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ABSTRACT: The presence and distribution of apoptotic cells during thymus development and in adult were studied by in situ end-labelling of fragmented DNA in three temperate species carp (Cyprinus carpio), sea bass (Dicentrarchus labrax) and dusky grouper (Epinephelus marginatus) and in the adult thymus of three Antarctic species belonging to the genus Trematomus spp During thymus development some few isolated apoptotic cell (AC) firstly appeared in the central-external part of the organ (carp: 5 days p.h; sea bass: 35 days p.h grouper: 43 days p.h). Initially the cells were isolated and then increased in number and aggregated in small groups in the outer-cortical region of the thymus larvae. The high density of apoptotic cells was observed in the junction between cortex and medulla from its appearance (border between cortex and medulla, BCM). ACs decreased in number in juveniles and adult as well as the ACs average diameter. In late juveniles and in adulthood, the apoptosis were restricted to the cortex. In Antarctic species the thymus is highly adapted to low temperature (high vascularisation to effort the circulation of glycoproteins enriched plasma and strongly compact parenchyma). The apoptosis process was more extended (4-7 fold) as compare with the thymus of temperate species, even if the distribution of ACs was similar in all examined species. Data suggested a common process of T lymphocyte negative-selection in BCM of thymus during the ontogeny. The selection process seems to be still active in adult polar fish, but restricted mainly in the cortex zone.
Fish & Shellfish Immunology 04/2013; · 3.32 Impact Factor
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ABSTRACT: All jawed vertebrates share lymphocyte receptors that allow the recognition of pathogens and the discrimination between self and non-self antigens. The T cell transmembrane receptor (TcR) has a central role in the maturation and function of T lymphocytes in vertebrates via an important role in positive selection of the variable region of TcR αβ/γδ chains. In this study, the TcRβ transcript expression and TcRβ(+) cell distribution during the ontogeny of the immune system of sea bass (Dicentrarchus labrax, L.) were analysed. RT-PCR analysis of larvae during early development demonstrated that the β chain transcript is expressed by 19 days post-fertilisation (p.f.). RNA probes specific for the β chain were synthesised and used for in situ hybridisation experiments on 30 day p.f. to 180 day old juvenile larvae. A parallel immunohistochemical study was performed using the anti-T cell monoclonal antibody DLT15 developed in our laboratory [Scapigliati et al., Fish Shellfish Immunol 1996; 6:383-401]. The first thymus anlage was detectable at 32-33 days p.f. (Corresponding to about 27 days post-hatch). DLT15(+) cells were detected at day 35 p.f. in the thymus whereas TcRβ(+) cells were recognisable at day 38 p.f. in the thymus and at day 41 p.f. in the gut. TcRβ(+) cells were observed in capillaries from 41 to 80 days p.f. At day 46 p.f., TcRβ(+) cells were identified in the head kidney and were detected in the spleen 4 days later. The present results demonstrate that TcRβ(+) cells can be differentiated first in the thymus and then in other organs/tissues, suggesting potential TcRβ(+) cell colonisation from the thymus to the middle gut. Once the epithelial architecture of the thymus is completed with the formation of the cortical-medullary border (around 70-75 days p.f.), DLT15(+) cells or TcRβ(+) cells are confined mainly to the cortex and cortical-medullary border. In particular, a large influx of TcRβ(+) cells was observed at the cortical-medullary border from 72 to 90 days p.f., suggesting a role in positive selection for this thymic region during the ontogeny of the fish immune system. This study provides novel information about the primary differentiation and distribution of TcRβ(+) cells in sea bass larvae and juveniles.
Fish & Shellfish Immunology 01/2011; 30(3):773-82. · 3.32 Impact Factor
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ABSTRACT: Real-time polymerase chain reaction (PCR) and in situ hybridization analyses were performed to investigate the occurrence and distribution of T-lymphocytes expressing TcRbeta in intestine and lymphoid tissues of the bony fish, Dicentrarchus labrax (sea bass). Immunohistochemistry with the monoclonal antibody DLT15 (pan-T-cell marker) was carried out to compare the cytology, distribution and number of T-cells and TcRbeta+ cells in the various sampled lymphoid organs. The highest TcRbeta expression was revealed by real-time PCR in the thymus, with high levels also being found in the gut. In the thymus, DLT15+ and TcRbeta+ cell populations were concentrated in the cortex and TcRbeta+ cells were notably reactive at the cortical-medullary border, suggesting a specialized role of this region in thymocyte selection. The density of DLT15+ T-cells increased from the anterior to posterior intestine, whereas TcRbeta+ lymphocytes were more numerous in the middle intestine compared with other segments. The existence, in fish thymus, of a medulla and a cortex comparable with those of mammals is revealed by this study. The concentration of TcRbeta+ cells in the sea bass midgut also strongly suggests a special role of this intestinal segment in antigen-specific cellular immunity. The large population of TcRbeta(-)/DLT15+ T-cells in the posterior gut can probably be ascribed to the TcRgammadelta phenotype fraction.
Cell and Tissue Research 10/2007; 329(3):479-89. · 3.11 Impact Factor
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ABSTRACT: Real-time polymerase chain reaction (PCR) and in situ hybridization analyses were performed to investigate the occurrence
and distribution of T-lymphocytes expressing TcRβ in intestine and lymphoid tissues of the bony fish, Dicentrarchus labrax (sea bass). Immunohistochemistry with the monoclonal antibody DLT15 (pan-T-cell marker) was carried out to compare the cytology,
distribution and number of T-cells and TcRβ+ cells in the various sampled lymphoid organs. The highest TcRβ expression was revealed by real-time PCR in the thymus, with
high levels also being found in the gut. In the thymus, DLT15+ and TcRβ+ cell populations were concentrated in the cortex and TcRβ+ cells were notably reactive at the cortical-medullary border, suggesting a specialized role of this region in thymocyte selection.
The density of DLT15+ T-cells increased from the anterior to posterior intestine, whereas TcRβ+ lymphocytes were more numerous in the middle intestine compared with other segments. The existence, in fish thymus, of a
medulla and a cortex comparable with those of mammals is revealed by this study. The concentration of TcRβ+ cells in the sea bass midgut also strongly suggests a special role of this intestinal segment in antigen-specific cellular
immunity. The large population of TcRβ-/DLT15+ T-cells in the posterior gut can probably be ascribed to the TcRγδ phenotype fraction.
Cell and Tissue Research 08/2007; 329(3):479-489. · 3.11 Impact Factor
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ABSTRACT: The ontogeny of carp (Cyprinus carpio L.) immune cells was studied in mucosal organs (intestine, gills and skin) using the monoclonal antibodies WCL38 (intraepithelial lymphocytes), WCL15 (monocytes/macrophages) and WCI12 (B cells). In addition, recombination activating gene 1 expression was examined in the intestine with real time quantitative PCR and in situ hybridization to investigate extrathymic generation of lymphocytes. WCL38(+) intraepithelial lymphocytes (putative T cells) appeared in the intestine at 3 days post-fertilization (dpf), which is shortly after hatching but before feeding, implying an important function at early age. These lymphoid cells appear in the intestine before the observation of the first thymocytes at 3-4 dpf, and together with the expression of recombination activating gene 1 in the intestine, suggests that similar to mammals at least part of these cells are generated in the intestine. WCL15(+)monocytes/macrophages appeared in the lamina propria of the intestine at 7 dpf, but considerably later in the epithelium, while WCI12(+) (B) cells appeared in intestine and gills at 6-7 weeks. From these results it can be concluded that putative T cells occur much earlier than B cells, and that B cells appear much later in the mucosae than in other internal lymphoid organs (2 wpf).
Anatomy and Embryology 02/2006; 211(1):19-29. · 1.42 Impact Factor
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ABSTRACT: Transfer of immunoglobulins (IgM-like) from the female to the teleost embryo has been demonstrated but mechanisms of uptake into and storage within the eggs remain to be clarified. The monoclonal antibody DLIg3 against Dicentrarchus labrax Ig light chain revealed an active role of both follicle cells and oocytes in the Ig uptake. The primordial follicular cells showed DLIg3 immunoreactivity even at a pre-vitellogenetic stage. Early vitellogenetic oocytes (lipid vesicle stages) had DLIg3 staining of pore canals, plasmalemma and outer cortex and of their follicular cells. In protein yolk granule oocytes, DLIg3 staining was also detected within vesicles of the outer-mid cortex and juxtanuclear yolk granules; therefore, a centripetal transport of Ig throughout oocyte development is apparently carried out. Immunoelectron microscopy confirmed the presence of Ig within thecal and granulosa cells (and in the interposed basement membrane) of pre-vitellogenic and vitellogenic follicles. Thus, the transport of Ig to the egg apparently occurs also by transcytosis across the follicle cells. Igs were localised in the pore canals surrounding the microvilli and in vesicles of outer-mid cortex of vitellogenic oocytes. Reverse transcription/polymerase chain reaction with primers designed for the constant region of sea bass Ig light chain detected Ig mRNA in hydrated oocytes, a smaller content in released eggs and no signal in larvae at day two post-hatching. These findings show that a significant level of Ig gene transcription in the oocyte and/or a transfer of transcripts may also occur.
Cell and Tissue Research 03/2004; 315(2):259-70. · 3.11 Impact Factor
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ABSTRACT: Transfer of immunoglobulins (IgM-like) from the female to the teleost embryo has been demonstrated but mechanisms of uptake into and storage within the eggs remain to be clarified. The monoclonal antibody DLIg3 against Dicentrarchus labrax Ig light chain revealed an active role of both follicle cells and oocytes in the Ig uptake. The primordial follicular cells showed DLIg3 immunoreactivity even at a pre-vitellogenetic stage. Early vitellogenetic oocytes (lipid vesicle stages) had DLIg3 staining of pore canals, plasmalemma and outer cortex and of their follicular cells. In protein yolk granule oocytes, DLIg3 staining was also detected within vesicles of the outer-mid cortex and juxtanuclear yolk granules; therefore, a centripetal transport of Ig throughout oocyte development is apparently carried out. Immunoelectron microscopy confirmed the presence of Ig within thecal and granulosa cells (and in the interposed basement membrane) of pre-vitellogenic and vitellogenic follicles. Thus, the transport of Ig to the egg apparently occurs also by transcytosis across the follicle cells. Igs were localised in the pore canals surronding the microvilli and in vesicles of outer-mid cortex of vitellogenic oocytes. Reverse transcription/polymerase chain reaction with primers designed for the constant region of sea bass Ig light chain detected Ig mRNA in hydrated oocytes, a smaller content in released eggs and no signal in larvae at day two post-hatching. These findings show that a significant level of Ig gene transcription in the oocyte and/or a transfer of transcripts may also occur.
Cell and Tissue Research 01/2004; 315(2):259-270. · 3.11 Impact Factor
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ABSTRACT: Naked circular plasmid DNA containing the cytomegalovirus (CMV)-promoter-driven lacZ reporter gene (pCMV-LacZ) was injected in the epaxial muscle of gilthead sea bream (Sparus aurata). A mosaic pattern of expression of beta-galactosidase (beta-gal) in the myofibres at the site of injection was visualised by in situ histochemical staining using 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside. As measured by o-nitrophenyl-beta-D-galactopyranoside assay, beta-gal enzymatic activity was found to steadily increase for at least 50 days post injection (p.i.) in pCMV-LacZ-injected muscle. In parallel, foreign DNA was detected by polymerase chain reaction in injected muscles (but not in other tissues) up to 60 days p.i., persisting most probably in an extrachromosomal, non-replicative, circular form. Neither beta-gal activity nor pCMV-LacZ-related amplification products were found 90 days p.i. Antibodies against beta-gal were demonstrated in pCMV-LacZ-injected fish sampled 45 days p.i. The results suggest that intramuscular delivery of foreign genes represents a realistic approach for DNA vaccine technology for the prevention of infectious diseases in gilthead sea bream.
Fish & Shellfish Immunology 11/2003; 15(4):283-95. · 3.32 Impact Factor
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ABSTRACT: Species that live in extreme conditions have specially adapted physiology and tissue/organ organisation. The adaptation of lymphoid organs to low temperatures in polar species could be an original field of study, indicating how the immune system works under extreme conditions. In fishes, the head kidney is a key organ for immunity and here the cytology of this organ is studied in two common Antarctic species: Trematomus bernacchii and Chionodraco hamatus. Ultrastructural analysis revealed heterogeneity of epithelial cells, with reticular cells, subcapsular- and perivascular-limiting cells. Differences in the size and morphology of epithelial cells were observed between the polar species and warm water species of fish. Intermingled with epithelial cell leucocytes, such as lymphocytes, thrombocytes and macrophages, had comparable morphology in both species, contrary to sharp differences observed in the morphology of erythrocytes and granulocytes. The functional adaptation of the head kidney to the low temperatures of polar water is discussed.
Tissue and Cell 05/2002; 34(2):63-72. · 1.04 Impact Factor
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ABSTRACT: Lymphomyeloid organs of two common species of Antarctic fish, Trematomus nicolai and Chionodraco hamatus, were studied with the aim of analysing some morphological aspects of these organs in relation to adaptation to low environmental
temperature. The thymuses of T. nicolai and C. hamatus were flattened, incompletely lobated, with numerous Hassall-like bodies, which were mainly located in the central part of
the organ in C. hamatus. In T. nicolai, thymocytes, erythroid and reticular epithelial cells filled the organ. In C. hamatus, the thymocytes intermingled with reticular epithelial cells were often close to groups of melano-macrophages. In both species,
the thymus did not show distinct compartmentalisation; however, the thymocytes had significantly different sizes in the outer
and inner portions of the thymus. The head kidney of both species was completely filled by haematopoietic tissue, highly vascularised
and mainly lymphopoietic in T. nicolai, while both erythropoietic and lymphopoietic in C. hamatus. The spleen appeared mainly erythropoietic in T. nicolai and mainly lymphopoietic in C. hamatus. Solitary melano-macrophages in T. nicolai were close to numerous small vascular ellipsoids where erythroid and lymphoid cells were intermingled without the formation
of red and white pulp areas. In C. hamatus, large lymphoid areas were organised around the capillaries. The possible adaptation of lymphoid organs to the low temperature
of polar water is discussed.
Polar Biology 03/2000; 23(5):321-328. · 1.66 Impact Factor
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ABSTRACT: Lymphomyeloid organs of the Antarctic fish Trematomus bernacchii were studied with the aim of analysing some morphological aspects related to adaptation to low environmental temperature.
The thymus of T. bernacchii was flattened, incompletely lobated and scarcely regionalised. It was filled by lymphoid elements intermingled with stromal
elements. The head kidney appeared highly vascularised and mainly lymphopoietic. The spleen appeared mainly erythropoietic,
with scarcely developed areas of white pulp.
Polar Biology 09/1997; 18(5):358-362. · 1.66 Impact Factor
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Italian Journal of Zoology 01/1996; 63(4):329-331. · 0.94 Impact Factor
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Italian Journal of Zoology 01/1996; 63(4):333-335. · 0.94 Impact Factor
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ABSTRACT: The monoclonal antibody DLT15 specifically recognised antigenic determinants expressed during development of thymic lymphoid cells of the sea bassDicentrarchus labrax(L.). They were first detected at day 30 post-hatching in the thymus of fish maintained at 16° C; thereafter, a marked increase in the number of immunoreactive cells occurred mostly in the outer region of the thymus. The appearance of DLT15-immunoreactive cells in the lymphoid organs was in the sequence thymus, head-kidney and spleen. The thymus thus appears to be a primary lymphoid organ in the sea bass. Infrequent cells sharing the antigenic determinants expressed on thymocytes were localised in the developing head-kidney and spleen.
Fish & Shellfish Immunology 6(7):493-505. · 3.32 Impact Factor
