Retinol binding protein 4 promotes the phenotypic transformation of vascular smooth muscle cells under high glucose condition via modulating RhoA/ROCK1 pathway.

Phenotypic switch of vascular smooth muscle cells (VSMCs) contributes to the pathogenesis of atherosclerosis (AS). High level of retinol binding protein 4 (RBP4) is regarded as a risk factor in cardiac-cerebral vascular disease. This study is performed to clarify the biological function of RBP4 in modulating the phenotypic switch of VSMCs induced via RhoA/ROCK1 signaling pathway. In vivo experiment, all the rats were divided into control group (NC), diabetic group (DM) and diabetic atherosclerosis group (DAS). The expressions of biochemical indicators, RhoA and Rho associated coiled-coil containing protein kinase 1 (ROCK1) were detected. In vitro experiment, VSMCs were cultured under high glucose condition, and ectogenic RBP4, HA-1100, rapamycin, or 3-methyladenine (3-MA) were supplemented to treat the VSMCs, respectively. The proliferation and migration of VSMCs were evaluated. The regulatory relationship between RBP4 and ROCK1 was predicted by bioinformatics analysis, and validated by qRT-PCR and Western blot. The regulatory effects of RBP4 on contractile phenotypic markers such as calponin, MYH11, α-SMA and autophagy markers including LC3II, LC3I, and Beclin-1 as well as mTOR were also detected. Moreover, VSMCs were cultured exposed to ROCK1 overexpressed plasmid or short hairpin RNA (shRNA), the proliferation and migration of VSMCs were evaluated and the regulatory effects of RhoA/ROCK1 signaling pathway on contractile phenotypic markers and autophagy markers were also detected. In vivo, RhoA, ROCK1, and mTOR were highly expressed in the rats intraperitoneally injected with RBP4. In vitro, the expressions of calponin, MYH11, α-SMA, LC3II, LC3I, and Beclin-1 were decreased in VSMCs treated with ROCK1-OA under high glucose condition, conversely, the expressions were increased in VSMCs exposed to ROCK1-shRNA. After incubated with rapamycin additionally, the expressions of calponin, MYH11, α-SMA, LC3II/I and Beclin-1 were up-regulated and the expression of p-mTOR was decreaed in VSMCs of HG+ROCK1-OA. Conversely, after incubated with 3-MA additionally, the expressions of calponin, MYH11, α-SMA, LC3II/I and Beclin-1 were down-regulated and the expression of p-mTOR was elevated in VSMCs of HG+ROCK1-shRNA. Ectogenic RBP4 facilitated high glucose-induced proliferation and migration of VSMCs, and it repressed the expression of calponin, MYH11, α-SMA, LC3II/I, and Beclin-1 in VSMCs. As expected, ROCK1 inhibit or counteracted the biological effects of RBP4 on VSMCs. In addition, the expressions of contractile phenotypic markers, LC3II/I, and Beclin-1 were promoted and mTOR were decreased after the VSMCs treated with autophagy agonist, whereas no significant difference was observed in the expressions of ROCK1, RhoA. RBP4 is an injurious factor in the pathogenesis of diabetic AS, and it promotes the phenotypic switch of VSMCs via activating RhoA/ROCK1 pathway and inhibiting autophagy.

Optimization of the extract from flower of Citrus aurantium L. var. amara Engl. and its inhibition of lipid accumulation.

Flower of Citrus aurantium L. var. amara Engl. (CAVA) has been confirmed to have promising anti-obesity effects. However, the regulation of alkaloid extracts from flower of CAVA (Al) on lipid metabolism remain unknown. In this study, Al was optimized by ultrasound-assisted extraction using response surface methodology. The optimal conditions were ultrasonic time 72 min, ethanol concentration 78% and liquid/solid ratio 30 ml/g with the maximum alkaloid yield 5.66%. LC-MS assay indicated that the alkaloid compounds were enriched in Al after optimization. Nine alkaloid compounds were identified in Al by LC-MS assay and stachydrine, caffeine and cathine appeared as the major alkaloid compounds. Bioactivity assay showed that Al treatment significantly increased superoxide dismutase (SOD) activity, and reduced malonaldehyde (MDA) and reactive oxygen species (ROS) levels. Al administration also reversed oleic acid-induced hepatic steatosis in Hep G2 cells by inhibiting the expression of lipogenesis-signaling genes including fatty acid synthase (FAS), peroxisome proliferator-activated receptor subtype γ (PPARγ), uncoupling protein 2 (UCP2), and retinol binding protein (RBP4). However, OA-induced reduction of lipolysis-related gene carnitine palmitoyl transferase 1A (CPT1A) in Hep G2 cells was not improved by Al supplementation. Moreover, the increased SOD activity and decreased MDA and ROS contents were also observed in Caenorhabditis elegans by Al addition. Al intervention exhibited the ability to inhibit lipid accumulation in C. elegans by suppressing expression of lipid metabolism-related genes. These results suggested that the alkaloid extracts from the flower of CAVA showed great potential to regulate lipid metabolism. PRACTICAL APPLICATIONS: The extraction of alkaloid extracts from the flower of CAVA was optimized with a maximum yield of 5.66%. The regulatory effects and mechanisms of Al on lipid metabolism of Hep G2 cells and Caenorhabditis elegans were also investigated. More clinical studies are required to evaluate the potential of using alkaloids from the flower of CAVA as therapeutic agents against lipid metabolic disorders.

Genetic dissection in mice reveals a dynamic crosstalk between the delivery pathways of vitamin A.

Vitamin A is distributed within the body to support chromophore synthesis in the eyes and retinoid signaling in most other tissues. Two pathways exist for the delivery of vitamin A: the extrinsic pathway transports dietary vitamin A in lipoproteins from intestinal enterocytes to tissues, while the intrinsic pathway distributes vitamin A from hepatic stores bound to serum retinol binding protein (RBP). Previously, the intestine-specific homeodomain transcription factor (ISX) and the RBP receptor STRA6 were identified as gatekeepers of these pathways; however, it is not clear how mutations in the corresponding genes affect retinoid homeostasis. Here, we used a genetic dissection approach in mice to examine the contributions of these proteins in select tissues. We observed that ISX deficiency increased utilization of both preformed and provitamin A. We found that increased storage of retinoids in peripheral tissues of ISX-deficient mice was dependent on STRA6 and induced by retinoid signaling. In addition, double-mutant mice exhibited a partial rescue of the Stra6 mutant ocular phenotype. This rescue came at the expense of a massive accumulation of vitamin A in other tissues, demonstrating that vitamin A is randomly distributed when present in excessive amounts. Remarkably, provitamin A supplementation of mutant mice induced the expression of the RBP receptor 2 in the liver and was accompanied by increased hepatic retinyl ester stores. Taken together, these findings indicate dynamic crosstalk between the delivery pathways for this essential nutrient and suggest that hepatic reuptake of vitamin A takes place when excessive amounts circulate in the blood.

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.

Vitamin A deficiency has declined in Malawi, but with evidence of elevated vitamin A in children.

BACKGROUND: Reduction of vitamin A deficiency (VAD) in Malawi coincided with introduction of vitamin A-fortified staple foods, alongside continued biannual high-dose vitamin A supplementation (VAS). OBJECTIVE: We describe coverage of vitamin A interventions and vitamin A status in the 2015-2016 Malawi Micronutrient Survey. METHODS: Food samples and biospecimens were collected within a representative household survey across 105 clusters. Retinol was measured using ultraviolet excitation fluorescence (sugar) and photometric determination (oil). Preschool children (PSC, aged 6-59 mo, n = 1102), school-age children (SAC, aged 5-14 y, n = 758), nonpregnant women (n = 752), and men (n = 219) were initially assessed for vitamin A status using retinol binding protein (RBP) and modified relative dose response (MRDR). Randomly selected fasted MRDR participants (n = 247) and nonfasted women and children (n = 293) were later assessed for serum retinol, retinyl esters, and carotenoids. Analyses accounted for complex survey design. RESULTS: We tested sugar and oil samples from 71.8% and 70.5% of the households (n = 2,112), respectively. All of the oil samples and all but one of the sugar samples had detectable vitamin A. National mean retinol sugar and oil contents were 6.1 ± 0.7 mg/kg and 6.6 ± 1.4 mg/kg, respectively. Receipt of VAS in the previous 6 mo was reported by 68.0% of PSC. VAD prevalence (RBP equivalent to <0.7µmol retinol/L) was 3.6% in PSC, and <1% in other groups. One woman and no children had MRDR ≥0.060 indicating VAD. Among fasted PSC and SAC, 18.0% (95% CI: 6.4, 29.6) and 18.8% (7.2, 30.5) had >5% of total serum vitamin A as retinyl esters, and 1.7% (0.0, 4.1) and 4.9% (0.0, 10.2) had >10% of total serum vitamin A as retinyl esters. Serum carotenoids indicated recent intake of vitamin A-rich fruits and vegetables. CONCLUSIONS: Near elimination of VAD in Malawi is a public health success story, but elevated levels of vitamin A among children suggests that vitamin A interventions may need modification.

Identification of MFRP and the secreted serine proteases PRSS56 and ADAMTS19 as part of a molecular network involved in ocular growth regulation.

Precise regulation of ocular size is a critical determinant of normal visual acuity. Although it is generally accepted that ocular growth relies on a cascade of signaling events transmitted from the retina to the sclera, the factors and mechanism(s) involved are poorly understood. Recent studies have highlighted the importance of the retinal secreted serine protease PRSS56 and transmembrane glycoprotein MFRP, a factor predominantly expressed in the retinal pigment epithelium (RPE), in ocular size determination. Mutations in PRSS56 and MFRP constitute a major cause of nanophthalmos, a condition characterized by severe reduction in ocular axial length/extreme hyperopia. Interestingly, common variants of these genes have been implicated in myopia, a condition associated with ocular elongation. Consistent with these findings, mice with loss of function mutation in PRSS56 or MFRP exhibit a reduction in ocular axial length. However, the molecular network and cellular processes involved in PRSS56- and MFRP-mediated ocular axial growth remain elusive. Here, we show that Adamts19 expression is significantly upregulated in the retina of mice lacking either Prss56 or Mfrp. Importantly, using genetic mouse models, we demonstrate that while ADAMTS19 is not required for ocular growth during normal development, its inactivation exacerbates ocular axial length reduction in Prss56 and Mfrp mutant mice. These results suggest that the upregulation of retinal Adamts19 is part of an adaptive molecular response to counteract impaired ocular growth. Using a complementary genetic approach, we show that loss of PRSS56 or MFRP function prevents excessive ocular axial growth in a mouse model of early-onset myopia caused by a null mutation in Irbp, thus, demonstrating that PRSS56 and MFRP are also required for pathological ocular elongation. Collectively, our findings provide new insights into the molecular network involved in ocular axial growth and support a role for molecular crosstalk between the retina and RPE involved in refractive development.

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.

Treatment With FoxP3+ Antigen-Experienced T Regulatory Cells Arrests Progressive Retinal Damage in a Spontaneous Model of Uveitis.

We specify the clinical features of a spontaneous experimental autoimmune uveitis (EAU) model, in which foreign hen-egg lysozyme (HEL) is expressed in the retina, controlled by the promoter for interphotoreceptor retinol binding protein (IRBP). We previously reported 100% P21 (post-partum day) IRBP:HEL single transgenic (sTg) mice, when crossed to transgenic T cell receptor mice (3A9) generating the double transgenic (dTg) genotype, develop EAU despite profound lymphopenia (thymic HEL-specific T cell deletion). In this work, we characterized the immune component of this model and found conventional dTg CD4+ T cells were less anergic than those from 3A9 controls. Furthermore, prior in vitro HEL-activation of 3A9 anergic T cells (Tan) rendered them uveitogenic upon adoptive transfer (Tx) to sTg mice, while antigen-experienced (AgX, dTg), but not naïve (3A9) T cells halted disease in P21 dTg mice. Flow cytometric analysis of the AgX cells elucidated the underlying pathology: FoxP3+CD25hiCD4+ T regulatory cells (Treg) comprised ∼18%, while FR4+CD73+FoxP3-CD25lo/-CD4+ Tan comprised ∼1.2% of total cells. Further Treg-enrichment (∼80%) of the AgX population indicated FoxP3+CD25hiCD4+ Treg played a key role in EAU-suppression while FoxP3-CD25lo/-CD4+ T cells did not. Here we present the novel concept of dual immunological tolerance where spontaneous EAU is due to escape from anergy with consequent failure of Treg induction and subsequent imbalance in the [Treg:Teffector] cell ratio. The reduced numbers of Tan, normally sustaining Treg to prevent autoimmunity, are the trigger for disease, while immune homeostasis can be restored by supplementation with AgX, but not naïve, antigen-specific Treg.

Tofacitinib inhibits the development of experimental autoimmune uveitis and reduces the proportions of Th1 but not of Th17 cells.

Purpose: Tofacitinib is a pan-Janus kinase (JAK) inhibitor that suppresses cytokine signaling and in turn, the cells that participate in inflammatory immunopathogenic processes. We examined the capacity of tofacitinib to inhibit the induction of experimental autoimmune uveitis (EAU) and related immune responses. Methods: EAU was induced in B10.A mice with immunization with bovine interphotoreceptor retinoid-binding protein (IRBP), emulsified in complete Freund's adjuvant (CFA), and a simultaneous injection of pertussis toxin. Tofacitinib, 25 mg/kg, was administered daily, and the vehicle was used for control. EAU development was assessed by histological analysis of the mouse eyes, and related immune responses were assessed by (i) the levels of interferon (IFN)-γ and interleukin (IL)-17, secreted by spleen cells cultured with IRBP; (ii) flow cytometric analysis of intracellular expression by spleen, or eye-infiltrating CD4 or CD8 cells of IFN-γ, IL-17, and their transcription factors, T-bet and RORγt. In addition, the inflammation-related cell markers CD44 and CD62L and Ki67, a proliferation marker, were tested. The proportions of T-regulatory cells expressing FoxP3 were determined by flow cytometric intracellular staining, while levels of antibody to IRBP were measured with enzyme-linked immunosorbent assay (ELISA). Results: Treatment with tofacitinib significantly suppressed the development of EAU and reduced the levels of secreted IFN-γ, but not of IL-17. Further, treatment with tofacitinib reduced in the spleen and eye-infiltrating cells the intracellular expression of IFN-γ and its transcription factor T-bet. In contrast, treatment with tofacitinib had essentially no effect on the intracellular expression of IL-17 and its transcription factor, RORγt. The selective effect of tofacitinib treatment was particularly evident in the CD8 population. Treatment with tofacitinib also increased the population of CD44, but reduced the populations of cells producing CD62L and Ki67. Treatment with tofacitinib had no effect on the proportion of FoxP3 producing regulatory cells and on the antibody production to IRBP. Conclusions: Treatment with tofacitinib inhibited the development of EAU, reduced the production of IFN-γ, but had essentially no effect on the production of IL-17.

Single particle cryo-EM of the complex between interphotoreceptor retinoid-binding protein and a monoclonal antibody.

Interphotoreceptor retinoid-binding protein (IRBP) is a highly expressed protein secreted by rod and cone photoreceptors that has major roles in photoreceptor homeostasis as well as retinoid and polyunsaturated fatty acid transport between the neural retina and retinal pigment epithelium. Despite two crystal structures reported on fragments of IRBP and decades of research, the overall structure of IRBP and function within the visual cycle remain unsolved. Here, we studied the structure of native bovine IRBP in complex with a monoclonal antibody (mAb5) by cryo-electron microscopy, revealing the tertiary and quaternary structure at sufficient resolution to clearly identify the complex components. Complementary mass spectrometry experiments revealed the structure and locations of N-linked carbohydrate post-translational modifications. This work provides insight into the structure of IRBP, displaying an elongated, flexible three-dimensional architecture not seen among other retinoid-binding proteins. This work is the first step in elucidation of the function of this enigmatic protein.

Parental Uveitis Influences Offspring With an Increased Susceptibility to the Experimental Autoimmune Uveitis.

Purpose: Previous studies have shown that parental abnormal physiological conditions such as inflammation, stress, and obesity can be transferred to offspring. The purpose of this study was to investigate the impact of parental uveitis on the development and susceptibility to experimental autoimmune uveitis (EAU) in offspring. Methods: Parental male and female B10RIII mice were immunized with interphotoreceptor retinoid binding protein (IRBP) 161-180 in complete Freund's adjuvant and were immediately allowed to mate. Gross examination of the offspring gestated with EAU was performed to determine the influence of parental uveitis on offspring development after birth. Gene expression profiles were analyzed in the affected eyes of offspring under EAU to identify differentially expressed genes (DEGs). Adult offspring were given 5, 25, and 50 µg IRBP161-180 to compare their susceptibility to EAU. Immunized mice were clinically and pathologically evaluated for the development of EAU. Ag-specific T-cell proliferation and IL-17 production from spleens and lymph nodes were evaluated on day 14 or 35 after immunization. Results: Hair loss, delay of eye opening, and swollen spleens in the offspring from parents with uveitis were observed from day 14 to 39 after birth. DEGs were involved in the immune system process, muscle system process, and cell development. The altered antigen processing and presentation, cell adhesion molecules, and phagosome in the eyes of the offspring from uveitis-affected parents were enriched. Offspring gestated with EAU showed a susceptibility to EAU and an earlier onset and higher severity of EAU compared to the control group mice. IRBP-specific lymphocyte proliferation and IL-17 production were observed in the EAU offspring with exposure to parental uveitis. Conclusions: The results suggest that mouse parents with uveitis can increase their offspring's susceptibility to EAU, probably through altering cell adhesion molecules and antigen processing and presentation related to the T-cell proliferation and Th17 response.

Matairesinol ameliorates experimental autoimmune uveitis by suppression of IRBP-specific Th17 cells.

We investigated the effects of matairesinol (MAT) in the experimental autoimmune uveitis (EAU), a classical animal model of uveitis. We found that treatment with MAT could alleviate intraocular inflammation of EAU. Notably, Th17 cells in eyes of EAU mice could be predominantly restrained by MAT. Furthermore, MAT could inhibit Th17 differentiation in vitro. In addition, MAT inhibited the signaling of MAPK and ROR-γt, a pivotal transcription factor for Th17 cell differentiation in vitro and in vivo. Taken together, these results suggested that MAT had immune-suppressive effects on autoimmune inflammation through Th17 cells.

Maternal-Fetal Transfer of Vitamin A and Its Impact on Mammalian Embryonic Development.

The placenta, a hallmark of mammalian embryogenesis, allows nutrients to be exchanged between the mother and the fetus. Vitamin A (VA), an essential nutrient, cannot be synthesized by the embryo, and must be acquired from the maternal circulation through the placenta. Our understanding of how this transfer is accomplished is still in its infancy. In this chapter, we recapitulate the early studies about the relationship between maternal dietary/supplemental VA intake and fetal VA levels. We then describe how the discovery of retinol-binding protein (RBP or RBP4), the development of labeling and detection techniques, and the advent of knockout mice shifted this field from a macroscopic to a molecular level. The most recent data indicate that VA and its derivatives (retinoids) and the pro-VA carotenoid, ß-carotene, are transferred across the placenta by distinct proteins, some of which overlap with proteins involved in lipoprotein uptake. The VA status and dietary intake of the mother influence the expression of these proteins, creating feedback signals that control the uptake of retinoids and that may also regulate the uptake of lipids, raising the intriguing possibility of crosstalk between micronutrient and macronutrient metabolism. Many questions remain about the temporal and spatial patterns by which these proteins are expressed and transferred throughout gestation. The answers to these questions are highly relevant to human health, considering that those with either limited or excessive intake of retinoids/carotenoids during pregnancy may be at risk of obtaining improper amounts of VA that ultimately impact the development and health of their offspring.

Vitamin A and iron status of children before and after treatment of uncomplicated severe acute malnutrition.

BACKGROUND & AIMS: Treatment of children with uncomplicated severe acute malnutrition (SAM) is based on ready-to-use therapeutic foods (RUTF) and aims for quick regain of lost body tissues while providing sufficient micronutrients to restore diminished body stores. Little evidence exists on the success of the treatment to establish normal micronutrient status. We aimed to assess the changes in vitamin A and iron status of children treated for SAM with RUTF, and explore the effect of a reduced RUTF dose. METHODS: We collected blood samples from children 6-59 months old with SAM included in a randomised trial at admission to and discharge from treatment and analysed haemoglobin (Hb) and serum concentrations of retinol binding protein (RBP), ferritin (SF), soluble transferrin receptor (sTfR), C-reactive protein (CRP) and α1-acid glycoprotein (AGP). SF, sTfR and RBP were adjusted for inflammation (CRP and AGP) prior to analysis using internal regression coefficients. Vitamin A deficiency (VAD) was defined as RBP < 0.7 µmol/l, anaemia as Hb < 110 g/l, storage iron deficiency (sID) as SF < 12 µg/l, tissue iron deficiency (tID) as sTfR > 8.3 mg/l and iron deficiency anaemia (IDA) as both anaemia and sID. Linear and logistic mixed models were fitted including research team and study site as random effects and adjusting for sex, age and outcome at admission. RESULTS: Children included in the study (n = 801) were on average 13 months of age at admission to treatment and the median treatment duration was 56 days [IQR: 35; 91] in both arms. Vitamin A and iron status markers did not differ between trial arms at admission or at discharge. Only Hb was 1.7 g/l lower (95% CI -0.3, 3.7; p = 0.088) in the reduced dose arm compared to the standard dose, at recovery. Mean concentrations of all biomarkers improved from admission to discharge: Hb increased by 12% or 11.6 g/l (95% CI 10.2, 13.0), RBP increased by 13% or 0.12 µmol/l (95% CI 0.09, 0.15), SF increased by 36% or 4.4 µg/l (95% CI 3.1, 5.7) and sTfR decreased by 16% or 1.5 mg/l (95% CI 1.0, 1.9). However, at discharge, micronutrient deficiencies were still common, as 9% had VAD, 55% had anaemia, 35% had sID, 41% had tID and 21% had IDA. CONCLUSION: Reduced dose of RUTF did not result in poorer vitamin A and iron status of children. Only haemoglobin seemed slightly lower at recovery among children treated with the reduced dose. While improvement was observed, the vitamin A and iron status remained sub-optimal among children treated successfully for SAM with RUTF. There is a need to reconsider RUTF fortification levels or test other potential strategies in order to fully restore the micronutrient status of children treated for SAM.

Effect of berberine on spleen transcriptome and gut microbiota composition in experimental autoimmune uveitis.

BACKGROUND: Berberine (BBR) was reported to have immunoregulatory and anti-inflammatory properties. In this study, we investigated whether BBR could exert its effects on the development of experimental autoimmune uveitis (EAU), and if so, what was the underlying mechanism? METHODS: EAU was induced in B10R.III mice by immunization with IRBP 161-180, followed by 100 mg/kg/d BBR intragastric administration. Disease severity was assessed by evaluation of clinical and histopathological scores. Blood-retinal barrier (BRB) breakdown was tested by Evans blue. Effector and regulatory T (Treg) cell balance was evaluated by quantitative real-time PCR and flow cytometry. Spleen transcriptome was characterized by RNA sequencing (RNA-seq). Gut microbiota composition was investigated by 16S rRNA analysis. RESULTS: BBR treatment significantly blocked EAU as shown by the decrease of the clinical and histological scores, as well as the inhibition of BRB breakdown. The frequency of splenic Th1 and Th17 cells was decreased, whereas Treg cells were increased in the BBR-treated group. RNA-seq of the spleen revealed 476 differentially expressed genes (DEGs) between the EAU and EAU-BBR group. GO functional classification, as well as KEGG analysis demonstrated that BBR treatment markedly influences genes belonging to chromatin remodeling and immune-related pathways. Intervention with BBR modified the gut microbiome in EAU mice, increasing the number of bacteria with immunomodulatory capacity. Depletion of gut microbiota affected the efficacy of BBR on EAU. Moreover, the altered bacterial strains showed a significant correlation with the expression of histones. CONCLUSIONS: BBR inhibited IRBP induced EAU, which was associated with a significant change in the spleen transcriptome and intestinal microbial composition.

Disruption of Intestinal Homeostasis and Intestinal Microbiota During Experimental Autoimmune Uveitis.

Purpose: We determine the changes in intestinal microbiota and/or disruptions in intestinal homeostasis during uveitis. Methods: Experimental autoimmune uveitis (EAU) was induced in B10.RIII mice with coadministration of interphotoreceptor retinoid-binding protein peptide (IRBP) and killed mycobacterial antigen (MTB) as an adjuvant. Using 16S rRNA gene sequencing, we looked at intestinal microbial differences during the course of uveitis, as well as intestinal morphologic changes, changes in intestinal permeability by FITC-dextran leakage, antimicrobial peptide expression in the gastrointstinal tract, and T lymphocyte prevalence before and at peak intraocular inflammation. Results: We demonstrate that increased intestinal permeability and antimicrobial peptide expression in the intestinal tract coincide in timing with increased effector T cells in the mesenteric lymph nodes, during the early stages of uveitis, before peak inflammation. Morphologic changes in the intestine were most prominent during this phase, but also occurred with adjuvant MTB alone, whereas increased intestinal permeability was found only in IRBP-immunized mice that develop uveitis. We also demonstrate that the intestinal microbiota were altered during the course of uveitis, and that some of these changes are specific to uveitic animals, whereas others are influenced by adjuvant MTB alone. Intestinal permeability peaked at 2 weeks, coincident with an increase in intestinal bacterial strain differences, peak lipocalin production, and peak uveitis. Conclusions: An intestinal dysbiosis accompanies a disruption in intestinal homeostasis in autoimmune uveitis, although adjuvant MTB alone promotes intestinal disruption as well. This may indicate a novel axis for future therapeutic targeting experimentally or clinically.

Nutrition and Inflammatory Biomarkers in Chronic Pancreatitis Patients.

BACKGROUND: Chronic pancreatitis (CP) patients frequently experience malabsorption and maldigestion, leading to micronutrient and macronutrient deficiencies. Comorbid diabetes and lifestyle habits, such as alcohol consumption, may impact nutrition status. METHODS: We compared micronutrient antioxidant, bone metabolism, serum protein, and inflammatory marker levels in 301 CP patients and 266 controls with no known pancreatic disease. We analyzed serum prealbumin and retinol binding protein; vitamins A, D, E, and B12; osteocalcin; tumor necrosis factor-α; and C-reactive protein (CRP). We also evaluated biomarkers among subsets of patients, examining factors including time since diagnosis, body mass index, alcohol as primary etiology, diabetes mellitus, vitamin supplementation, and pancreatic enzyme replacement. RESULTS: After correcting for multiple comparisons, CP patients had significantly lower levels than controls of the following: vitamin A (40.9 vs 45.4 µg/dL) and vitamin E (α-tocopherol [8.7 vs 10.3 mg/L] and γ-tocopherol [1.8 vs 2.2 mg/L]), as well as osteocalcin (7.9 vs 10 ng/mL) and serum prealbumin (23 vs 27 mg/dL). Both patients and controls who took vitamin supplements had higher serum levels of vitamins than those not taking supplements. Compared with controls, in controlled analyses, CP patients had significantly lower levels of vitamins A, D, and E (both α-tocopherol and γ-tocopherol). CP patients also had significantly lower levels of osteocalcin, serum prealbumin, and retinol binding protein, and higher CRP. CONCLUSIONS: CP patients demonstrated lower levels of selected nutrition and bone metabolism biomarkers than controls. Diabetes and alcohol did not impact biomarkers. Vitamin supplements and pancreatic enzyme replacement therapy improved nutrition biomarkers in CP patients.

Ligand binding properties of two Brugia malayi fatty acid and retinol (FAR) binding proteins and their vaccine efficacies against challenge infection in gerbils.

Parasitic nematodes produce an unusual class of fatty acid and retinol (FAR)-binding proteins that may scavenge host fatty acids and retinoids. Two FARs from Brugia malayi (Bm-FAR-1 and Bm-FAR-2) were expressed as recombinant proteins, and their ligand binding, structural characteristics, and immunogenicities examined. Circular dichroism showed that rBm-FAR-1 and rBm-FAR-2 are similarly rich in α-helix structure. Unexpectedly, however, their lipid binding activities were found to be readily differentiated. Both FARs bound retinol and cis-parinaric acid similarly, but, while rBm-FAR-1 induced a dramatic increase in fluorescence emission and blue shift in peak emission by the fluorophore-tagged fatty acid (dansyl-undecanoic acid), rBm-FAR-2 did not. Recombinant forms of the related proteins from Onchocerca volvulus, rOv-FAR-1 and rOv-FAR-2, were found to be similarly distinguishable. This is the first FAR-2 protein from parasitic nematodes that is being characterized. The relative protein abundance of Bm-FAR-1 was higher than Bm-FAR-2 in the lysates of different developmental stages of B. malayi. Both FAR proteins were targets of strong IgG1, IgG3 and IgE antibody in infected individuals and individuals who were classified as endemic normal or putatively immune. In a B. malayi infection model in gerbils, immunization with rBm-FAR-1 and rBm-FAR-2 formulated in a water-in-oil-emulsion (®Montanide-720) or alum elicited high titers of antigen-specific IgG, but only gerbils immunized with rBm-FAR-1 formulated with the former produced a statistically significant reduction in adult worms (68%) following challenge with B. malayi infective larvae. These results suggest that FAR proteins may play important roles in the survival of filarial nematodes in the host, and represent potential candidates for vaccine development against lymphatic filariasis and related filarial infections.

Significance of retinol binding protein and prealbumin in neonatal nutritional evaluation.

Neonatal nutritional status means great significance for health, safety and thriving growth of neonates. Therefore, clinical evaluation of neonatal nutritional status is of great significance. In order to find effective judgment basis and curative effect evaluation index that reflect nutritional status of neonate. In this study randomly selected 2400 cases of premature infants from neonates born during June 2014 - June 2015 as study group, and selected 2000 normal neonates born in the same period as reference group. A comparative study was done on basic situation of the two indexes of retinol binding protein and prealbumin of the two groups of neonates. Results showed that retinol binding protein index of observation group infants was relatively low, when compared to reference group neonates, differences between the two groups P<0.05, with statistical significance. Medical personnel timely provided intravenous nutrition therapy for observation group infants and measure their index of retinol binding protein and prealbumin after 7 days of treatment, finding obvious improvement compared to the previous one. Neonatal nutritional status improved significantly, with difference between the two groups P<0.05, with statistical value. Hence, it is not difficult to conclude that timely index detection of retinol binding protein and prealbumin of neonates means important significance for neonatal nutrition evaluation, improvement in quality level of neonatal life, and thus is recommended to be promoted in clinical application.

Iron, zinc, vitamin A and selenium status in a cohort of Indonesian infants after adjusting for inflammation using several different approaches.

Inflammation confounds the interpretation of several micronutrient biomarkers resulting in estimates that may not reflect the true burden of deficiency. We aimed to assess and compare the micronutrient status of a cohort of Indonesian infants (n 230) at aged 6, 9 and 12 months by ignoring inflammation (unadjusted) and adjusting four micronutrient biomarkers for inflammation with C-reactive protein (CRP) and α-1-glycoprotein (AGP) using the following methods: (1) arithmetic correction factors with the use of a four-stage inflammation model; and (2) regression modelling. Prevalence of infants with any inflammation (CRP>5 mg/l and/or AGP>1 g/l) was about 25% at each age. Compared with unadjusted values, regression adjustment at 6, 9 and 12 months generated the lowest (P50 % across all ages. In conclusion, without inflammation adjustment, Fe deficiency was grossly under-estimated and vitamin A and Zn deficiency over-estimated, highlighting the importance of correcting for the influence of such, before implementing programmes to alleviate micronutrient malnutrition. However, further work is needed to validate the proposed approaches with a particular focus on assessing the influence of varying degrees of inflammation (i.e. recurrent acute infections and low-grade chronic inflammation) on each affected nutrient biomarker.