A chimeric protein-based vaccine elicits a strong IgG antibody response and confers partial protection against Shiga toxin-producing Escherichia coli in mice.

Background: Shiga toxin-producing Escherichia coli (STEC) is a foodborne pathogen that causes gastrointestinal infections, ranging from acute diarrhea and dysentery to life-threatening diseases such as Hemolytic Uremic Syndrome. Currently, a vaccine to prevent STEC infection is an unmet medical need. Results: We developed a chimeric protein-based vaccine targeting seven virulence factors of STEC, including the Stx2B subunit, Tir, Intimin, EspA, Cah, OmpT, and AggA proteins. Immunization of mice with this vaccine candidate elicited significant humoral and cellular immune responses against STEC. High levels of specific IgG antibodies were found in the serum and feces of immunized mice. However, specific IgA antibodies were not detected in either serum or feces. Furthermore, a significantly higher percentage of antigen-specific CD4+ T cells producing IFN-γ, IL-4, and IL-17 was observed in the spleens of immunized mice. Notably, the immunized mice showed decreased shedding of STEC O157:H7 and STEC O91:H21 strains and were protected against weight loss during experimental infection. Additionally, infection with the STEC O91:H21 strain resulted in kidney damage in control unimmunized mice; however, the extent of damage was slightly lower in immunized mice. Our findings suggest that IgG antibodies induced by this vaccine candidate may have a role in inhibiting bacterial adhesion and complement-mediated killing. Conclusion: This study provides evidence that IgG responses are involved in the host defense against STEC. However, our results do not rule out that other classes of antibodies also participate in the protection against this pathogen. Additional work is needed to improve the protection conferred by our vaccine candidate and to elucidate the relevant immune responses that lead to complete protection against this pathogen.

Basal Sodium-Dependent Vitamin C Transporter 2 polarization in choroid plexus explant cells in normal or scorbutic conditions.

Vitamin C is incorporated into the cerebrospinal fluid (CSF) through choroid plexus cells. While the transfer of vitamin C from the blood to the brain has been studied functionally, the vitamin C transporter, SVCT2, has not been detected in the basolateral membrane of choroid plexus cells. Furthermore, it is unknown how its expression is induced in the developing brain and modulated in scurvy conditions. We concluded that SVCT2 is intensely expressed in the second half of embryonic brain development and postnatal stages. In postnatal and adult brain, SVCT2 is highly expressed in all choroidal plexus epithelial cells, shown by colocalization with GLUT1 in the basolateral membranes and without MCT1 colocalization, which is expressed in the apical membrane. We confirmed that choroid plexus explant cells (in vitro) form a sealed epithelial structure, which polarized basolaterally, endogenous or overexpressed SVCT2. These results are reproduced in vivo by injecting hSVCT2wt-EYFP lentivirus into the CSF. Overexpressed SVCT2 incorporates AA (intraperitoneally injected) from the blood to the CSF. Finally, we observed in Guinea pig brain under scorbutic condition, that normal distribution of SVCT2 in choroid plexus may be regulated by peripheral concentrations of vitamin C. Additionally, we observed that SVCT2 polarization also depends on the metabolic stage of the choroid plexus cells.

Functional Analyses of Embryonic Cerebrospinal Fluid Proteins.

The embryonic cerebrospinal fluid (eCSF) influences neuroepithelial cell behavior, affecting proliferation, differentiation, and survival. One major question to resolve in the field is to precisely describe the eCSF molecules responsible and to understand how these molecules interact in order to exert their functions. Here we describe an in vitro protocol to analyze the influence of eCSF components on neuroepithelium development.

Anatomical and histological characteristics of the nervous system of the Chilean giant mussel, Choromytilus chorus (Molina 1782) (Bivalvia, Mytilidae) / Características anatómicas e histológicas del sistema nervioso del mejillón gigante, Choromytilus chorus (Molina 1782) (Bivalvia, Mytilidae)

The anatomy and histology of the nervous system in the mussel Choromytilus chorus were studied. Juvenile specimens of C. chorus and adult broodstock were collected in Laraquete Cove, Chile (37°09'S; 37°11'O). The juveniles were used for histological analysis and the adults for a macroscopic description of anatomical. The histological description was carried out by Gallego´s trichrome technique. The macroscopic analysis showed that nervous system network includes three pairs of ganglia of orange color and little size (20-40 mm) (cerebral, pedal and visceral) located in the anterior, middle and posterior zone of the specimen, respectively. The histological analysis showed many type de cells inside the ganglia (neurosecretory, granulated and glial cells). The ganglia network could be involving in regulating several physiological processes in the mussels through of their neurosecretions.

Se estudió la anatomía e histología del sistema nervioso en el coro Choromytilus del mejillón. Se recolectaron especímenes juveniles de C. coros y reproductores adultos en Laraquete Cove, Chile (37 ° 09'S, 37 ° 11'O). Los especímenes juveniles se utilizaron para el análisis histológico y los adultos para una descripción macroscópica de anatómica. La descripción histológica se realizó mediante la técnica de tricrómico de Gallego. El análisis macroscópico mostró que la red del sistema nervioso incluye tres pares de ganglios de color naranjo y poco tamaño (20-40 mm) (cerebral, pedal y visceral) localizados en la zona anterior, media y posterior de la muestra, respectivamente. El análisis histológico mostró muchos tipos de células dentro de los ganglios (células neurosecretoras, granuladas y gliales). La red de ganglios podría estar involucrada en la regulación de varios procesos fisiológicos en los mejillones a través de sus neurosecreciones.

Expression Patterns of Extracellular Matrix Proteins during Posterior Commissure Development.

Extracellular matrix (ECM) molecules are pivotal for central nervous system (CNS) development, facilitating cell migration, axonal growth, myelination, dendritic spine formation, and synaptic plasticity, among other processes. During axon guidance, the ECM not only acts as a permissive or non-permissive substrate for navigating axons, but also modulates the effects of classical guidance cues, such as netrin or Eph/ephrin family members. Despite being highly important, little is known about the expression of ECM molecules during CNS development. Therefore, this study assessed the molecular expression patterns of tenascin, HNK-1, laminin, fibronectin, perlecan, decorin, and osteopontin along chick embryo prosomere 1 during posterior commissure development. The posterior commissure is the first transversal axonal tract of the embryonic vertebrate brain. Located in the dorso-caudal portion of prosomere 1, posterior commissure axons primarily arise from the neurons of basal pretectal nuclei that run dorsally to the roof plate midline, where some turn toward the ipsilateral side. Expressional analysis of ECM molecules in this area these revealed to be highly arranged, and molecule interactions with axon fascicles suggested involvement in processes other than structural support. In particular, tenascin and the HNK-1 epitope extended in ventro-dorsal columns and enclosed axons during navigation to the roof plate. Laminin and osteopontin were expressed in the midline, very close to axons that at this point must decide between extending to the contralateral side or turning to the ipsilateral side. Finally, fibronectin, decorin, and perlecan appeared unrelated to axonal pathfinding in this region and were instead restricted to the external limiting membrane. In summary, the present report provides evidence for an intricate expression of different extracellular molecules that may cooperate in guiding posterior commissure axons.

Interaction between SCO-spondin and low density lipoproteins from embryonic cerebrospinal fluid modulates their roles in early neurogenesis.

During early stages of development, encephalic vesicles are composed by a layer of neuroepithelial cells surrounding a central cavity filled with embryonic cerebrospinal fluid (eCSF). This fluid contains several morphogens that regulate proliferation and differentiation of neuroepithelial cells. One of these neurogenic factors is SCO-spondin, a giant protein secreted to the eCSF from early stages of development. Inhibition of this protein in vivo or in vitro drastically decreases the neurodifferentiation process. Other important neurogenic factors of the eCSF are low density lipoproteins (LDL), the depletion of which generates a 60% decrease in mesencephalic explant neurodifferentiation. The presence of several LDL receptor class A (LDLrA) domains (responsible for LDL binding in other proteins) in the SCO-spondin sequence suggests a possible interaction between both molecules. This possibility was analyzed using three different experimental approaches: (1) Bioinformatics analyses of the SCO-spondin region, that contains eight LDLrA domains in tandem, and of comparisons with the LDL receptor consensus sequence; (2) Analysis of the physical interactions of both molecules through immunohistochemical colocalization in embryonic chick brains and through the immunoprecipitation of LDL with anti-SCO-spondin antibodies; and (3) Analysis of functional interactions during the neurodifferentiation process when these molecules were added to a culture medium of mesencephalic explants. The results revealed that LDL and SCO-spondin interact to form a complex that diminishes the neurogenic capacities that both molecules have separately. Our work suggests that the eCSF is an active signaling center with a complex regulation system that allows for correct brain development.

Complementary expression of EphA7 and SCO-spondin during posterior commissure development.

Bilaterally symmetric organisms need to exchange information between the two sides of their bodies in order to integrate sensory inputs and coordinate motor control. This exchange occurs through commissures formed by neurons that project axons across the midline to the contralateral side of the central nervous system. The posterior commissure is the first transversal axonal tract of the embryonic vertebrate brain. It is located in the dorsal portion of the prosomere 1, at the caudal diencephalon. The axons of the posterior commissure principally come from neurons of ventrolateral and dorsolateral pretectal nuclei (parvocellular and magnocellular nucleus of the posterior commissure, respectively) that extend their axons toward the dorsal region. The trajectory of these axons can be divided into the following three stages: (1) dorsal axon extension towards the lateral roof plate; (2) fasciculation in the lateral roof plate; and (3) midline decision of turning to the ipsilateral side or continuing to the opposite side. The mechanisms and molecules that guide the axons during these steps are unknown. In the present work, immunohistochemical and in situ hybridization analyses were performed, with results suggesting the participation of EphA7 in guiding axons from the ventral to the dorsal region of the prosomere 1 through the generation of an axonal corridor limited by repulsive EphA7 walls. At the lateral roof plate, the axons became fasciculated in presence of SCO-spondin until reaching the midline. Finally, EphA7 expression was observed in the diencephalic midline roof plate, specifically in the region where some axons turn to the ipsilateral side, suggesting its participation in this decision. In summary, the present work proposes a mechanism of posterior commissure formation orchestrated by the complementary expression of the axon guidance cues SCO-spondin and EphA7.

Role of porosity and pore architecture in the in vivo bone regeneration capacity of biodegradable glass scaffolds.

The aim of this work is to shed light on the role of porosity and pore architecture in the in vivo bone regeneration capacity of biodegradable glass scaffolds. A calcium phosphate glass in the system P2O5-CaO-Na2O-TiO2 was foamed using two different porogens, namely albumen and hydrogen peroxide (H2O2); the resulting three-dimensional porous structures were characterized and implanted in New Zealand rabbits to study their in vivo behavior. Scaffolds foamed with albumen displayed a monomodal pore size distribution centered around 150 µm and a porosity of 82%, whereas scaffolds foamed with H2O2 showed lower porosity (37%), with larger elongated pores, and multimodal size distribution. After 12 weeks of implantation, histology results revealed a good osteointegration for both types of scaffolds. The quantitative morphometric analysis showed the substitution of the biomaterial by new bone in the case of glasses foamed with albumen. In contrast, bone neoformation and material resorption were significantly lower in the defects filled with the scaffolds foamed with H2O2. The results obtained in this study showed that both calcium phosphate glass scaffolds were osteoconductive, biocompatible, and biodegradable materials. However, differences in porosity, pore architecture, and microstructure led to substantially different in vivo response.

Subdivisions of chick diencephalic roof plate: implication in the formation of the posterior commissure.

The subcommissural organ (SCO) is a roof plate differentiation located in the caudal diencephalon under the posterior commissure (PC). A role for SCO and its secretory product, SCO-spondin, in the formation of the PC has been proposed. Here, we provide immunohistochemical evidence to suggest that SCO is anatomically divided in a bilateral region positive for SCO-spondin that surrounds a negative medial region. Remarkably, axons contacting the lateral region are highly fasciculated, in sharp contrast with the defasciculated axons of the medial region. In addition, lateral axon fascicles run toward the midline inside of tunnels limited by the basal prolongations of SCO cells and extracellular SCO-spondin. Our in vitro data in collagen gel matrices show that SCO-spondin induces axonal growth and fasciculation of pretectal explants. Together, our findings support the idea that SCO-spondin participates in the guidance and fasciculation of axons of the PC.

Polarized expression of integrin beta1 in diencephalic roof plate during chick development, a possible receptor for SCO-spondin.

The roof plate of the caudal diencephalon is formed by the posterior commissure (PC) and the underlying secretory ependyma, the subcommissural organ (SCO). The SCO is composed by radial glial cells bearing processes that cross the PC and attach to the meningeal basement membrane. Since early development, the SCO synthesizes SCO-spondin, a glycoprotein that shares similarities to axonal guidance proteins. In vitro, SCO-spondin promotes neuritic outgrowth through a mechanism mediated by integrin beta1. However, the secretion of SCO-spondin toward the extracellular matrix that surrounds the PC axons and the expression of integrins throughout PC development have not been addressed. Here we provide immunohistochemical evidence to suggest that during chick development SCO cells secrete SCO-spondin through their basal domain, where it is deposited into the extracellular matrix in close contact with axons of the PC that express integrin beta1. Our results suggest that SCO-spondin has a role in the development of the PC through its interaction with integrin beta1.

The Na+-dependent L-ascorbic acid transporter SVCT2 expressed in brainstem cells, neurons, and neuroblastoma cells is inhibited by flavonoids.

Ascorbic acid (AA) is best known for its role as an essential nutrient in humans and other species. As the brain does not synthesize AA, high levels are achieved in this organ by specific uptake mechanisms, which concentrate AA from the bloodstream to the CSF and from the CSF to the intracellular compartment. Two different isoforms of sodium-vitamin C co-transporters (SVCT1 and SVCT2) have been cloned. Both SVCT proteins mediate high affinity Na(+)-dependent L-AA transport and are necessary for the uptake of vitamin C in many tissues. In the adult brain the expression of SVCT2 was observed in the hippocampus and cortical neurons by in situ hybridization; however, there is no data regarding the expression and distribution of this transporter in the fetal brain. The expression of SVCT2 in embryonal mesencephalic neurons has been shown by RT-PCR suggesting an important role for vitamin C in dopaminergic neuronal differentiation. We analyze SVCT2 expression in human and rat developing brain by RT-PCR. Additionally, we study the normal localization of SVCT2 in rat fetal brain by immunohistochemistry and in situ hybridization demonstrating that SVCT2 is highly expressed in the ventricular and subventricular area of the rat brain. SVCT2 expression and function was also confirmed in neurons isolated from brain cortex and cerebellum. The kinetic parameters associated with the transport of AA in cultured neurons and neuroblastoma cell lines were also studied. We demonstrate two different affinity transport components for AA in these cells. Finally, we show the ability of different flavonoids to inhibit AA uptake in normal or immortalized neurons. Our data demonstrates that brain cortex and cerebellar stem cells, neurons and neuroblastoma cells express SVCT2. Dose-dependent inhibition analysis showed that quercetin inhibited AA transport in cortical neurons and Neuro2a cells.

Differential distribution of the Sodium-vitamin C cotransporter-1 along the proximal tubule of the mouse and human kidney.

Vitamin C is reabsorbed from the renal lumen by one isoform of sodium-vitamin C co-transporters that mediate high affinity sodium-dependent L-ascorbic acid transport. Sodium-vitamin C cotransporter-1 mRNA has been detected in intestine and liver and the S3 segment of the renal proximal tubule. Here, we found that its distribution was broader and all three proximal tubule segments of mouse and human expressed the transporter but the S3 segment had the highest expression. Sodium-vitamin C co-transporter-1 expression was also found in the renal epithelial-derived LLC-PK1 cell line. Ascorbic acid transport in these cells was regulated by a single kinetic component that depended on the sodium concentration, pH and temperature. Reducing ascorbate concentration increased the apical expression of the transporter suggesting the presence of a feedback system for regulation of transporter abundance at the luminal membrane.

Plasma membrane destination of the classical Xenopus laevis progesterone receptor accelerates progesterone-induced oocyte maturation.

Xenopus laevis oocyte maturation is induced by the steroid hormone progesterone through a non-genomic mechanism initiated at the cell membrane. Recently, two Xenopus oocyte progesterone receptors have been cloned; one is the classical progesterone receptor (xPR-1) involved in genomic actions and the other a putative seven-transmembrane-G-protein-couple receptor. Both receptors are postulated to be mediating the steroid-induced maturation process in the frog oocyte. In this study, we tested the hypothesis that the classical progesterone receptor, associated to the oocyte plasma membrane, is participating in the reinitiation of the cell cycle. Addition of a myristoilation and palmytoilation signal at the amino terminus of xPR-1 (mp xPR-1), increased the amount of receptor associated to the oocyte plasma membrane and most importantly, significantly potentiated progesterone-induced oocyte maturation sensitivity. These findings suggest that the classical xPR-1, located at the plasma membrane, is mediating through a non-genomic mechanism, the reinitiation of the meiotic cell cycle in the X. laevis oocyte.

Sodium vitamin C cotransporter SVCT2 is expressed in hypothalamic glial cells.

Kinetic analysis of vitamin C uptake demonstrated that different specialized cells take up ascorbic acid through sodium-vitamin C cotransporters. Recently, two different isoforms of sodium-vitamin C cotransporters (SVCT1/SLC23A1 and SVCT2/SLC23A2) have been cloned. SVCT2 was detected mainly in choroidal plexus cells and neurons; however, there is no evidence of SVCT2 expression in glial and endothelial cells of the brain. Certain brain locations, including the hippocampus and hypothalamus, consistently show higher ascorbic acid values compared with other structures within the central nervous system. However, molecular and kinetic analysis addressing the expression of SVCT transporters in cells isolated from these specific areas of the brain had not been done. The hypothalamic glial cells, or tanycytes, are specialized ependymal cells that bridge the cerebrospinal fluid with different neurons of the region. Our hypothesis postulates that SVCT2 is expressed selectively in tanycytes, where it is involved in the uptake of the reduced form of vitamin C (ascorbic acid), thereby concentrating this vitamin in the hypothalamic area. In situ hybridization and optic and ultrastructural immunocytochemistry showed that the transporter SVCT2 is highly expressed in the apical membranes of mouse hypothalamic tanycytes. A newly developed primary culture of mouse hypothalamic tanycytes was used to confirm the expression and function of the SVCT2 isoform in these cells. The results demonstrate that tanycytes express a high-affinity transporter for vitamin C. Thus, the vitamin C uptake mechanisms present in the hypothalamic glial cells may perform a neuroprotective role concentrating vitamin C in this specific area of the brain.

Hypothalamic ependymal-glial cells express the glucose transporter GLUT2, a protein involved in glucose sensing.

The GLUT2 glucose transporter and the K-ATP-sensitive potassium channels have been implicated as an integral part of the glucose-sensing mechanism in the pancreatic islet beta cells. The expression of GLUT2 and K-ATP channels in the hypothalamic region suggest that they are also involved in a sensing mechanism in this area. The hypothalamic glial cells, known as tanycytes alpha and beta, are specialized ependymal cells that bridge the cerebrospinal fluid and the portal blood of the median eminence. We used immunocytochemistry, in situ hybridization and transport analyses to demonstrate the glucose transporters expressed in tanycytes. Confocal microscopy using specific antibodies against GLUT1 and GLUT2 indicated that both transporters are expressed in alpha and beta tanycytes. In addition, primary cultures of mouse hypothalamic tanycytes were found to express both GLUT1 and GLUT2 transporters. Transport studies, including 2-deoxy-glucose and fructose uptake in the presence or absence of inhibitors, indicated that these transporters are functional in cultured tanycytes. Finally, our analyses indicated that tanycytes express the K-ATP channel subunit Kir6.1 in vitro. As the expression of GLUT2 and K-ATP channel is linked to glucose-sensing mechanisms in pancreatic beta cells, we postulate that tanycytes may be responsible, at least in part, for a mechanism that allows the hypothalamus to detect changes in glucose concentrations.

Reissner fiber binds and transports away monoamines present in the cerebrospinal fluid.

The subcommissural organ (SCO) is a brain gland that secretes glycoproteins into the cerebrospinal fluid (CSF), where they subsequently aggregate to form Reissner fiber (RF). By addition of newly released glycoproteins to its cephalic end, RF grows constantly through the Sylvian aqueduct, fourth ventricle and central canal of the spinal cord. Disaggregation of RF-material and passage to blood occur when RF reaches the terminal ventricle at the filum. The present investigation was designed to test the hypothesis that RF binds, transports and clears away monoamines present in the CSF. Four experimental protocols were applied: (i) in vivo binding of labeled monoamines to the rat RF, studied by pulse and chase, and after perfusion for 7 days; (ii) identification of monoamines, by high-performance liquid chromatography (HPLC), naturally occurring in the bovine RF; (iii) in vitro binding of labeled and unlabeled monoamines to the isolated bovine RF; and (iv) tentative identification of the amine binding site(s) in RF-proteins by use of specific antibodies. The results obtained indicate that RF participates in the regulation of the CSF concentration of monoamines either by binding and transporting them away throughout the central canal of the spinal cord (L-DOPA, noradrenaline, adrenaline), or by transiently binding them and releasing them back to the CSF (serotonin). Furthermore, evidence was obtained that (i) adrenaline and noradrenaline share the same binding site, and that this site would correspond to a repeated sequence present in the SCO-spondin, the major protein component of RF; and (ii) serotonin has its own binding site in RF.