Vaccination and immune responses of European sea bass (Dicentrarchus labrax L.) against betanodavirus.

This review summarizes the available knowledge on the immune defences of European sea bass against antigenic preparations derived from the viral encephalopathy and retinopathy virus (betanodavirus), which represents a major threat to the health of this fish species. The nodavirus is widely present and differentiates into several strains that infect invertebrates (in insects, alphanodavirus) and teleost fish, and thus may represent a great problem for farmed fish species. Many efforts have been directed to discovering new immunizations to induce protection in sea bass, especially at young stages, and these efforts have included employing diverse betanodavirus strains, antigen preparation, vaccination routes, and the addition of adjuvants and/or immunostimulants. The obtained results showed that inactivated preparations of betanodavirus that were administered intraperitoneally may induce both immune recognition and protection. Attempts at performing mucosal immunization by immersion and/or oral administration, which is a vaccination route that is highly preferred for sea bass, have shown intriguing results, and more studies are necessary for its improvement. Overall, the objective of identifying a reliable vaccine that also cross-protects against different genotypes or reassortant viruses for use in European sea bass against betanodavirus appears to be an attainable goal in the near future.

Evolution of Th2 responses: characterization of IL-4/13 in sea bass (Dicentrarchus labrax L.) and studies of expression and biological activity.

Th2 immunity is a primary host defence against metazoan pathogens and two of the important cytokines involved in this immune response in mammals are IL-4 and IL-13. Recently the origin and evolution of Th2 immune responses have been investigated in fish where a molecule with relatedness to both IL-4 and IL-13 is present, termed IL-4/13. Different IL-4/13 paralogues (IL-4/13 A and IL-4/13B) exist in teleost fish. In this paper, we have focused on the IL-4/13 isoforms found in the European sea bass (Dicentrarchus labrax L.). Two tandem duplicated but divergent IL-4/13 A isoforms and one IL-4/13B are present, a unique situation compared to other teleosts. These genes were studied in terms of their in vitro and in vivo transcript levels after different treatments and their biological activities after production of the recombinant isoforms. The results show that the presence of these three paralogues is associated with different activities, both in terms of their expression profiles and the ability of the proteins to modulate the expression of immune genes in head kidney leukocytes. It is clear that the initiation and control of type-2 responses in seabass is complex due to the presence of multiple IL-4/13 isoforms with overlapping but distinct activities.

Reproductive biology in Anophelinae mosquitoes (Diptera, Culicidae): Fine structure of the female accessory gland.

The morphology and ultrastructure of female accessory reproductive glands of Anopheles maculipennis s.s., Anopheles labranchiae and Anopheles stephensi were investigated by light and electron microscopy. The reproductive system in these species is characterized by two ovaries, two lateral oviducts, a single spermatheca and a single accessory gland. The gland is globular and has a thin duct which empties into the vagina, near the opening of the spermathecal duct. Significant growth of the accessory reproductive gland is observed immediately after blood meal, but not at subsequent digestion steps. At ultrastructural level, the gland consists of functional glandular units belonging to type 3 ectodermal glands. The secretory cells are elongated and goblet shaped, with most of their cytoplasm and large nucleus in the basal part, close to the basement lamella. Finely fibrous electron-transparent material occupies the secretory cavity that is in contact with the end of a short efferent duct (ductule) emerging from the gland duct. The present study is the first detailed description of female accessory gland ultrastructure in Anophelinae and provides insights into the gland's functional role in the reproductive biology of these insects.

Tubal sterilization by laparoscopy or hysteroscopy: which is the most cost-effective procedure?

By using the activity-based cost/management (ABC/M) system we computed and compared costs needed for laparoscopic tubal sterilization (LTS) and Essure hysteroscopic tubal occlusion (EHTO). We found that total health costs related to consultation and presurgery did not differ between LTS and EHTO; EHTO has low recovery unit costs but is more costly for the operating theater, mainly due to Essure microinserts.

Compartmentalisation of T cells expressing CD8alpha and TCRbeta in developing thymus of sea bass Dicentrarchus labrax (L.).

Eggs, larvae, post-larvae and sexually immature juveniles of the teleost Dicentrarchus labrax (L.) were assayed for the expression of genes encoding the T cell receptor beta and CD8alpha. RT-PCR of RNA extracted from larvae revealed TCRbeta transcripts from day 25 post-hatching (ph) and CD8alpha transcripts from 26 days later. At day 51 ph, CD8alpha and TCRbeta mRNAs were localised by in situ hybridisation in thymocytes of the outer and lateral zones of the thymic paired glands. From day 75 ph onwards the signal was mainly detected in the outer region, drawing a cortex-medulla demarcation. In 1-year-old fish, CD8alpha+ and TCRbeta+ thymocytes almost filled the cortex and extended in large cords in the medulla. A CD8alpha(-)TCRbeta+ subcapsular lymphoid zone was evident near the septa coming from the inner connective capsule that delimited the thymus. The localisation of CD8alpha and TCRbeta transcripts demonstrated a compartmentalisation of the juvenile thymus due to distinct localisation of thymocytes at different developmental stages.

T cell receptor beta chain from sea bream (Sparus aurata): molecular cloning, expression and modelling of the complexes with MHC class I.

The T cell receptor is a fundamental mediator of the adaptive immune responses, since TR alphabeta on T cells recognize foreign structures (peptides derived from processed antigens) bound to the major histocompatibility complex (MHC) on APC cells. In the present study, we report the cloning of six TRB chains cDNA sequences from gilthead sea bream (Sparus aurata), a fish of high economical impact in South Mediterranean aquaculture. The V-BETA domains have the canonical features of known teleost and mammalian TR V-BETA domains and have been divided in four different subgroups. A multiple alignment of the six sea bream TRB chains with other known TRB sequences was assembled and showed the conservation of the four cysteine residues involved in disulphide bonds and of some amino acids with an important role in the assembly and signalling of the TR alphabeta/CD3 complex. Real-time PCR analysis was used to investigate TRB basal expression, that was maximum in the thymus followed by gut, and TRB in vitro expression after stimulation with LPS or PHA-L at 4 and 24h (only the 4h stimulation with LPS gave a significant effect). Moreover, the 3D structures of sea bream TRB chains and MHC-I were predicted by homology modelling with the final aim to investigate the interaction surface in the V-BETA/MHC-I complexes.

Single serum activin a testing to predict ectopic pregnancy.

CONTEXT: Ectopic pregnancy (EP) is an important cause of maternal deaths in early pregnancy because most fatal cases result from delayed diagnosis and inappropriate investigation. OBJECTIVE: We evaluated whether the measurement of activin A may be useful in the diagnosis of EP in women with unknown pregnancy location. DESIGN: The study was designed as an open observational study. SETTING: The study was set in a tertiary referral center for obstetric care. PATIENTS: Patients were women with unknown pregnancy location (n = 536) who had complaints of bleeding, pain, or cramping. INTERVENTIONS: Interventions included clinical examination; transvaginal ultrasound scan; human chorionic gonadotropin (hCG), progesterone, and activin A measurements; laparoscopy; uterine curettage; and histological examination. MAIN OUTCOME MEASURES: Main outcome measures were pregnancy outcomes and evaluation of sensitivity, specificity, and predictive values of hCG, progesterone, and activin A as diagnostic tests for the detection of EP. RESULTS: Pregnancy outcomes included 155 (28.9%) viable intrauterine pregnancies (IUP), 305 (56.9%) first-trimester spontaneous abortion (SAB), and 76 (14.2%) EP. SAB had the lowest (P < 0.0001) hCG and progesterone concentrations, significantly lower than EP (P < 0.001) and IUP (P < 0.001). In EP, levels were significantly (P < 0.001) lower than in IUP. On the contrary, activin A levels were lowest (P < 0.0001) in EP, significantly lower than in SAB (P < 0.001) and IUP (P < 0.001). IUP had significantly (P < 0.001) lower activin A levels than SAB. When evaluated by the receiver operating curve analysis, activin A at the cutoff of 0.37 ng/ml combined a sensitivity and a specificity of 100 and 99.6%, respectively, for prediction of EP. When activin A concentrations were below the cutoff, the positive predictive value for EP was 97.43%, and 0% for concentrations higher than 0.37 ng/ml. CONCLUSIONS: Activin A measurement may identify patients at risk of EP with a high sensibility and specificity.

Effects of administration of probiotic strains on GALT of larval gilthead seabream: Immunohistochemical and ultrastructural studies.

Two bacterial strains Lactobacillus fructivorans (AS17B), isolated from adult seabream (Sparus aurata L.) gut, and Lactobacillus plantarum (906), isolated from human faeces, were administered contemporaneously during seabream development using Brachionus plicatilis and/or Artemia salina and dry feed as vectors. Experimental group A received the probiotic strains already via rotifers from day 5 post-hatch (ph), whereas treatment of group B began with Artemia feeding from day 27 ph. Fish were sampled at day 28 ph (group A and control) and day 99 ph (groups A, B and control) for electron microscopy, histology and immunohistochemistry with the polyclonal antiserum ORa against homologous serum Ig and the mAb G7 specific for seabream acidophilic granulocytes. In all groups, timing and pattern of differentiation of the digestive tract did not differ. Furthermore, neither tissue damage nor manifest inflammation was provoked by probiotic administration. At day 28 ph, the developing GALT already housed mucosal leucocytes, including Ig(+) cells but no acidophilic granulocytes. No differences were seen between experimental groups. At day 99 ph, the density of Ig(+) cells (+51%) and acidophilic granulocytes (+284%) was significantly higher (p<0.05) in group A than in controls. Also group B had a higher density of Ig(+) cells (+17%) and acidophilic granulocytes (+130%) compared with controls, although less pronounced. Light and electron microscopy observations detailed the occurrence of heterogeneous populations of lymphocytes and granulocytes in the developing intestinal mucosa, and highlighted the net expansion of G7(+) acidophilic granulocytes (A +536%, B +292% vs. control) due to probiotic administration. Evidence is provided that early feeding with probiotic-supplemented diet increased the number of Ig(+) cells and acidophilic granulocytes in seabream gut and that the effects were more pronounced when administration started during gut metamorphosis. These results point to a stimulatory effect of probiotics on the gut immune system that correlates with improvement of fry survival.

Use of a progestogen only preparation containing desogestrel in the treatment of recurrent pelvic pain after conservative surgery for endometriosis.

OBJECTIVE: To assess the effect of a new progestin progestogen only pill (desogestrel) versus an oral contraceptive in the treatment of recurrent endometriosis. STUDY DESIGN: A randomized prospective clinical study. A group of women with endometriosis (n=40) who showed recurrent dysmenorrhea and/or pelvic pain after conservative surgery, and did not desire a pregnancy. Continuous treatment for 6 months with desogestrel (75 microg/d) (n=20) versus a combined oral contraceptive (ethinyl estradiol 20 microg plus desogestrel 150 microg) (n=20) was performed. RESULTS: A significant improvement of both pelvic pain and dysmenorrhea was observed following each type of treatment (P<0.001). The use of desogestrel progestogen only pill was associated with a breakthrough bleeding in 20% patients, while a significant body weight increase was observed in 15% after oral contraceptive. CONCLUSIONS: Both desogestrel and an oral estro-progestinic were effective, safe and low cost therapy of pain symptoms after endoscopic surgery for endometriosis, the former showing an impact on breakthrough bleeding, the later an incidence on body weight increase.

Immunoglobulin protein and gene transcripts in sea bream (Sparus aurata L.) oocytes.

Cellular mechanisms of Ig transfer to Sparus aurata developing oocytes were analysed. Homologous serum Ig was purified and used to raise the rabbit polyclonal antiserum ORa. Immunohistochemistry revealed an active role of both follicular cells (already at a pre-vitellogenetic stage) and oocytes in the Ig uptake. Early vitellogenetic oocytes (lipid vesicle stages) had ORa staining of outer cortex and oolemma as well as of their follicular cells. In protein yolk granule oocytes, ORa staining was notably found in the pore canals crossing the egg envelope and at the periphery of yolk platelets. A transfer of Ig from the blood to the follicles appears likely. In addition, RT-PCR using specific primers for the constant region of sea bream Ig L chain detected Ig mRNA in released eggs and no signal in post-hatching larvae. These findings show that a significant level of Ig gene transcription in the oocyte and/or a transfer of transcripts may also occur.

The CD8alpha from sea bass (Dicentrarchus labrax L.): Cloning, expression and 3D modelling.

In this paper we describe the cloning, expression and structural study by modelling techniques of the CD8alpha from sea bass (Dicentrarchus labrax L.). The sea bass CD8alpha cDNA is comprised of 1490 bp and is translated in one reading frame to give a protein of 217 amino acids, with a predicted 26 amino acids signal peptide, a 88 bp 5'-UTR and a 748 bp 3'-UTR. A multiple alignment of CD8alpha from sea bass with other known CD8alpha sequences shows the conservation of most amino acid residues involved in the peculiar structural domains found within CD8alpha's. Cysteine residues that are involved in disulfide bonding to form the V domain are conserved. In contrast, an extra cysteine residue found in most mammals in this region is not present in sea bass. The transmembrane and cytoplasmic regions are the most conserved regions within the molecule in the alignment analysis. However, the motif (CXCP) that is thought to be responsible for binding p56lck is missing in the sea bass sequence. Phylogenetic analysis conducted using amino acid sequences showed that sea bass CD8alpha grouped with other known teleost sequences and that three different clusters were formed by the mammalian, avian and fish CD8alpha sequences. The thymus was the tissue with the highest CD8alpha expression, followed by gut, gills, peripheral blood leukocytes and spleen. Lower CD8alpha mRNA levels were found in head kidney, liver and brain. It was possible to create a partial 3D model using the human and mouse structures as template. The CD8alpha 11-120 amino acid region was taken into consideration and the best obtained 3D model shows the presence of ten beta-strands, involving about 50% of the sequence. The global structure was defined as an immunoglobulin-like beta-sandwich made of two anti-parallel sheets. Two cysteines were present in this region and they were at a suitable distance to form an S-S bond as seen in the template human and mouse structures.

Production and characterization of a continuous embryonic cell line from sea bass (Dicentrarchus labrax L.).

Continuous cell lines represent an important tool both for biological studies and for their applications in marine biotechnology. In this article we describe the production and characterization of a continuous adherent cell line, named DLEC, derived from early embryos of the European sea bass Dicentrarchus labrax L. (Actinopterygii, Moronidae). Cells were obtained by disrupting 2- to 12-hour-old embryos and culturing resulting cells at 18 degrees C in RPMI medium containing 5% fetal calf serum (FCS) and 10% supernatant fraction of the embryo homogenate. After 8 weeks culture medium was replaced with Liebovitz's L15 medium containing 10% FCS and DLEC cells started proliferation. Subsequently, they were continuously cultured until the 50th passage without evident changes in their morphology. DLEC cells show a fibroblast-like shape and a modal chromosome number of 48, as do the wild-type cells; conversely the constant presence of six to nine meta-submetacentric elements in the karyotype (vs. zero to two in the wild-type) indicates the occurrence of chromosomal rearrangements during stabilization. DLEC cells are sensitive to substances known to induce differentiation of mammalian cells such as retinoic acid and phorbol esters. They have been transfected using liposomes with a commercial plasmid vector containing a reporter gene, thus suggesting a possible importance as an alternative expression system of recombinant vertebrate proteins in teleost cells.

Biological activity of sea bass (Dicentrarchus labrax L.) recombinant interleukin-1beta.

Biological activities of a putative mature sea bass interleukin-1beta peptide, produced as a recombinant protein (rIL-1beta) in Escherichia coli, have been investigated. The rIL-1beta contains a 6-histidine tag at the N-terminus, and protein purification has been achieved through this tag by affinity chromatography. Biological activities have been investigated both at the cellular and gene expression levels. In in vitro assays sea bass rIL-1beta induced the proliferation of murine D10.G4.1 cells and increased yeast phagocytosis by sea bass head kidney leukocytes. The purified cytokine was also tested in a lymphocyte-activation factor assay, where it induced the proliferation of sea bass thymocytes. Finally, in an in vivo assay, rIL-1beta administered intraperitoneally increased expression levels of the IL-1beta gene and activated macrophages to produce a cyclooxygenase 2 homologue (COX-2) gene in the head kidney.

Yolk sac tumor in a young girl: a case report.

BACKGROUND: Yolk sac tumor is a rare neoplasm characterized by high malignancy given its premature metastasis, that is frequent in adolescence. CASE REPORT: A 21-year-old woman came to our observation for an ovarian cyst (13 cm in diameter). Following salpingo-oophorectomy, it was revealed as a yolk sac tumor by histological diagnosis. The patient exhibited a highly elevated level of alpha1-fetoprotein (AFP) (1156 UI/ml). She is now undergoing chemotherapy treatment. CONCLUSION: This is an interesting case of yolk sac tumor in a young girl, at an age typical for germ cell tumor. AFP represents a valid marker resulting in a useful diagnostic tool.

The treatment with a COX-2 specific inhibitor is effective in the management of pain related to endometriosis.

OBJECTIVE: To evaluate the efficacy and safety of a cyclooxygenase (COX)-2 specific inhibitors versus placebo in the treatment of endometriosis-associated pelvic pain. STUDY DESIGN: A group of women (n = 28) with pelvic pain after conservative surgery for symptomatic endometriosis (Stage I and II) were enrolled at the Department of Pediatric, Obstetrics and Reproductive Medicine of University of Siena. A treatment with a COX-2 specific inhibitors (rofecoxib, 25mg per day) (n = 16) or placebo (n = 12) was given for 6 months. Pelvic pain quantification with a clinical evaluation, including Visual Analogue Scale (VAS) for pain, was performed before and up to 6 months after treatment. RESULTS: A significant improvement of both pelvic pain and dyspareunia was observed after a 6 months persisting since the end of the treatment (P < 0.0001). The efficacy of rofecoxib was higher than placebo and no recurrence occurred, while in the placebo-treatment a 16% (2/12) occurred. No significant side effects have been found with the use of rofecoxib. CONCLUSIONS: The use of COX-2 specific inhibitors was effective, safe and low cost therapy in the management of pelvic pain associated to endometriosis and might be also proposed in early stage of endometriosis.

Immunoglobulin protein and gene transcripts in ovarian follicles throughout oogenesis in the teleost Dicentrarchus labrax.

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.

Expression in Escherchia coli and purification of sea bass (Dicentrarchus labrax) interleukin 1beta, a possible immunoadjuvant in aquaculture.

Interleukin 1beta (IL-1beta) is a pleiotropic cytokine that plays a pivotal role in regulating immune responses. Our group has recently cloned IL-1beta from sea bass (Dicentrarchus labrax), one of the main Mediterranean aquacultured fish species. The cDNA is 1292 bp and codes for a deduced peptide of 29.4 kDa with a pI of 5.1. As for trout and carp IL-1beta precursor sequence, no candidate cut site for ICE (IL-1beta converting enzyme) enzyme was apparent in the alignments of sea bass IL-1beta with other mammalian IL-1betas. Nevertheless, a possible mature peptide could start at Ala86, giving a protein of 176 amino acids. The nucleotide sequence coding for this polypeptide was cloned into a pQE-30 expression vector. The plasmid was then transformed in Escherichia coli, and the recombinant protein was purified. Finally, we demonstrated that this purified recombinant IL-1beta was able to induce IL-1beta gene expression in a dose-dependent manner on cells purified from sea bass head kidney and could have immunoadjuvant effects in sea bass vaccination experiments.

Vitellin cleavage products are proteolytically degraded by ubiquitination in stick insect embryos.

Vitellin polypeptides are proteolytically processed in ovarian follicles and embryos of the stick insect Carausius morosus. Data show that vitellin polypeptide A(3) of 54kDa is processed to yield polypeptide A(3)(*) of about 48kDa upon completion of ovarian development, whereas vitellin polypeptide A(2) of 90kDa yields polypeptide E(9) during embryonic development. As vitellin polypeptides are processed, polypeptides A(3)(*) and E(9) are transferred from the yolk granules to the cytosolic space of the vitellophages and start to express a ubiquitin reactivity. At the confocal microscope, anti-ubiquitin antibodies label specifically numerous small yolk granules and the cytosolic space of vitellophages. During embryonic development, ubiquitin carrying granules undergo acidification in much the same way as larger yolk granules. However, only these latter organelles are capable of converting a latent cysteine pro-protease into an active yolk protease upon acidification of their luminal space. These data are interpreted as indicating that ubiquitin-like polypeptides are restricted to small granules throughout ovarian and embryonic development, and that vitellin cleavage products are ubiquitinated following acidification of large yolk granules and transfer to the cytosolic space of the vitellophages.

Morphological study of the larval spiracular system in eight Lutzomyia species (Diptera: Psychodidae)

The morphology of the spiracles of fourth instar larva in eight sandfly species were examined by light and scanning electron microscopy. Species studied were: Lutzomyia longipalpis (Lutz & Neiva) L. ovallesi (Ortiz), L. youngi Feliciangeli & Murillo, L. evansi (Nuñez-Tovar), L. trinidadensis (Newstead), L. migonei (França), L. absonodonta Feliciangeli, and L. venezuelensis (Floch & Abonnenc). In larvae of all eight species both thoracic and abdominal spiracles are located at the top of a globular bulge. Their structure consists of a spiracular plate with a sclerotized central portion and a rose-like peripheral portion. The latter has circularly arranged papillae, separated from each other by elongated septa. Each papilla is longitudinally crossed by a fine cleft dividing it into two identical parts. The taxonomic and adaptative value of spiracular morphology is discussed.

An Ultrastructural Investigation on Vitellophage Invasion of the Yolk Mass during and after Germ Band Formation in Embryos of the Stick Insect Carausius morosus Br.: (embryogenesis/yolk/vitellophage/Carausius morosus/scanning and transmission electron microscopy).

Developing embryos of the stick insect Carausius morosus were examined ultrastructurally with a view to studying vitellophage invasion of the yolk mass during and after germ band formation. Newly laid eggs in C.morosus have a unique yolk fluid compartment surrounded by a narrow fringe of cytoplasm comprising several small yolk granules. Vitellophages originate mainly from a thin layer of stem cells, the so-called yolk cell membrane, interposed between the germ band and the yolk mass. Throughout development, a thin basal lamina separates the yolk cell membrane from the overlying embryo. Vitellophages extend from the yolk cell membrane with long cytoplasmic processes or filopodia to invade the central yolk mass. Along their route of entrance, filopodia engulf portions of the yolk mass and sequester it into membrane-bounded granules. As this process continues, the yolk mass is gradually partitioned into a number of yolk granules inside the vitellophages. Later in development, the yolk cell membrane is gradually replaced by the endodermal cells that emerge from the anterior and posterior embryonic rudiments. From this stage of development onwards, vitellophages remain attached to the basal lamina through long filopodia extending between the endodermal cells. Yolk confined in different vitellophagic cells appears heterogeneous both in density and texture, suggesting that yolk degradation may be spatially differentiated.