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G Scapigliati

Publications and source records attributed to G Scapigliati.

At least 19 recordsLinked to original sources
PubMed2006

Evolution of cytokine responses: IL-1beta directly affects intracellular Ca2+ concentration of teleost fish leukocytes through a receptor-mediated mechanism.

In this work we studied the biological activities of recombinant IL-1beta from the teleosts sea bass (Dicentrarchus labrax) and rainbow trout (Oncorhynchus mykiss) by investigating the effects induced on intracellular Ca2+ concentrations ([Ca2+]i) of spleen leucocytes. Splenocytes were loaded with the Ca2+-permeant Fura-2AM, and then stimulated with rIL-1beta. The emitted fluorescence was read for 5 min at 1 min intervals on a dual excitation fluorescence fluorimeter. Results showed that rIL-1beta induced in both species a rise in [Ca2+]i, and a subsequent decrease until 5 min after stimulation. The stimulating effect was dose-dependent in both species reaching a plateau at 200 ng/ml of rIL-1beta, was abolished by heat-treatment of rIL-1beta, and affected in a dose-dependent fashion by treatment of leucocytes with trypsin. These features suggested a functional IL-1 receptor was involved in the binding. The observed rise in [Ca2+]i was not detected in human PBMC and was species-specific, since rIL-1beta from sea bass, trout, and human were unable to interfere each other in the assay. Moreover, incubation of splenocytes with rIL-1beta induced a rapid tyrosine phosphorylation of a 24 kDa polypeptide in both species. This work represents the first evidence of a direct effect on [Ca2+]i induced by IL-1beta and suggests that in the evolution of IL-1 activities, teleost fishes display a peculiar IL-1-associated behaviour that is lacking in mammals.

Animals↗
PubMed2006

Transmission electron microscopy, immunocytochemical and fluorescence in situ hybridisation studies in a case of 100% necrozoospermia: case report.

We present the ultrastructural, functional and chromosomal analyses of sperm from an infertile man with a normal karyotype and 100% necrozoospermia. Tests for microbial infection showed the presence of Escherichia coli in seminal and urethral fluid. Semen analysis was performed concomitantly with the infection and repeated twice after the therapy, in the absence of infection, to control the possible recovery of necrozoospermia. Morphological sperm evaluation was performed by light, fluorescent and electron microscopy; meiotic segregation was examined by fluorescence in situ hybridisation (FISH) technique. Polymerase chain reaction (PCR) was carried out on DNA from peripheral blood lymphocytes to analyse partial sequences of the Akap4 and Akap3 genes. AKAP3 protein is synthesised in round spermatids, incorporated into the fibrous sheath and is involved in organising the basic structure of the fibrous sheath. AKAP4 protein is incorporated late in spermatid development and plays a major role in completing fibrous sheath assembly. The whole sperm population was immotile and non-viable. The ultrastructural characteristics of sperm necrosis were identified. Immunostaining of tubulin and AKAP4 proteins was negative in sperm tails. PCR did not reveal any deletions in the investigated regions. FISH sperm analysis highlighted an altered meiotic segregation. After recovery from infection, 100% necrozoospermia persisted and chromosomal sperm aneuploidies were still present.

A Kinase Anchor Proteins↗
PubMed2006

Biological activity of cytokines: an evolutionary perspective.

It appears evident that teleost fish are at present the vertebrate group in which, excluding mammals, most information on the immune system is available. However, despite the great impetus on the discovery of genes homologous to mammalian immunomodulatory molecules, the knowledge on biological activities exerted by cytokines is meager. This review reports the present knowledge on the biological activities of cytokine-like and cytokines in invertebrates and cold-blooded vertebrates.

Amphibians↗
PubMed2005

Ultrastructural studies of spermatozoa from infertile males with Robertsonian translocations and 18, X, Y aneuploidies.

BACKGROUND: In order to clarify the relationship between chromosomal rearrangements, sperm morphology and interchromosomal effects (ICE), we studied the spermatogenetic defects in seven infertile Robertsonian translocation carriers. METHODS: Lymphocyte karyotypes were evaluated using Giemsa-Trypsin-Giemsa banding and fluorescence in-situ hybridization (FISH). Semen analysis was performed by light and transmission electron microscopy. FISH of sperm nuclei was carried out to detect possible ICE. RESULTS: Lymphocyte karyotype analysis revealed five t(13;14), one t(13;21) and one t(14;22) carriers. Sperm ultrastructural examination highlighted a higher percentage of immaturity, apoptosis and necrosis than in controls. Aneuploidies of gonosomes were detected in sperm from five out of six carriers of Robertsonian translocation, whereas aneuploidy of chromosome 18 was evident in three out of six carriers. The frequencies of diploidy were altered in all cases. CONCLUSIONS: Since these infertile patients showed severe spermatogenetic impairment from the morphological and meiotic points of view, we recommend detailed sperm ultrastructural and chromosomal analysis before undertaking ICSI cycles in Robertsonian translocation carriers.

Adult↗
PubMed2005

Short- and long-term effects of a dietary yeast beta-glucan (Macrogard) and alginic acid (Ergosan) preparation on immune response in sea bass (Dicentrarchus labrax).

The present study investigated the immunomodulatory activity of Ergosan, an algal extract containing alginic acid, and Macrogard, a yeast extract containing beta-glucans, on innate and specific immunity in sea bass (Dicentrarchus labrax). Four cycles of experimental feeding using normal fish feed formulation (control group) supplemented with Ergosan (0.5%) or Macrogard (0.1%) were performed at 60-day intervals (15 days of treatment+45 days of suspension). Serum complement, lysozyme, total proteins and heat shock protein (HSP) concentrations were measured at 15, 30 and 45 days from the end of the first 15-day feeding cycle (short term) and 45 days after the end of each feeding cycle over a 35-week period (long term). The percentage of B- and T-lymphocytes in peripheral blood leucocytes and gut were measured over long-term trial. Significant elevation (P < 0.05) in serum complement activity occurred in sea bass fed with alginic acid and glucans, at 15 days from the end of first cycle of treatment. Significant elevation (P < 0.05) in serum lysozyme, gill and liver HSP concentration were observed in the same experimental groups at 30 days from the end of treatment, whereas a significant increase (P < 0.05) of complement activity was only observed in fish that received an Ergosan diet. At 45 days from the end of treatment, complement, lysozyme and HSP concentration did not differ among groups. Over the long-term period, no significant differences were observed in innate and specific immune parameters, survival, growth performances and conversion index in treated and control fish. A dramatic decrease of both innate and acquired immune parameters was observed during the winter season in all groups, followed by a partial recovery when water temperature increased. Reduction in complement and lysozyme activities was significatively correlated (p < 0.01) to water temperature variation. The results suggested the potential of alginic acid and beta-glucans to activate some innate immune responses in sea bass, and particularly under conditions of immunodepression related to environmental stress.

Alginates↗
PubMed2005

Phylogeny and ontogeny of fish leucocytes.

In contrast to higher vertebrates, most fish species hatch at the embryonic stage of life. Consequently, they have to defend against a variety of micro-organisms living in their aquatic environment. This paper is focussed on the development of leucocytes functioning within this early innate system and later on in the acquired immune system (B and T cells). Most of the data are derived from cyprinid fish (zebrafish, carp), which are excellent models to study early ontogeny. Attention is also paid to the phylogeny of leucocytes, with special attention to early chordates. It is clear that young fish use innate mechanisms during the first weeks/months of their development. In zebrafish, a variety of hematopoietic genes have been sequenced which allow a detailed picture of the development of the distinct leucocytes and their precursors. In cyprinids and sea bass, the thymus is the first lymphoid organ and T cells appear to be selected there much earlier than the first detection of T cell-dependent antibody responses. The first B cells are most probably generated in head kidney. Although T cells are selected earlier than B cells, T cell independent responses occur earlier than the T cell-dependent responses. The very early (pre-thymic) appearance of T-like cells in gut of sea bass and carp suggests an extra-thymic origin of these cells. However, B cells populate the GALT much later than spleen or kidney, indicating a rather late appearance of mucosal immunity. The first plasma cells are found long after the intake of food in cyprinids, but in many marine fish they appear around the first food intake. In general, acquired immunity is not correlated to food intake.

Age Factors↗
PubMed2005

'Round head' sperm defect. Ultrastructural and meiotic segregation study.

The sperm 'round head' defect, also known as globozoospermia, is an uncommon alteration of sperm morphology generally characterised by 100% round headed sperm totally lacking an acrosome. This alteration is a genetic sperm defect as demonstrated by analysing the incidence of these alterations in a population of infertile men showing a history of consanguinity and cases belonging to the same family. Ultrastructural characteristics and meiotic segregation in spermatozoa from two patients affected by 'round head' sperm defect were investigated. The sperm quality was examined by light and transmission electron microscopy (TEM) and fluorescence in situ hybridization (FISH) analysis was performed in order to investigate the meiotic behavior of chromosomes namely gonosomes and chromosome 18. TEM analysis, mathematically elaborated, clearly diagnosed the 'round head' genetic sperm defect and highlighted at the same time the presence of other phenotypic alterations belonging to pathologies such as immaturity, apoptosis and necrosis. It is possible to hypothesize that round headed sperm could be a 'weak phenotype' allowing the sperm pathologies to overlap with a sperm defect of genetic origin, further compromising fertilizing potential. FISH analysis revealed a positive correlation between globozoospermia and higher disomies of sex chromosomes and diploidies suggesting a higher risk of creating an aneuploid embryo after intracytoplasmic sperm injection.

Adult↗
PubMed2004

Formation of the egg envelope of a teleost, Dicentrarchus labrax (L.): immunochemical and cytochemical detection of multiple components.

The formation of the egg envelope in a teleost, Dicentrarchus labrax (L.), was analysed at histological and ultrastructural level. The sequential deposition of three main layers (Z1, Z2 and Z3) constitutes the extracellular matrix throughout oocyte development. Various findings indicate that these subunits are biochemically distinct: (1) periodate- and phosphotungstic acid-reactive carbohydrates are obviously detected only in the Z1, that constitutes the initial deposit of the egg envelope in early lipidic oocytes; (2) a monoclonal antibody (DLE7) against egg envelope polypeptides did not immunostain the Z1 and the underlying Z2; (3) the antigenic determinants recognised by DLE7, thought to be exogenous in origin (synthesised in the liver), are incorporated in the inner layer (Z3). In addition, DLE7 immunostained a thin layer, assembled together with Z3. This line has not yet been described in teleost eggs and was named Z1a. This study first describes at fine cytological level the contribution of exogenous proteins to formation of the different egg envelope layers. Results obtained with conventional, immunochemical and cytochemical techniques suggest multiple synthetic sources (exogenous and follicular) of egg envelope proteins.

Animals↗
PubMed2003

Functional characterisation of the recombinant tumor necrosis factors in rainbow trout, Oncorhynchus mykiss.

Tumor necrosis factor (TNF) is a key mediator in regulating the inflammatory response. Previously two TNF genes have been cloned and sequenced from rainbow trout, Oncorhynchus mykiss. In this study, the mature peptides of the two TNF molecules were produced in bacteria, purified under native conditions and their bioactivities evaluated in vitro. Both trout rTNF1 and rTNF2 induced gene expression of a number of proinflammatory factors including IL1beta, TNF1, TNF2, IL8 and COX2 in freshly isolated head kidney leucocytes and the macrophage cell line RTS11. The stimulatory doses of both rTNFs were >or=10 ng/ml. Moreover, leucocyte migration and phagocytic activity were enhanced in vitro by the rTNFs in a dose dependent manner. Western blot analysis revealed the presence of multiple forms of rTNF structures including monomeric, dimeric and trimeric forms, suggesting that formation of a homotrimeric structure may be essential for the TNF bioactivities.

Adjuvants, Immunologic↗
PubMed2002

The immune system of sea bass, Dicentrarchus labrax, reared in aquaculture.

The sea bass Dicentrarchus labrax is one the most important seawater fish species of south Europe and Mediterranean aquaculture, and studies on its immune system are important for both scientific and applied purposes. In this paper, we summarise the results obtained in studies of the immune system in this species, and present original data on cell-mediated acquired immune response.

Animals↗
PubMed2001

The production and bioactivity of rainbow trout (Oncorhynchus mykiss) recombinant IL-1 beta.

The predicted rainbow trout mature interleukin-1 beta (IL-1 beta) peptide has been produced as a recombinant protein in E. coli. The bioactivity of this molecule has been studied using trout head kidney cell preparations and a trout macrophage cell line (RTS11). Trout rIL-1 beta was shown to increase the expression level of IL-1 beta, cyclooxygenase (COX2) and MHC class II beta chain transcription, as determined by Northern blot analysis. Stimulatory doses of rIL-1 beta were typically > or =10 ng/ml. Induction of IL-1 beta expression occurred within 1h post-stimulation with trout rIL-1 beta and was maximal 3-6h post-stimulation. Trout rIL-1 beta was also able to increase murine D10.G4.1 cell proliferation and trout head kidney leukocyte phagocytic activity, in a dose-dependent manner. However, equivalent D10.G4.1 cell proliferation was induced with approximately 1000-fold lower doses of human rIL-1 beta. That LPS contamination did not contribute to the effects seen was confirmed by determining its concentration in the trout rIL-1 beta preparation, and demonstrating that the rIL-1 beta activity was inhibited by heating or pre-incubation with a polyclonal anti-trout rIL-1 beta antibody.

Animals↗
PubMed2001

Phylogeny of cytokines: molecular cloning and expression analysis of sea bass Dicentrarchus labrax interleukin-1beta.

In this paper the cloning of interleukin-1beta (IL-1beta) from the fish Dicentrarchus labrax (sea bass) is described. Using degenerate primers designed from known IL-1beta sequences, a cDNA fragment was amplified by PCR and elongated by 3' and 5' RACE to give the full-length coding sequence for sea bass IL-1beta. The cDNA is 1292 bp, lacks a putative ICE cut site, and codes for a deduced peptide of 29.4 kDa with a pI of 5.1. Sequence analysis showed highest amino acid similarity with rainbow trout (62%), Xenopus (46%), and carp (45.5%) IL-1beta sequences. Expression studies show that sea bass IL-1beta can be upregulated by bacterial lipopolysaccharide both in vitro and in vivo in leucocytes from blood, head-kidney, spleen, gills and liver, whereas the IL-1beta transcript was not detectable in thymus and gut-associated lymphoid tissue. Northern blot analysis with head-kidney leucocyte RNA showed a main LPS-upregulated band at 1.3 kb, and two minor bands at 0.9 and 3.0 kb, respectively. Phylogenetic comparisons with IL-1beta from other vertebrates is presented.

Amino Acid Sequence↗
PubMed2000

Immunopurification of T-cells from sea bass Dicentrarchus labrax (L.).

The monoclonal antibody DLT15, specific for thymocytes and peripheral T-cells of the teleost fish Dicentrarchus labrax (sea bass), was used to purify immunoreactive cells from blood and gut-associated lymphoid tissue. The purification was performed by immuno-magnetic sorting of leucocyte fractions enriched by Percoll density gradient centrifugation, and the purity of the isolated cells was estimated by cytofluorimetric analysis. Following a single step, the percentage of DLT15-purified cells was 88 +/- 10% for gut-associated lymphoid tissue and 79 +/- 18% for blood leucocytes. DLT15-purified cells from gut-associated lymphoid tissue were employed for RNA extraction and cDNA synthesis. In RT-PCR experiments using as primers degenerate oligonucleotides corresponding to the peptide sequence MYWY and VYFCA of the trout TcR beta chain, a 203 bp product was amplified. When sequenced, the cDNA was found to show 60% nucleotide identity to the trout TcRV beta 3. By 3'-RACE the cDNA was elongated to obtain the TcR constant region, with high similarity to other fish TcR sequences. These results strongly suggest that cells recognised by DLT15 are putative T lymphocytes.

Amino Acid Sequence↗
PubMed2000

Invertebrate and fish cytokines.

Cytokine-like molecules are well described in invertebrates, although most recent studies have revealed that there is analogy, rather than homology, between invertebrate and vertebrate cytokine-like activities. Cytokines certainly appeared early in the evolution of vertebrates, dating back some 400 millions years. Here, evidence will be reviewed and updated of the presence of these molecules in jawed fish and in particular, in bony fish, which represent the oldest group displaying true functionality of immune system as known in modern vertebrates. Many studies during the last ten years have confirmed the presence of functional homologues of mammalian cytokines in fish. In this review, particular attention will be focussed on IL-1beta, a very ancient defence cytokine recently sequenced in two species, rainbow trout (Oncorhynchus mykiss) and carp (Cyprinus carpio). Original data on the partial peptide sequence of IL-1beta in the mediterranean sea bass Dicentrarchus labrax are also presented.

Amino Acid Sequence↗
PubMed1999

Immunodetection of lymphocyte subpopulations involved in allograft rejection in a teleost, Dicentrarchus labrax (L.).

Monoclonal antibodies which recognize antigenic determinants expressed by T-cells and Ig-bearing cells, respectively, allowed lymphocyte subpopulations involved in allograft rejection of muscle transplants to be identified in the teleost fish Dicentrarchus labrax (L.). The monoclonal antibody DLT15 first allowed recognizing T-cells involved in an in vivo antigen-driven cellular response in teleosts. Immunohistochemical studies showed a high density of lymphocytes in allografts and provided evidence of predominance of T-cells. The heterogeneity of the cell populations recognized by the antibodies was evidenced by the different size, cytology, and staining patterns of T-cells and Ig-bearing cells.

Animals↗
PubMed1999

Histological and cytological studies on the developing thymus of sharpsnout seabream, Diplodus puntazzo.

The structure of the developing thymus of the marine teleost, Diplodus puntazzo, was studied by light and transmission electron microscopy. The first anlage of the thymus developed by d 20 postfertilisation (p.f.) as a group of undifferentiated cells dorsal to the epithelium of the branchial chamber. The organ increased significantly in size around d 51-66 p.f. and differentiation of cortex and medulla occurred concomitantly. On the basis of their localisation, 4 main types of epithelial cell were distinguished: (1) limiting, adjacent to the connective capsule; (2) medullary and cortical reticular cells; (3) nurse cells, located in the corticomedullary boundary; (4) Hassall-like corpuscles. The majority of medium to large blast-like lymphoid cells were localised in the medulla, while small lymphocytes were housed in the cortical region. These morphological features were maintained at later stages. However, in juveniles in the medulla we observed reticular epithelial cells with cysts and rare Hassall-like corpuscles. The study was designed to obtain more information concerning the histology of the developing thymus of sharpsnout seabream and give a concise description of the differentiation of epithelial cells and lymphoid cells in the thymic parenchyma.

Animals↗
PubMed1999

A monoclonal antibody against chorion proteins of the sea bass Dicentrarchus labrax (Linnaeus, 1758): studies of chorion precursors and applicability in immunoassays.

The monoclonal antibody DLE7 was obtained against 44- to 50-kDa polypeptides solubilized from the vitelline envelope of the Mediterranean sea bass Dicentrarchus labrax. In Western blot analysis of chorion lysates it recognized cross-reactive bands at 44 kDa, 48 kDa, and 110 kDa. Previous affinity blotting with concanavalin-A showed that most of solubilized bands were glycosylated. Enzymatic deglycosylation of chorion proteins followed by Western blot analysis with DLE7 showed that the 48-kDa and 110-kDa antigens were differentially affected by endoglycosidase-F treatment. When DLE7 was employed in immunofluorescence analysis, isolated chorions and ovarian cryosections stained intensely. Positivity was also observed in liver cryosections of spawning females but not in liver of males and nonspawning females. To study the origin and delivery of chorion proteins, DLE7 was used in Western blot analysis of liver homogenates and blood serum of spawning females. Cross-reacting bands were detected in liver (90 kDa) and serum (180 kDa, 50 kDa). DLE7 was also used for the first time to set up an indirect ELISA assay to detect egg antigens in the blood of egg-producing females, raising the possibility of using DLE7 as a female-specific marker of spawning for sea bass.

Animals↗