Intestinal immunity of dogfish Scyliorhinus canicula spiral valve: A histochemical, immunohistochemical and confocal study.

The present study describes histochemical and immunohistochemical characteristics of the spiral valve and its associated lymphoid tissue (GALT) in the dogfish Scyliorhinus canicula. The mucosal surface of the spiral valve represents the first line of defense against pathogens coming from the external environment through food. Epithelial, mucus and immune cells play a key role in controlling the inflammatory response. Valve intestine of S. canicula had many folds lined by simple columnar cells and goblet cells, which later reacted positive to PAS, AB and AB-PAS, histochemical stains differentiated the different types of mucins; lectin histochemistry (PNA and WGA), detected neutral and acid mucins secreted that plays an important role in protection against invading pathogens. Integrin α5ß1 was expressed in enterocytes that line the valve's folds with greater marking in the apical part of the cells. Laminin was found on the apical side of the epithelium, in fibrillar and cellular elements of the lamina propria and in the muscularis mucosa. In the spiral valve gut-associated lymphoid tissue (GALT) has been studied. For the first time, massive leucocytes aggregates were identified by confocal immunofluorescence techniques, using the following antibodies: TLR2, S100, Langerin/CD207. Our results expand knowledge about Dogfish valve intestine giving important news in understanding comparative immunology.

Tolerability to dogfish in children with fish allergy.

BACKGROUND: Fish is a potent food allergen. The aim of this work is to demonstrate that dogfish, a small shark, has low allergenicity in both its clinical tolerance as well as its molecular structure. METHODS: We present a study of 34 paediatric patients with IgE-mediated immediate reactions after eating fish. The diagnosis of several fish allergies was demonstrated by skin prick techniques and determination of specific IgE, in all the cases excluding sensitisation to Anisakis simplex. Open oral challenge test was checked with dogfish. Analysis was by SDS-PAGE of dogfish and other fish (megrim, shark, hake, sole, cod, anchovy and tuna) and Western-blot with "pool" of patients polysensitised sera against extracts of dogfish and other fish, and ELISA - inhibition with the "pool" sera. RESULTS: The prick-prick with raw dogfish was slightly positive in six patients, however cooked was negative in 34 cases. The specific IgE showed in the 34 cases class ≥2 for megrim, hake, sole, cod and anchovy, class 0 for tuna in 26 patients, class 0 for emperor in 18 patients and class 0 to Anisakis simplex in all cases. The IgE binding capacity for proteins of allergenic extracts of tested fish revealed, in immunoblotting, the absence of IgE-mediated recognition abstract dogfish by the "pool" of polysensitised patient sera. CONCLUSIONS: Testing in vivo and in vitro demonstrated the low allergenicity of dogfish. Dogfish brings an alternative to eating fish in polysensitised patients.

Selection of cholera toxin specific IgNAR single-domain antibodies from a naïve shark library.

Shark immunoglobulin new antigen receptor (IgNAR, also referred to as NAR) variable domains (Vs) are single-domain antibody (sdAb) fragments containing only two hypervariable loop structures forming 3D topologies for a wide range of antigen recognition and binding. Their small size ( approximately 12kDa) and high solubility, thermostability and binding specificity make IgNARs an exceptional alternative source of engineered antibodies for sensor applications. Here, two new shark NAR V display libraries containing >10(7) unique clones from non-immunized (naïve) adult spiny dogfish (Squalus acanthias) and smooth dogfish (Mustelus canis) sharks were constructed. The most conserved consensus sequences derived from random clone sequence were compared with published nurse shark (Ginglymostoma cirratum) sequences. Cholera toxin (CT) was chosen for panning one of the naïve display libraries due to its severe pathogenicity and commercial availability. Three very similar CT binders were selected and purified soluble monomeric anti-CT sdAbs were characterized using Luminex(100) and traditional ELISA assays. These novel anti-CT sdAbs selected from our newly constructed shark NAR V sdAb library specifically bound to soluble antigen, without cross reacting with other irrelevant antigens. They also showed superior heat stability, exhibiting slow loss of activity over the course of one hour at high temperature (95 degrees C), while conventional antibodies lost all activity in the first 5-10min. The successful isolation of target specific sdAbs from one of our non-biased NAR libraries, demonstrate their ability to provide binders against an unacquainted antigen of interest.

Molecular cloning and preliminary expression analysis of banded dogfish (Triakis scyllia) CC chemokine cDNAs by use of suppression subtractive hybridization.

Suppression subtractive hybridization was carried out by using cDNAs of peripheral white blood cells (PWBCs) of banded dogfish (Triakis scyllia) after phorbol 12-myristate 13-acetate (PMA) stimulation. The Trsc-SCYA107, MIP3alpha1 and MIP3alpha2 clones contained an open reading frame encoding 97, 99 and 97 amino acids, respectively. Comparison of the deduced amino acids showed that the banded dogfish MIP3alpha1 and MIP3alpha2 sequences shared 42.3% and 40.0% identity with human SCYA20, respectively, while the Trsc-SCYA107 sequence shared 50.6, 44.2 and 42.0% identity with the catshark (Scyliorhinus canicula) Scca-SCYA107, rainbow trout (Oncorhynchus mykiss) CK4A and CK4B, respectively. The genomic sequences of banded dogfish Trsc-SCYA107, MIP3alpha1 and MIP3alpha2 contain four exons and three introns, and MIP3alpha1 and MIP3alpha2 shared the same intron/exon organization with that of human. The MIP3alpha1 and MIP3alpha2 genes of lipopolysaccharide (LPS)-unstimulated banded dogfish were expressed in gill, kidney and liver, while Trsc-SCYA107 mRNA was detected in various tissues except for brain. However, the constitutive expression of MIP3alpha2 gene was much lower than the Trsc-SCYA107 and MIP3alpha1 genes. RT-PCR analysis of the Trsc-SCYA107 expression in tissues of LPS-stimulated fish showed enhanced expression at 24 h poststimulation in the gill, heart, leydig, spleen and testes, while the expression of MIP3alpha1 and MIP3alpha2 was not influenced by LPS-stimulation in vivo. Furthermore, a relative increase in the expression of the Trsc-SCYA107 and MIP3alpha2 genes in PWBCs was observed at 1-12 h poststimulation with PMA and LPS, with maximal expression observed at 3 h, while MIP3alpha1 expression was observed at 3-12 h poststimulation only with PMA.

Molecular cloning and sequencing of the banded dogfish (Triakis scyllia) interleukin-8 cDNA.

The dogfish (Triakis scyllia) interleukin-8 (IL-8) cDNA was isolated from mitogen-stimulated peripheral white blood cells (WBCs) utilising the polymerase chain reaction (PCR). The cDNA sequence showed that the dogfish IL-8 clones contained an open reading frame encoding 101 amino acids. A short 5' untranslated region (UTR) of 70 nucleotides and a long 3' UTR of 893 nucleotides were also present in this 1.2-kb cDNA. Furthermore, the 3' UTR of the mRNA contained the AUUUA sequence that has been implicated in shortening of the half-life of several cytokines and growth factors. The predicted IL-8 peptide had one potential N-linked glycosylation site (Asn-72-Thr-74) that is not conserved in other vertebrates. It also contained four cysteine residues (Cys-34, 36, 61 and 77), which are characteristic of CXC subfamily cytokines and found in all vertebrates, to date. The dogfish IL-8 lacked an ELR motif as found in the lamprey and trout. Comparison of the deduced amino acids showed that the dogfish IL-8 sequence shared 50.5, 41.2, 37.1 and 40.4-45.5% identity with the chicken, lamprey, trout and mammalian IL-8 sequences, respectively.

Distinct patterns of IgH structure and organization in a divergent lineage of chrondrichthyan fishes.

Immunoglobulin heavy chain (IgH) genes in representative chondrichthyan fishes (sharks and skates) consist of independently functioning clusters, containing separate variable (VH), diversity (DH), and joining (JH) region elements and constant (CH) region exons. IgH loci have been characterized in Hydrolagus colliei (spotted ratfish), a modern representative of a major independent chondrichthyan lineage. Three distinct families of IgH gene clusters were identified. The most numerous genes consist of unjoined VH-D1-D2-JH segments that correspond to the most abundant Hydrolagus spleen (cDNA) transcripts which apparently arise from a diversified gene family. In the second cluster type, VH, DH, and JH segments are germline-joined, whereas the CH exons exhibit typical organization. This gene type is found in only a few copies per haploid genome and both transmembrane and secretory transcripts have been identified. A third cluster type has been identified that consists of unjoined VH elements but lacks a typical CH1 exon, which is substituted with a second CH2-like exon. Transcripts from this third cluster type also appear to derive from a diversified gene family. Genomic D regions of the two unjoined clone types exhibit structural differences that are consistent with incorporation of recombination machinery-mediated events. Genomic library screening indicates that 90% of VH+ clones are truncated, nearly identical pseudogenes (lacking JH and CH). These studies demonstrate an early phylogenetic origin for the cluster type of gene organization and document extensive organizational diversification within an apparent single class of IgH genes.

Rostral floor plate (flexural organ) secretes glycoproteins immunologically similar to subcommissural organ glycoproteins in dogfish (Scyliorhinus canicula) embryos.

The subcommissural organ of vertebrates secretes glycoproteins into the cerebrospinal fluid of the third cerebral ventricle. This material polymerizes in Reissner's fiber. During ontogenetic development, besides the subcommissural organ, the ependyma lining the pontine flexure constitutes an additional Reissner's fiber-secreting gland named flexural organ. We have studied the secretion of the flexural organ and the subcommissural organ in dogfish (Scyliorhinus canicula) embryos using three different antisera and the lectins concanavalin A and wheat germ agglutinin. AFRU is an antiserum against the bovine Reissner's fiber, Ab-600 is an antiserum against 600 kDa dogfish subcommissural organ glycoproteins; and APSO is an antiserum against immunoaffinity purified bovine subcommissural organ secretory glycoproteins. These three antisera immunostained the flexural organ indicating that it contains epitopes similar to those present in bovine and dogfish subcommissural organ glycoproteins. It seems highly probable that the flexural organ and the subcommissural organ of dogfish embryos secrete similar compound(s). Other ependymal regions were also immunostained with Ab-600 and APSO antisera. Then, Reissner's fiber-like glycoproteins were transiently expressed by most embryonary ependymal cells. These glycoproteins might play a role in the development of the central nervous system of vertebrates.

Neural cells from dogfish embryos express the same subtype-specific antigens as mammalian neural cells in vivo and in vitro.

Neural cells are classically identified in vivo and in vitro by a combination of morphological and immunocytochemical criteria. Here, we demonstrate that antibodies used to identify mammalian oligodendrocytes, neurons, and astrocytes recognize these cell types in the developing spiny dogfish central nervous system and in cultures prepared from this tissue. Oligodendrocyte-lineage-specific antibodies O1, O4, and R-mAb labeled cells in the 9 cm dogfish brain stem's medial longitudinal fascicle (MLF) and in areas lateral to it. Process-bearing cells, cultured from the dogfish brain stem, were also labeled with these antibodies. An anti-lamprey neurofilament antibody (LCM), which recognized 60 and 150 kDa proteins in dogfish brain stem homogenates, labeled axons and neurons in the brain stem and axons in the cerebellum of the dogfish embryo. It also labeled cell bodies and/or processes of some cultured cerebellar cells. An anti-bovine glial fibrillary acidic protein antibody, which recognized 42-44 kDa protein(s) in dogfish brain stem homogenates, labeled astrocyte-like processes in the brain stem and cerebellum of the dogfish embryo and numerous large and small flat cells in the cerebellar cultures. These results demonstrate that dogfish oligodendrocytes, neurons, and astrocytes express antigens that are conserved in mammalian neural cells. The ability to culture and identify neural cell types from cartilaginous fish sets the stage for studies to determine if proliferation, migration, and differentiation of these cell types are regulated in a similar fashion to mammalian cells.

Macrophage-lymphocyte cell clusters in the hypothalamic ventricle of some elasmobranch fish: ultrastructural analysis and possible functional significance.

BACKGROUND: Previous studies have demonstrated the existence of lympho-haemopoietic tissue in the meninges and choroid plexuses of various primitive vertebrates, including the stingray Dasyatis akajei and in early human embryos. In the present study, we extend these results analyzing macrophage-lymphocyte cell clusters found in the floor of the hypothalamic ventricle of several specimens of elasmobranchs. METHODS: After aseptical isolation of the brain from several specimens of smooth dogfish Triakis scyllia, cloudy dogfish Scyliorhinus torazame, gummy shark Mustelus manazo, and stingray Dasyatis akajei their hypothalamic regions were processed routinely by light, scanning, and transmission electron microscopy. RESULTS: The study of serial histological sections demonstrated that the macrophage-lymphocyte cell clusters proceeded from the meningeal lymphohaemopoietic tissue, reaching the ventricular lumen along large blood vessels. In this tissue, macrophages, different sized lymphocytes, lymphoblasts, granulocytes, monocytes, and developing and mature plasma cells were closely packed among a meshwork of fibroblastic reticular cell processes. It never invaded the brain parenchyma. A cell layer of glial elements and a continuous basement membrane interposed between the lymphoid tissue and the neural elements although some macrophages had migrated across the ependymal cell layer. In the ventricular lumen very irregular macrophages with long cell processes and containing abundant engulfed material of unknown origin formed big cell clusters with neighboring lymphocytes, lymphoblasts, and plasma cells, similar to those described during the immune response. Moreover, electron lucent cells which resembled the antigen-presenting cells of higher vertebrates established intimate surface cell contacts with the surrounding lymphocytes. In the third ventricle of several specimens of gummy shark, Mustelus manazo, morphologically similar cell clusters appeared but these were not connected to the meningeal lympho-haemopoietic tissue. No intraventricular cell aggregates were found in the stingray brain. CONCLUSIONS: Although we cannot rule out that these macrophage-lymphocyte cell clusters represent a permanent structure in the elasmobranch brain they rather seem to be only established after specific stimulation for preventing the entrance of noxious, foreign materials into the elasmobranch brain parenchyma.

Neuronal localization of immunoreactive adrenocorticotropin-like substance in the hypothalamus of elasmobranch fishes.

The occurrence and localization of adrenocorticotropin (ACTH)-like material in the brain of two species of elasmobranch fishes, Scyliorhinus canicula and Squalus acanthias, were studied by immunohistochemical techniques using an antiserum generated against human ACTH1-24. In both species immunopositive neurons and fibers were recognized in the basal hypothalamus mainly distributed in the nucleus lateralis tuberis, in which many of these elements were of the liquor-contacting type. A few ACTH-like positive cells were also found in the nucleus lobi lateralis hypothalami. The features of this peptidergic system, its topographic distribution to form intrinsic circuits within the posterior hypothalamic area and probably connections with the pituitary, suggest implications in brain neuromodulatory activities and hypophyseal regulation for this peptide.

Ontogeny of gut-associated lymphoid tissue (GALT) in the dogfish Scyliorhinus canicula L.

Cartilaginous fish occupy a fundamental position in vertebrate phylogeny and it is likely that this group has retained some of the ancestral immune mechanisms. The ontogeny of GALT has received little attention in elasmobranchs and this study correlates this development with morphological differentiation, development of other lymphoid organs, exposure to seawater and transition from yolk dependence to exogenous food as a source of nutrient. GALT was first represented by individual lymphocyte-like and macrophage-like cells in the lamina propria. In later stages accumulations and intraepithelial leucocytes were recorded prior to hatching. The size of accumulations and the number of lymphocyte and macrophage-like cells infiltrating the lamina propria and epithelium increased in fish as they became dependent upon an exogenous diet. Although GALT developed after the thymus and lymphoid-like tissue in the kidney and at approximately the same time as the epigonal, Leydig and spleen, the source of cells populating the gut is unknown. Plasma cells and granulocytes were not observed in the developing fish until 6 months post-hatch after which the fish had a similar GALT distribution and content to the adult fish.