Promotion of Orange-Fleshed Sweet Potato Increased Vitamin A Intakes and Reduced the Odds of Low Retinol-Binding Protein among Postpartum Kenyan Women.

Background: Orange-fleshed sweet potato (OFSP) improves vitamin A (VA) status of young children; research with pregnant and lactating women is limited.Objective: We examined the effectiveness of the Mama SASHA (Sweetpotato Action for Security and Health in Africa) program to improve nutrition knowledge, diets, and nutritional status of pregnant and lactating women (PLW) in Western Kenya.Methods: Eight health facilities were allocated to the Mama SASHA intervention or comparison arms. PLW in intervention facilities received enhanced nutrition counseling at health clinics, were linked with community-based maternal support groups, and received vouchers for OFSP vine cuttings. Control PLW received clinic-based nutrition counseling only. A total of 505 women in early and midpregnancy, attending their first antenatal care visit, and with no previous engagement in project activities were enrolled from the 8 facilities. Nutrition and health-seeking knowledge, food security, dietary patterns, and anthropometric measurements were collected at 4 time points at ≤9 mo postpartum. VA intakes were assessed with multipass 24-h recalls in a subsample of 206 mothers at 8-10 mo postpartum. VA status was assessed by using serum retinol-binding protein (RBP). Impacts were estimated with multilevel mixed models adjusted for clustering and differences at enrollment.Results: At enrollment, 22.9% of women had RBP <1.17 µmol/L. By 9 mo postpartum, intervention women had significantly higher intakes of VA [adjusted difference = 297.0 retinol activity equivalent (RAE) units; 95% CI: 82, 513 RAE units; P = 0.01; n = 206], greater consumption of VA-rich fruit and vegetables in the previous 7 d (difference-in-difference estimate: 0.40 d; 95% CI: 0.23, 0.56 d; P < 0.01), and a 45% reduction in the odds of RBP <1.17 µmol/L (OR: 0.55; 95% CI: 0.33, 0.92; P = 0.01).Conclusion: Promotion of OFSP to PLW through health services is a feasible strategy to improve women's nutrition knowledge, VA intakes, and maternal RBP.

IRBP deficiency permits precocious ocular development and myopia.

PURPOSE: Interphotoreceptor retinoid-binding protein (IRBP) is abundant in the subretinal space and binds retinoids and lipophilic molecules. The expression of IRBP begins precociously early in mouse eye development. IRBP-deficient (KO) mice show less cell death in the inner retinal layers of the retina before eyelid opening compared to wild-type C57BL/6J (WT) controls and eventually develop profound myopia. Thus, IRBP may play a role in eye development before visually-driven phenomena. We report comparative observations during the course of the natural development of eyes in WT and congenic IRBP KO mice that suggest IRBP is necessary at the early stages of mouse eye development for correct function and development to exist in later stages. METHODS: We observed the natural development of congenic WT and IRBP KO mice, monitoring several markers of eye size and development, including haze and clarity of optical components in the eye, eye size, axial length, immunohistological markers of differentiation and eye development, visually guided behavior, and levels of a putative eye growth stop signal, dopamine. We conducted these measurements at several ages. Slit-lamp examinations were conducted at post-natal day (P)21. Fundus and spectral domain optical coherence tomography (SD-OCT) images were compared at P15, P30, P45, and P80. Enucleated eyes from P5 to P10 were measured for weight, and ocular dimensions were measured with a noncontact light-emitting diode (LED) micrometer. We counted the cells that expressed tyrosine hydroxylase (TH-positive cells) at P23-P36 using immunohistochemistry on retinal flatmounts. High-performance liquid chromatography (HPLC) was used to analyze the amounts of dopamine (DA) and 3,4-dihydroxyphenylacetic acid (DOPAC) at P7-P60. Monocular form deprivation in the right eye was induced using head-mounted goggles from P28 to P56. RESULTS: Eye elongation and eye size in the IRBP KO mice began to increase at P7 compared to the WT mice. This difference increased until P12, and the difference was maintained thereafter. SD-OCT images in live mice confirmed previously reported retinal thinning of the outer nuclear layer in the IRBP KO mice compared to the WT mice from P15 to P80. Slit-lamp and fundoscopy examination outcomes did not differ between the WT and KO mice. SD-OCT measurements of the optical axis components showed that the only factor contributing to excess optical axis length was the depth of the vitreous body. No other component of optical axis length (including corneal thickness, anterior chamber depth, and lens thickness) was different from that of the WT mouse. The refractive power of the IRBP KO mice did not change in response to form deprivation. The number of retinal TH-positive cells was 28% greater in the IRBP KO retinas compared to the WT mice at P30. No significant differences were observed in the steady-state retinal DA or DOPAC levels or in the DOPAC/DA ratios between the WT and IRBP KO mice. CONCLUSIONS: The IRBP KO mouse eye underwent precocious development and rapid eye size growth temporally about a day sooner than the WT mouse eye. Eye size began to differ between the WT and KO mice before eyelid opening, indicating no requirement for focus-dependent vision, and suggesting a developmental abnormality in the IRBP KO mouse eye that precedes form vision-dependent emmetropization. Additionally, the profoundly myopic KO eye did not respond to form deprivation compared to the non-deprived contralateral eye. Too much growth occurred in some parts of the eye, possibly upsetting a balance among size, differentiation, and focus-dependent growth suppression. Thus, the loss of IRBP may simply cause growth that is too rapid, possibly due to a lack of sequestration or buffering of morphogens that normally would bind to IRBP but are unbound in the IRBP KO eye. Despite the development of profound myopia, the DA levels in the IRBP KO mice were not statistically different from those in the WT mice, even with the excess of TH-positive cells in the IRBP KO mice compared to the WT mice. Overall, these data suggest that abnormal eye elongation in the IRBP KO mouse is independent of, precedes, and is epistatic to the process(es) of visually-driven refractive development.

Interphotoreceptor Retinoid-Binding Protein Mitigates Cellular Oxidative Stress and Mitochondrial Dysfunction Induced by All-trans-Retinal.

PURPOSE: Point and null mutations in interphotoreceptor retinoid-binding protein (IRBP) cause retinal dystrophy in affected patients and IRBP-deficient mice with unknown mechanism. This study investigated whether IRBP protects cells from damages induced by all-trans-retinal (atRAL), which was increased in the Irbp(-/-) retina. METHODS: Wild-type and Irbp(-/-) mice retinal explants in buffer with or without purified IBRP were exposed to 800 lux light for different times and subjected to retinoid analysis by high-performance liquid chromatography. Purity of IRBP was determined by Coomassie Brilliant Blue staining and immunoblot analysis. Cellular damages induced by atRAL in the presence or absence of IRBP were evaluated in the mouse photoreceptor-derived 661W cells. Cell viability and death were measured by 3-(4,5-dimethyl-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) and TUNEL assays. Expression and modification levels of retinal proteins were determined by immunoblot analysis. Intracellular reactive oxygen species (ROS) and nitric oxide (NO) were detected with fluorogenic dyes and confocal microscopy. Mitochondrial membrane potential was analyzed by using JC-1 fluorescent probe and a flow cytometer. RESULTS: Content of atRAL in Irbp(-/-) retinal explants exposed to light for 40 minutes was significantly higher than that in wild-type retinas under the same light conditions. All-trans-retinal caused increase in cell death, tumor necrosis factor activation, and Adam17 upregulation in 661W cells. NADPH oxidase-1 (NOX1) upregulation, ROS generation, NO-mediated protein S-nitrosylation, and mitochondrial dysfunction were also observed in 661W cells treated with atRAL. These cytotoxic effects were significantly attenuated in the presence of IRBP. CONCLUSIONS: Interphotoreceptor retinoid-binding protein is required for preventing accumulation of retinal atRAL, which causes inflammation, oxidative stress, and mitochondrial dysfunction of the cells.

Micronutrient status and neurodevelopment in internationally adopted children.

AIM: To assess the status of nutrients relevant for brain development in internationally adoptees from disparate global regions and determine whether identified deficiencies are associated with neurodevelopment. METHODS: Participants included children adopted from Post-Soviet States (n = 15), Ethiopia (n = 26) or China (n = 17), ages 8-18 months. A comprehensive nutritional battery and a neurodevelopmental assessment were completed at baseline (within one month of arrival) and follow-up (six months later). RESULTS: At baseline, 35% were stunted, and 68% had at least one abnormal nutritional biochemical marker. The most common were low retinol-binding protein (33%), zinc deficiency (29%), vitamin D insufficiency/deficiency (21%), and iron deficiency (15%). There was significant catch-up growth in height and weight at follow-up, but little improvement in micronutrient deficiencies. Iron deficiency was associated with lower cognitive scores on the Bayley Scales of Infant Development-III, p = 0.027, and slower speed of processing, p = 0.012. Zinc deficiency was associated with compromised memory functioning, p = 0.001. CONCLUSION: Nutrient deficiencies were common during the early adoption period in internationally adoptees from three global regions, and iron and zinc deficiencies were associated with poorer neurodevelopmental outcomes. Results emphasise the importance of monitoring micronutrient status at arrival and during the early adoption period, irrespective of country of origin.

Trastornos de la conducta alimentaria en la edad pediátrica: una patología en auge / Eating disorders in pediatric age: a boom pathology

Objetivos: determinar las características de los pacientes en edad pediátrica afectados de trastornos de la conducta alimentaria que requirieron ingreso en el Servicio de Pediatría del Complejo Hospitalario Universitario de Canarias (CHUC). Material y método: se realizó un estudio retrospectivo de una cohorte de pacientes pediátricos diagnosticados de trastornos de la conducta alimentaria e ingresados en planta de hospitalización pediátrica durante los últimos siete años. Resultados: de los 35 pacientes de la muestra, un 85,7% fueron mujeres, con una edad media al debut de 13,5 años. En el 77,1% de los casos el diagnóstico principal fue el de anorexia nerviosa de tipo restrictivo. Las alteraciones analíticas más frecuentes detectadas en el momento del ingreso consistieron en descenso de los niveles plasmáticos de proteína fijadora del retinol (RBP), presente en el 57,6% de los casos, e hipovitaminosis D, que en esta muestra estaba presente en el 46,9% de los casos. Se requirió el uso de suplementos hipercalóricos en el 71,4% de los pacientes durante la hospitalización. La ganancia ponderal media durante la estancia fue mayor cuanto menor era el índice de masa corporal (IMC) al ingreso (p = 0,006). Conclusiones: los TCA son enfermedades con incidencia creciente en la edad pediátrica. La pubertad constituye un momento de especial vulnerabilidad para el desarrollo de los TCA (así como de complicaciones médicas secundarias a la desnutrición). En muchos casos el ingreso hospitalario constituye una herramienta necesaria para un correcto manejo, instaurándose las medidas de control necesarias para la recuperación ponderal, la prevención de complicaciones del soporte nutricional y el abordaje de la psicopatología subyacente. El diagnóstico y tratamiento precoz resultan cruciales para evitar una excesiva pérdida ponderal y mayor incidencia de complicaciones (AU)

Aims: to determine the characteristics of pediatric patients suffering from eating disorders that were hospitalized at Hospital Universitario de Canarias. Materials and methods: a retrospective study in a cohort of pediatric patients diagnosed with eating disorders and admitted in our area was developed during the last seven years. Results: out of 35 patients in our study, 85.7 % were women, onset average age 13.5. 77.1% of the cases were diagnosed as anorexia nervosa- restrictive type. The most frequent analytical alterations, detected when patients were in hospital, consisted in a drop in plasma levels in retinol binding protein (RBP)- in 57.6% of the cases- and D hypovitaminosis- in 46.9 % of them: the use of high - calories supplements was required in 71.4% of patients during hospitalization. The average weight gain was higher when the body mass index (BMI) was smaller at patient's admission to hospital (p = 0,006). Conclussions: eating disorders are increasing in pediatric age: puberty is a special vulnerable period for its development, as well as medical complications secondary to malnutrition. Admission to hospital is an essential tool for handling many cases; taking the necessary monitoring leading to a weight increase, preventing complications in nutritional support and tackling the underlying psychopathology. Diagnosis and a precocious treatment are crucial to avoid an excessive weight loss and more complications (AU)

Vitamin A and retinol-binding protein deficiency among chronic liver disease patients.

OBJECTIVE: Vitamin A deficiency (VAD) is associated with the progression of chronic liver disease (CLD). The aim in this study was to assess levels of serum retinol and retinol-binding protein (RBP) as well as liver vitamin A stores in the presence of liver cirrhosis and hepatocellular carcinoma. METHODS: We ascertained the serum retinol and RBP levels of randomly selected CLD patients divided into two groups, one given 1500 UI (n = 89) and the other receiving 2500 UI (n = 89) doses of retinyl palmitate for the relative dose response test. Blood samples were collected in a fasting state and 5 and 7 h after supplementation. RESULTS: The prevalence of VAD was 62.4%. There was a progressive drop in serum retinol (P < 0.001) and RBP (P = 0.002) according to the severity of the liver disease, and a greater prevalence of severe VAD was noted in cirrhosis Child & Pugh C (52.8%). Fifty percent of the patients presented a low availability of RBP relative to retinol concentration, and there was no peak in RBP levels regardless of the dose of retinyl palmitate administered. CONCLUSIONS: Our findings suggest serum retinol and RBP are relevant as indicators of vitamin A nutritional status in the presence of CLD. Liver vitamin A store cannot be evaluated using the RDR test because CLD causes a reduction in RBP synthesis and interferes with the mobilization of endogenous vitamin A. Considering how the patients already showed a drop in RBP relative to retinol concentrations, it is reasonable to assume vitamin A supplementation may trigger harmful effects in CLD patients.

Methodologies for analysis of patterning in the mouse RPE sheet.

PURPOSE: Our goal was to optimize procedures for assessing shapes, sizes, and other quantitative metrics of retinal pigment epithelium (RPE) cells and contact- and noncontact-mediated cell-to-cell interactions across a large series of flatmount RPE images. METHODS: The two principal methodological advances of this study were optimization of a mouse RPE flatmount preparation and refinement of open-access software to rapidly analyze large numbers of flatmount images. Mouse eyes were harvested, and extra-orbital fat and muscles were removed. Eyes were fixed for 10 min, and dissected by puncturing the cornea with a sharp needle or a stab knife. Four radial cuts were made with iridectomy scissors from the puncture to near the optic nerve head. The lens, iris, and the neural retina were removed, leaving the RPE sheet exposed. The dissection and outcomes were monitored and evaluated by video recording. The RPE sheet was imaged under fluorescence confocal microscopy after staining for ZO-1 to identify RPE cell boundaries. Photoshop, Java, Perl, and Matlab scripts, as well as CellProfiler, were used to quantify selected parameters. Data were exported into Excel spreadsheets for further analysis. RESULTS: A simplified dissection procedure afforded a consistent source of images that could be processed by computer. The dissection and flatmounting techniques were illustrated in a video recording. Almost all of the sheet could be routinely imaged, and substantial fractions of the RPE sheet (usually 20-50% of the sheet) could be analyzed. Several common technical problems were noted and workarounds developed. The software-based analysis merged 25 to 36 images into one and adjusted settings to record an image suitable for large-scale identification of cell-to-cell boundaries, and then obtained quantitative descriptors of the shape of each cell, its neighbors, and interactions beyond direct cell-cell contact in the sheet. To validate the software, human- and computer-analyzed results were compared. Whether tallied manually or automatically with software, the resulting cell measurements were in close agreement. We compared normal with diseased RPE cells during aging with quantitative cell size and shape metrics. Subtle differences between the RPE sheet characteristics of young and old mice were identified. The IRBP(-/-) mouse RPE sheet did not differ from C57BL/6J (wild type, WT), suggesting that IRBP does not play a direct role in maintaining the health of the RPE cell, while the slow loss of photoreceptor (PhR) cells previously established in this knockout does support a role in the maintenance of PhR cells. Rd8 mice exhibited several measurable changes in patterns of RPE cells compared to WT, suggesting a slow degeneration of the RPE sheet that had not been previously noticed in rd8. CONCLUSIONS: An optimized dissection method and a series of programs were used to establish a rapid and hands-off analysis. The software-aided, high-sampling-size approach performed as well as trained human scorers, but was considerably faster and easier. This method allows tens to hundreds of thousands of cells to be analyzed, each with 23 metrics. With this combination of dissection and image analysis of the RPE sheet, we can now analyze cell-to-cell interactions of immediate neighbors. In the future, we may be able to observe interactions of second, third, or higher ring neighbors and analyze tension in sheets, which might be expected to deviate from normal near large bumps in the RPE sheet caused by druse or when large frank holes in the RPE sheet are observed in geographic atrophy. This method and software can be readily applied to other aspects of vision science, neuroscience, and epithelial biology where patterns may exist in a sheet or surface of cells.

Silencing of murine transthyretin and retinol binding protein genes has distinct and shared behavioral and neuropathologic effects.

The murine genes encoding transthyretin (TTR) and retinol binding protein (RBP) were independently silenced by targeted disruption more than 10 years ago. Studies of both strains showed surprisingly little impact on either thyroid function or retinoid metabolism. Silencing TTR led to a relatively mild behavioral phenotype. In order to gain insight into the behavioral effect and determine if it was related to TTR's function as the carrier of RBP we carried out simultaneous studies with homozygous Rbp4(-/-) and Ttr(-/-) animals 4-7 months of age. Both strains showed behavioral differences relative to Ttr and Rbp4 wild-type animals and each other. The patterns were discrete for each knockout although there was some overlap. Neuropathologic examination of the cortex and hippocampus revealed cortical and hippocampal (CA3) neuronal loss in both and some degree of gliosis, more pronounced in the Rbp4(-/-) mice. There also appeared to be a major reduction in proliferating neuroblasts in the subventricular zone in both strains, which was also more severe in the Rbp4(-/-) mice. This is the first description of behavioral abnormalities in Rbp4(-/-)mice. The data also indicate that it is unlikely that the behaviors seen in Ttr(-/-) mice are related to its function as an RBP carrier.

Abrogation of Rbpj attenuates experimental autoimmune uveoretinitis by inhibiting IL-22-producing CD4+ T cells.

Experimental autoimmune uveoretinitis (EAU) is an organ-specific T cell-mediated disease induced by immunizing mice with interphotoreceptor retinoid binding protein (IRBP). Autoaggressive CD4(+) T cells are the major pathogenic population for EAU. We investigated the contribution of Notch signaling in T cells to EAU pathogenesis because Notch signaling regulates various aspects of CD4(+) T cell functions. Rbpj is required for Notch signaling, and Rbpj deficiency in T cells inhibited EAU disease severity. The amelioration of EAU in T cell-specific Rbpj-deficient mice correlated with low levels of IL-22 production from CD4(+) T cells, although IRBP-specific CD4(+) T cell proliferation and Th17 differentiation were unaffected. Administration of recombinant IL-22 during the late phase, but not the early phase, of EAU increased EAU clinical scores in T cell-specific Rbpj-deficient mice. Notch inhibition in mice immunized with IRBP with a γ-secretase inhibitor (GSI) suppressed EAU progression, even when GSI was administered as late as 13 days after IRBP immunization. Our data demonstrate that Rbpj/Notch-mediated IL-22 production in T cells has a key pathological role in the late phase of EAU, and suggest that Notch blockade might be a useful therapeutic approach for treating EAU.

Receptor interacting protein kinase-mediated necrosis contributes to cone and rod photoreceptor degeneration in the retina lacking interphotoreceptor retinoid-binding protein.

Interphotoreceptor retinoid-binding protein (IRBP) secreted by photoreceptors plays a pivotal role in photoreceptor survival with an unknown mechanism. A mutation in the human IRBP has been linked to retinitis pigmentosa, a progressive retinal degenerative disease. Mice lacking IRBP display severe early and progressive photoreceptor degeneration. However, the signaling pathway(s) leading to photoreceptor death in IRBP-deficient mice remains poorly understood. Here, we show that amounts of tumor necrosis factor-α (TNF-α) in the interphotoreceptor matrix and retinas of Irbp(-/-) mice were increased more than 10-fold and fivefold, respectively, compared with those in wild-type mice. Moreover, TNF-α receptor 1, an important membrane death receptor that mediates both programmed apoptosis and necrosis, was also significantly increased in Irbp(-/-) retina, and was colocalized with peanut agglutinin to the Irbp(-/-) cone outer segments. Although these death signaling proteins were increased, the caspase-dependent and independent apoptotic pathways were mildly activated in the Irbp(-/-) retinas, suggesting that other cell death mechanism(s) also contributes to the extensive photoreceptor degeneration in Irbp(-/-) retina. We found that receptor interacting protein 1 and 3 (RIP1 and RIP3) kinases, the intracellular key mediators of TNF-induced cellular necrosis, were elevated at least threefold in the Irbp(-/-) retinas. Moreover, pharmacological inhibition of RIP1 kinase significantly prevented cone and rod photoreceptor degeneration in Irbp(-/-) mice. These results reveal that RIP kinase-mediated necrosis strongly contributes to cone and rod degeneration in Irbp(-/-) mice, implicating the TNF-RIP pathway as a potential therapeutic target to prevent or delay photoreceptor degeneration in patients with retinitis pigmentosa caused by IRBP mutation.

PDA (prolonged depolarizing afterpotential)-defective mutants: the story of nina's and ina's--pinta and santa maria, too.

Our objective is to present a comprehensive view of the PDA (prolonged depolarizing afterpotential)-defective Drosophila mutants, nina's and ina's, from the discussion of the PDA and the PDA-based mutant screening strategy to summaries of the knowledge gained through the studies of mutants generated using the strategy. The PDA is a component of the light-evoked photoreceptor potential that is generated when a substantial fraction of rhodopsin is photoconverted to its active form, metarhodopsin. The PDA-based mutant screening strategy was adopted to enhance the efficiency and efficacy of ERG (electroretinogram)-based screening for identifying phototransduction-defective mutants. Using this strategy, two classes of PDA-defective mutants were identified and isolated, nina and ina, each comprising multiple complementation groups. The nina mutants are characterized by allele-dependent reduction in the major rhodopsin, Rh1, whereas the ina mutants display defects in some aspects of functions related to the transduction channel, TRP (transient receptor potential). The signaling proteins that have been identified and elucidated through the studies of nina mutants include the Drosophila opsin protein (NINAE), the chaperone protein for nascent opsin (NINAA), and the multifunctional protein, NINAC, required in multiple steps of the Drosophila phototransduction cascade. Also identified by the nina mutants are some of the key enzymes involved in the biogenesis of the rhodopsin chromophore. As for the ina mutants, they led to the discovery of the scaffold protein, INAD, responsible for the nucleation of the supramolecular signaling complex. Also identified by the ina mutants is one of the key members of the signaling complex, INAC (ePKC), and two other proteins that are likely to be important, though their roles in the signaling cascade have not yet been fully elucidated. In most of these cases, the protein identified is the first member of its class to be so recognized.

A single dose of c9,t11 or t10,c12 conjugated linoleic acid isomers perturbs vitamin A metabolism in mice.

Conjugated linoleic acid (CLA) is a polyunsaturated fatty acid that has numerous biologic activities. Previous studies in rodents demonstrated that chronic intake of CLA t10,c12 or CLA c9,t11 isomers perturbs the metabolism of retinoids (vitamin A and its derivatives). Specifically, although both isomers increased liver retinoid levels, only CLA t10,c12 also stimulated hepatic retinol secretion into the bloodstream. Given that retinoid homeostasis in mammalian serum and tissues is crucial to maintain health, it is important to gain more insights into the mode of action of this nutrient-nutrient interaction. Here we hypothesized that an acute administration of either CLA isomer may also influence vitamin A metabolism. By gavaging wild-type and retinol-binding protein knockout mice with an oral bolus of radiolabeled retinol containing 1 of these 2 isomers, we showed that both CLA t10,c12 and CLA c9,t11 rapidly enhance hepatic uptake of dietary vitamin A and its resecretion from the liver in the form of retinol bound to retinol-binding protein. Indeed, in mice lacking this protein, the sole specific carrier for retinol in the circulation, this latter effect was blunted. In addition, by using a pharmacologic inhibitor of the clearance of chylomicrons, which distribute recently ingested vitamin A and lipids throughout the body, we provided evidence that CLA intake might rapidly enhance intestinal absorption of dietary vitamin A. These data demonstrate the existence of multiple levels of interaction between dietary CLA and retinoid metabolism and warrant further studies to understand the molecular mechanisms underlying these effects and their implications for human health.

Retinoic acid influences neuronal migration from the ganglionic eminence to the cerebral cortex.

The ganglionic eminence contributes cells to several forebrain structures including the cerebral cortex, for which it provides GABAergic interneurons. Migration of neuronal precursors from the retinoic-acid rich embryonic ganglionic eminence to the cerebral cortex is known to be regulated by several factors, but retinoic acid has not been previously implicated. We found retinoic acid to potently inhibit cell migration in slice preparations of embryonic mouse forebrains, which was reversed by an antagonist of the dopamine-D(2) receptor, whose gene is transcriptionally regulated by retinoic acid. Histone-deacetylase inhibitors, which amplify nuclear receptor-mediated transcription, potentiated the inhibitory effect of retinoic acid. Surprisingly, when retinoic acid signalling was completely blocked with a pan-retinoic acid receptor antagonist, this also decreased cell migration into the cortex, implying that a minimal level of endogenous retinoic acid is necessary for tangential migration. Given these opposing effects of retinoic acid in vitro, the in vivo contribution of retinoic acid to migration was tested by counting GABAergic interneurons in cortices of adult mice with experimental reductions in retinoic acid signalling: a range of perturbations resulted in significant reductions in the numerical density of some GABAergic interneuron subpopulations. These observations suggest functions of retinoic acid in interneuron diversity and organization of cortical excitatory-inhibitory balance.

Exaggerated eye growth in IRBP-deficient mice in early development.

PURPOSE: Because interphotoreceptor retinoid-binding protein (IRBP) is expressed before being needed in its presumptive role in the visual cycle, we tested whether it controls eye growth during development. METHODS: The eyes of congenic IRBP knockout (KO) and C57BL/6J wild-type (WT) mice ranging in age from postnatal day (P)2 to P440 were compared by histology, laser micrometry, cycloplegic photorefractions, and partial coherence interferometry. RESULTS: The size and weight of IRBP KO mouse eyes were greater than those of the WT mouse, even before eye-opening. Excessive ocular enlargement started between P7 and P10, with KO retinal arc lengths becoming greater compared with WT from P10 through P30 (18%; P < 0.01). The outer nuclear layer (ONL) of KO retinas became 20% thinner between P12 to P25, and progressed to 38% thinner at P30. At P30, there were 30% fewer cones per vertical section in KO than in WT retinas. Bromodeoxyuridine (BrdU) labeling indicated the same number of retinal cells were born in KO and WT mice. A spike in apoptosis was observed in KO outer nuclear layer at P25. These changes in size were accompanied by a large decrease in hyperopic refractive error, which reached -4.56 ± 0.70 diopters (D) versus +9.98 ± 0.993 D (mean ± SD) in WT, by postnatal day 60 (P60). CONCLUSIONS; In addition to its role in the visual cycle, IRBP is needed for normal eye development. How IRBP mediates ocular development is unknown.

The interphotoreceptor retinoid binding (IRBP) is essential for normal retinoid processing in cone photoreceptors.

11-cis Retinal is the light-sensitive component in rod and cone photoreceptors, and its isomerization to all-trans retinal in the presence of light initiates the visual response. For photoreceptors to function normally, all-trans retinal must be converted back into 11-cis retinal through the visual cycle. While rods are primarily responsible for dim light vision, the ability of cones to function in constant light is essential to human vision and may be facilitated by cone-specific visual cycle pathways. The interphotoreceptor retinoid-binding protein (IRBP) is a proposed retinoid transporter in the visual cycle, but rods in Irbp ( -/- ) mice have a normal visual cycle. However, there is evidence that IRBP has cone-specific functions. Cone electroretinogram (ERG) responses are reduced, despite having cone densities and opsin levels similar to C57Bl/6 (WT) mice. Treatment with 9-cis retinal rescues the cone response in Irbp ( -/- ) mice and shows that retinoid deficiency underlies cone dysfunction. These data indicate that IRBP is essential to normal cone function and demonstrate that differences exist in the visual cycle of rods and cones.

A hypoplastic retinal lamination in the purpurin knock down embryo in zebrafish.

Recently, we cloned a photoreceptor-specific purpurin cDNA from axotomized goldfish retina. In the present study, we investigate the structure of zebrafish purpurin genomic DNA and its function during retinal development. First, we cloned a 3.7-kbp genomic DNA fragment including 1.4-kbp 5'-flanking region and 2.3-kbp full-length coding region. In the 1.4-kbp 5'-upstream region, there were some cone-rod homeobox (crx) protein binding motifs. The vector of the 1.4-kbp 5'-flanking region combined with the reporter GFP gene showed specific expression of this gene only in the photoreceptors. Although the first appearance time of purpurin mRNA expression was a little bit later (40 hpf) than that of crx (17-24 hpf), the appearance site was identical to the ventral part of the retina. Next, we made purpurin or crx knock down embryos with morpholino antisense oligonucleotides. The both morphants (purpurin and crx) showed similar abnormal phenotypes in the eye development; small size of eyeball and lacking of retinal lamination. Furthermore, co-injection of crx morpholino and purpurin mRNA significantly rescued these abnormalities. These data strongly indicate that purpurin is a key molecule for the cell differentiation during early retinal development in zebrafish under transcriptional crx regulation.

Normal cone function requires the interphotoreceptor retinoid binding protein.

11-cis-retinal is the light-sensitive component in rod and cone photoreceptors, and its isomerization to all-trans retinal in the presence of light initiates the visual response. For photoreceptors to function normally, all-trans retinal must be converted back into 11-cis-retinal through a series of enzymatic steps known as the visual cycle. The interphotoreceptor retinoid-binding protein (IRBP) is a proposed retinoid transporter in the visual cycle, but rods in Irbp(-/-) mice have a normal visual cycle. While rods are primarily responsible for dim light vision, the ability of cones to function in constant light is essential to human vision and may be facilitated by cone-specific visual cycle pathways. We analyzed the cones in Irbp(-/-) mice to determine whether IRBP has a cone-specific visual cycle function. Cone electroretinogram (ERG) responses were reduced in Irbp(-/-) mice, but similar responses from Irbp(-/-) mice at all ages suggest that degeneration does not underlie cone dysfunction. Furthermore, cone densities and opsin levels in Irbp(-/-) mice were similar to C57BL/6 (wild-type) mice, and both cone opsins were properly localized to the cone outer segments. To test for retinoid deficiency in Irbp(-/-) mice, ERGs were analyzed before and after intraperitoneal injections of 9-cis-retinal. Treatment with 9-cis-retinal produced a significant recovery of the cone response in Irbp(-/-) mice and shows that retinoid deficiency underlies cone dysfunction. These data indicate that IRBP is essential to normal cone function and demonstrate that differences exist in the visual cycle of rods and cones.

Retinoids and spermatogenesis: lessons from mutant mice lacking the plasma retinol binding protein.

Using Rbp4-null mice as models, we have established for the first time the kinetics of the spermatogenetic alterations during vitamin A deficiency (VAD). Our data demonstrate that the VAD-induced testicular degeneration arises through the normal maturation of germ cells in a context of spermatogonia differentiation arrest. They indicate that retinoic acid (RA) appears dispensable for the transition of premeiotic to meiotic spermatocytes, meiosis, and spermiogenesis. They confirm that RA plays critical roles in controlling spermatogonia differentiation, spermatid adhesion to Sertoli cells, and spermiation, and suggest that the VAD-induced arrest of spermatogonia differentiation results from simultaneous blocks in RA-dependent events mediated by RA receptor gamma (RARgamma) in spermatogonia and by RARalpha in Sertoli cells. They also provide evidence that expression of major RA-metabolizing enzymes is increased in mouse Sertoli cells upon VAD and that vitamin A-deficient A spermatogonia differ from their RA-sufficient counterparts by the expression of the Stra8 gene.

Endogenous IRBP can be dispensable for generation of natural CD4+CD25+ regulatory T cells that protect from IRBP-induced retinal autoimmunity.

Susceptibility to experimental autoimmune uveitis (EAU), a model for human uveitis induced in mice with the retinal antigen interphotoreceptor retinoid-binding protein (IRBP), is controlled by "natural" CD4+CD25+ regulatory T (T reg) cells. To examine whether endogenous expression of IRBP is necessary to generate these T reg cells, we studied responses of IRBP knockout (KO) versus wild-type (WT) mice. Unexpectedly, not only WT but also IRBP KO mice immunized with a uveitogenic regimen of IRBP in complete Freund's adjuvant (CFA) exhibited CD25+ regulatory cells that could be depleted by PC61 treatment, which suppressed development of uveitogenic effector T cells and decreased immunological responses to IRBP. These EAU-relevant T reg cells were not IRBP specific, as their activity was not present in IRBP KO mice immunized with IRBP in incomplete Freund's adjuvant (IFA), lacking mycobacteria (whereas the same mice exhibited normal T reg cell activity to retinal arrestin in IFA). We propose that mycobacterial components in CFA activate T reg cells of other specificities to inhibit generation of IRBP-specific effector T cells in a bystander fashion, indicating that effective T reg cells can be antigen nonspecific. Our data also provide the first evidence that generation of specific T reg cells to a native autoantigen in a mouse with a diverse T cell repertoire requires a cognate interaction.

Retinoid status and responsiveness to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in mice lacking retinoid binding protein or retinoid receptor forms.

We have investigated the role of Vitamin A (retinoid) proteins in hepatic retinoid processing under normal conditions and during chemical stress induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a chemical known to interfere with retinoid turnover and metabolism. Three separate studies were performed in wildtype control mice and transgenic mice that lack one or more isoforms of retinoic acid receptors (RAR), retinoid X receptors (RXR), or intracellular retinoid-binding proteins (CRABP I, CRABP II, CRBP I). Body and organ weight development was monitored from 2 weeks of age to adult, and hepatic levels of retinyl esters, retinol, and retinoic acid were investigated. In addition, hepatic concentrations of 9-cis-4-oxo-13,14-dihydro-retinoic acid, a recently discovered retinoid metabolite that has proven sensitive to both TCDD exposure and Vitamin A status, were also determined. Mice absent in the three proteins CRBP I, CRABP I, and CRABP II (CI/CAI/CAII-/-) displayed significantly lower hepatic retinyl ester, retinol, and all-trans-retinoic acid levels compared to wildtype mice, whereas the liver concentrations of 9-cis-4-oxo-13,14-dihydro-retinoic acid was considerably higher. After treatment with TCDD, hepatic total retinoids were almost entirely depleted in the CI/CAI/CAII-/- mice, whereas wildtype mice and mice lacking CRABP I, and CRABP II (CAI/CAII-/-) retained approximately 60-70% of their Vitamin A content compared to controls at 28 days. RAR and RXR knockout mice responded similarly to wildtype mice with respect to TCDD-induced retinoid disruption, with the exception of RXRbeta-/- mice which showed no decrease in hepatic Vitamin A concentration, suggesting that the role of RXRbeta in TCDD-induced retinoid disruption should be further investigated. Overall, the abnormal retinoid profile in the triple knockout mice (CI/CAI/CAII-/-), but not double knockout (CAI/CAII-/-) mice, suggests that a loss of CRBP I may account for the difference in retinoid profile in CI/CAI/CAII-/- mice, and is likely to result in an increased susceptibility to hepatic retinoid depletion following dioxin exposure.