Cd concentration was calculated. RESULTS: As the sensitive biomarkers of renal injury, ß2-MG and NAG were selected to estimate the 95% confidence interval lower limit of the U-Cd benchmark dose (BMDL5) to be 3.07 and 2.98 µg/g Cr, respectively. Dietary Cd exposure limit was calculated to be 0.28 µg/kg bw/day (16.8 µg/day, based on the body weight of 60 kg), which was lower than the average Chinese Cd exposure (30.6 µg/day) by the China National Nutrient and Health Survey. CONCLUSION: This study established an overall association between U-Cd and renal injury biomarkers, and explored the Chinese dietary Cd exposure limits, which helps improve Chinese Cd exposure risk assessment and provides a reference basis for formulating reasonable exposure standards.

Albumin inhibits the nuclear translocation of Smad3 via interleukin-1beta signaling in hepatic stellate cells.

Activation of quiescent hepatic stellate cells (HSCs) to myofibroblasts plays a key role in liver fibrosis. We had previously shown that albumin and its derivative, R-III (a retinol-binding protein-albumin domain III fusion protein), inhibited HSC activation by sequestering retinoic acid (RA) and that R-III administration reduced carbon tetrachloride (CCl4)-induced liver fibrosis. In this study, we aimed to elucidate the mechanism of action of albumin downstream of RA sequestration. Nuclear factor-κB p65 was evenly distributed in the cytoplasm in activated mouse HSCs, whereas albumin expression or R-III treatment (albumin/R-III) caused the nuclear translocation of p65, probably via RA sequestration, resulting in a dramatic increase in interleukin-1beta (IL-1ß) expression. Albumin/R-III in turn induced the phosphorylation of Smad3 at the linker region, inhibiting its nuclear import in an IL-1ß-dependent manner. Consistent with the in vitro results, the level of IL-1ß mRNA expression was higher in CCl4/R-III-treated livers than in CCl4-treated livers. These findings reveal that albumin/R-III inhibits the transforming growth factor-ß-Smad3 signaling as well as the retinoic acid receptor-mediated pathway, which probably contributes to the inhibition of HSC activation, and suggest that R-III may be an anti-fibrotic drug candidate.

Measuring the serum level of retinol-binding protein can enhance the treatment of recalcitrant warts.

BACKGROUND: Warts are common benign (60%-65%) self-limited tumors of the epidermis caused by human papillomaviruses (HPVs). However, some warts fail to resolve despite of different treatments and become recalcitrant. Vitamin A has antiproliferative and antikeratinizing properties by which the disruption of HPV replication can be occurred. Concentrations of retinol-binding protein (RBP) and retinol in the circulation highly correlate with each others. AIM: To assess the serum level of RBP in patients with resistant warts to evaluate the possible role of retinol in the disease pathogenesis. PATIENTS: This case-control study included 30 patients with resistant cutaneous warts (defined as failure of cure after conventional treatment as 12 weeks of salicylic acid application, 4 or more cycles of cryotherapy or electrocautery and/or other physical treatment modalities) and 30 age- and sex-matched healthy controls. RBP level in the serum was measured by ELISA. RESULTS: There was a significant difference between cases and controls regarding the level of serum RBP (P = .001). However, serum RBP level did not differ significantly regarding sociodemographic or clinical data (P > .05 each). RBP is a good biomarker for significant early detection and discrimination between cases and controls (P = .001) at a cutoff point < 563.3 mg/l with sensitivity (93%) and specificity (80%). CONCLUSION: Low serum RBP level in our studied patients may suggest an important role of retinol in the resistant warts pathogenesis. Thus measuring serum RBP will help to identify patients who are going to have resistant warts in the future.

Chitosan Oligosaccharides Suppress Nuclear Factor-Kappa B Activation and Ameliorate Experimental Autoimmune Uveoretinitis in Mice.

We investigated the therapeutic potential and mechanism of chitosan oligosaccharides (COS) for experimental autoimmune uveoretinitis (EAU) in mice. EAU was induced in C57/BL6 mice by injection of human interphotoreceptor retinoid-binding protein (IRBP) peptides. At the same time, a high or low dose (20 or 10 mg/kg) of COS or phosphate-buffered saline (PBS) was given to mice daily after EAU induction. We found that mouse EAU is ameliorated by the high-dose COS treatment when compared with PBS treatment. In the retinas of high-dose COS-treated mice, the nuclear translocation of NF-κB subunit (p65) was suppressed, and the expression of several key EAU inflammatory mediators, IFN-γ, TNF-α, IL-1α, IL-4, IL-5, IL-6, IL-10, IL-17 and MCP-1 was lowered. These results suggest that COS may be a potential treatment for posterior uveitis.