Search

The CD3 coreceptor is a master T cell surface marker, and genes encoding CD3ζ, γδ, and e chains have been reported in several teleost fish. Here, a complete cDNA sequence of CD3ɛ chain was identified from a sea bass (Dicentrarchus labrax L.) gill transcriptome. Its basal expression was quantified in both lymphoid and non-lymphoid organs of sea bass juveniles with real-time qPCR analysis. After either in vitro stimulation of head kidney leukocytes with the T-cell mitogen phytohaemagglutinin or in vivo stimulation with an orally administered Vibrio anguillarum vaccine, CD3e expression levels increased in head kidney leukocytes, confirming that CD3e T cells may play important roles in fish systemic protection against pathogens. Further, three peptides were designed on the CD3ɛ cytoplasmic tail region and employed as immunogens for antibody production in rabbit. One antiserum so obtained, named RACD3/1, immunostained a band of the expected size in a western blot of a sea bass thymocyte lysate. The distribution of CD3e+ lymphocyte population in the lymphoid organs and mucosal tissues was addressed in healthy fish by IHC. In decreasing percentage order, CD3e+ lymphocytes were detected by flow cytometry in thymus, peripheral blood leukocytes, gills, head kidney, gut, and spleen. Finally, a significant in vivo enhancement of CD3e+ T intestinal lymphocytes was found in fish fed on diets in which 100% fish meal was replaced by the microalgae Nannochloropsis sp. biomass. These results indicate that CD3e+ T cells are involved in nutritional immune responses.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive disease with an overall 5-year survival rate of just 5%. A better understanding of the carcinogenesis processes and the mechanisms of the progression of PDAC is mandatory. Fifty-two PDAC patients treated with surgery and adjuvant therapy, with available primary tumors, normal tissue, preneoplastic lesions (PanIN), and/or lymph node metastases, were selected for the study. Proteins were extracted from small punches and analyzed by LC-MS/MS using data-independent acquisition. Proteomics data were analyzed using probabilistic graphical models, allowing functional characterization. Comparisons between groups were made using linear mixed models. Three proteomic tumor subtypes were defined. T1 (32% of patients) was related to adhesion, T2 (34%) had metabolic features, and T3 (34%) presented high splicing and nucleoplasm activity. These proteomics subtypes were validated in the PDAC TCGA cohort. Relevant biological processes related to carcinogenesis and tumor progression were studied in each subtype. Carcinogenesis in the T1 subtype seems to be related to an increase of adhesion and complement activation node activity, whereas tumor progression seems to be related to nucleoplasm and translation nodes. Regarding the T2 subtype, it seems that metabolism and, especially, mitochondria act as the motor of cancer development. T3 analyses point out that nucleoplasm, mitochondria and metabolism, and extracellular matrix nodes could be involved in T3 tumor carcinogenesis. The identified processes were different among proteomics subtypes, suggesting that the molecular motor of the disease is different in each subtype. These differences can have implications for the development of future tailored therapeutic approaches for each PDAC proteomics subtype., Cancers, 14 (10), ISSN:2072-6694

In jawed vertebrates, adaptive immune responses are enabled by T cells. Two lineages were characterized based on their T cell receptor (TcR) heterodimers, namely αβ or γδ peptide chains, which display an Ig domain-type sequence that is somatically rearranged. γδ T cells have been less extensively characterized than αβ and teleost fish, in particular, suffer from a severe scarcity of data. In this paper, we worked on the well-known model, the European sea bassDicentrarchus labrax,to broaden the understanding of teleost γδ-T cells. The T cell receptor chain (TR) γ transcript was expressed at a later developmental stage than TRβ, suggesting a layered appearance of fish immune cells, and the thymus displayed statistically-significant higher mRNA levels than any other organ or lymphoid tissue investigated. The polyclonal antibody developed against the TRγ allowed the localization of TRγ-expressing cells in lymphoid organs along the ontogeny. Cell positivity was investigated through flow cytometry and the highest percentage was found in peripheral blood leukocytes, followed by thymus, gut, gills, spleen and head kidney. Numerous TRγ-expressing cells were localized in the gut mucosa, and the immunogold labelling revealed ultrastructural features that are typical of T cells. At last, microalgae-based diet formulations significantly modulated the abundance of TRγ+cells in the posterior intestine, hinting at a putative involvement in nutritional immunity. From a comparative immunological perspective, our results contribute to the comprehension of the diversity and functionalities of γδ T cells during the development of a commercially relevant marine teleost model.

This article belongs to the Special Issue Stemness and Differentiation in Cancer., Colon and rectal tumors, often referred to as colorectal cancer, show different gene expression patterns in studies that analyze whole tissue biopsies containing a mix of tumor and non-tumor cells. To better characterize colon and rectal tumors, we investigated the gene expression profile of organoids generated from endoscopic biopsies of rectal tumors and adjacent normal colon and rectum mucosa from therapy-naive rectal cancer patients. We also studied the effect of vitamin D on these organoid types. Gene profiling was performed by RNA-sequencing. Organoids from a normal colon and rectum had a shared gene expression profile that profoundly differed from that of rectal tumor organoids. We identified a group of genes of the biosynthetic machinery as rectal tumor organoid-specific, including those encoding the RNA polymerase II subunits POLR2H and POLR2J. The active vitamin D metabolite 1α,25-dihydroxyvitamin D3/calcitriol upregulated stemness-related genes (LGR5, LRIG1, SMOC2, and MSI1) in normal rectum organoids, while it downregulated differentiation marker genes (TFF2 and MUC2). Normal colon and rectum organoids share similar gene expression patterns and respond similarly to calcitriol. Rectal tumor organoids display distinct and heterogeneous gene expression profiles, with differences with respect to those of colon tumor organoids, and respond differently to calcitriol than normal rectum organoids., This research was founded by Agencia Estatal de Investigación (PID2019-104867RB-I00/AEI/10.13039/501100011033), Agencia Estatal de Investigación-Fondo Europeo de Desarrollo Regional (SAF2016-76377-R, MINECO/AEI/FEDER, UE), Ministerio de Economía y Competitividad (SAF2017-90604-REDT), Instituto de Salud Carlos III—Fondo Europeo de Desarrollo Regional (CIBERONC, CB16/12/00273; CIBERES, CB15/00037), and Mutua Madrileña.

Cigarette smoking is associated with increased risk for all histologic types of lung cancer, but why the strength of this association is stronger for squamous cell carcinoma than adenocarcinoma of the lung (SQC-L, ADC-L) is not fully understood. Because nicotine and tobacco-specific nitrosamines contribute to carcinogenesis by activating nicotinic acetylcholine receptors (nAChRs) on lung tumors and epithelial cells, we investigated whether differential expression of nAChR subtypes in these tumors could explain their different association with smoking. Expression of nAChR subunit genes in paired tumor and non-tumor lung specimens from 40 SQC-L and 38 ADC-L patients was analyzed by quantitative PCR. Compared to normal lung, both tumors share: i) transcriptional dysregulation of CHRNA3/CHRNA5/CHRNB4 (α3, α5, β4 subunits) at the chromosomal locus that predisposes to lung cancer; and ii) decreased expression of CHRFAM7A (dupα7 subunit); this last subunit negatively modulates α7-nAChR activity in oocytes. In contrast, CHRNA7 (α7 subunit) expression was increased in SQC-L, particularly in smokers and non-survivors, while CHRNA4 (α4 subunit) expression was decreased in ADC-L. Thus, over-representation of cancer-stimulating α7-nAChR in SQC-L, also potentiated by smoking, and underrepresentation of cancer-inhibiting α4β2-nAChR in ADC-L could explain the different tobacco influences on the tumorigenic process in each cancer type, This study was supported by grants to C. Montiel and F. Arnalich from the Ministry of Economy, Industry and Competitiveness, Government of Spain (SAF2014-56623-R) and Foundation “Mutua Madrileña Investigación Biomédica” (FMM2011), Spain. A.B. is recipient of a fellowship (Beca FPI, Universidad Autónoma Madrid). J.L.C. and C.M.S. are recipients of fellowships (Beca FPU from Ministerio de Educación, Cultura y Deporte and Beca FPI from Ministry of Economy, Industry and Competitiveness, Government of Spain, respectively)

Immunoglobulin T (IgT) is one of the key effector molecules of jawed vertebrate's adaptive immune system, and in this work we describe the quantitative distribution of IgT-expressing and IgT-producing cells in tissues of the European seabass Dicentrarchus labrax by using mRNA riboprobes and a specific anti-IgT antibody. A polyclonal antiserum (pAb) was prepared by immunizing rabbits with three synthetic peptides deduced from the full length IgT cDNA sequence and located in a surface-exposed CH3 domain of IgT constant region. The obtained antiserum, named RAIgT1, was able to recognize by ELISA immunization antigens and IgT from intestinal mucus and serum. In western blots of head kidney leukocytes lysates the antiserum recognized a 180 kDa polypeptide in non-reducing, and a 75 kDa peptide in reducing conditions. Interestingly, the RAIgT1 pAb crossreacted intensely in western blots with rainbow trout IgT purified from mucus and serum. Antisense mRNA IgT oligonucleotide sequences were employed in in situ hybridization to detect IgT-expressing cells in sections from lymphoid tissues, and positive cells were observed in head kidney, spleen, intestine and gills. By employing RAIgT1 in quantitative immunohistochemistry, the highest number of IgT-producing cells was observed in the gills (9.5 ± 0.7%), followed by intestine (8.4 ± 1.2%), head kidney (6.2 ± 1.4%), and spleen (4.1 ± 0.7%). Interestingly, the number of IgT-B cells showed a regionalization in the intestine, increasing from the proximal to the terminal part. By immunofluorescence and flow cytometry of live leukocytes, the percentages of RAIgT1 stained cells were 34 ± 11% in the intestine, 22 ± 5% in head kidney, 16 ± 7% in spleen, and 9 ± 5% in gills. At the fluorescence microscope, live cells from these tissues showed a typical membrane-associated positivity and a lymphocytic morphology, and no IgT/IgM double positive cells were detected. Immunoreactive cells have been purified from head kidney using magnetic beads, and IgT-enriched cells showed by RT-PCR an enhanced expression of the IgT gene, whereas IgT-depleted cells had an highest expression of IgM and TRβ genes. These data describe for the first time a quantitative panel of IgT-expressing and IgT-immunoreactive cells in tissues of a teleost fish species.

The secretory region of the salivary glands in Glossina pallidipes Austen (Diptera: Glossinidae) is characterized by an external muscle layer. Scanning electron microscopy and transmission electron microscopy investigations provide a detailed description of the longitudinal muscle fibres and a comparison of their structure when affected by salivary gland hypertrophy virus. The virus is responsible for hypertrophy of the salivary glands in symptomatic flies, specifically of the muscle fibres, the cytoarchitecture of which is completely altered. Although observations did not reveal viral particles in the muscle cells of either asymptomatic or symptomatic flies, muscle fibres were enlarged and detached from one another and their associated basement membrane only in symptomatic flies. A decrease in type IV collagen labelling in the basement membrane of the muscles in symptomatic flies is reported and is considered a potential cause of the salivary gland muscle alteration and, possibly, myopathy. The maintenance of an organized muscular layer is essential for the normal secretion of saliva and hence its pathology in symptomatic tsetse flies could affect the normal transmission of the trypanosome that develops inside the salivary gland epithelium. Therefore, a better understanding of the possible role of the virus is essential in order to elucidate its impact on salivary deployment in symptomatic flies.

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.

Background The analysis of tumour-infiltrating immune cells within patients’ tumour samples in colorectal cancer (CRC) has become an independent predictor of patient survival. The tumour microenvironment and the immune checkpoints, such as PD-L1/PD-1, are relevant to the prognoses and also appear to be relevant for further CRC therapies. Methods We analysed the presence and features of the infiltrated monocyte/macrophage and lymphocyte populations in both tumour and peritumour samples from patients with CRC (n = 15). Results We detected a large number of CD14+ monocytes/macrophages with an alternative phenotype (CD64+CD163+) and CD4+ lymphocytes that infiltrated the tumour, but not the peritumour area. The monocytes/macrophages expressed PD-L1, whereas the lymphocytes were PD-1+; however, we did not find high PD-L1 levels in the tumour cells. Coculture of circulating naïve human monocytes/macrophages and lymphocytes with tumour cells from patients with proficient mismatch repair CRC induced both an alternative phenotype with higher expression of PD-L1 in CD14+ cells and the T-cell exhaustion phenomenon. The addition of an α-PD-1 antibody restored lymphocyte proliferation. Conclusion These results emphasise the interesting nature of immune checkpoint shifting therapies, which have potential clinical applications in the context of colorectal cancer.

Metabolic alterations encountered in tumors are well recognized and considered as a hallmark of cancer. In addition to Warburg Effect, epidemiological and experimental studies support the crucial role of lipid metabolism in colorectal cancer (CRC). The overexpression of four lipid metabolism-related genes (ABCA1, ACSL1, AGPAT1 and SCD genes) has been proposed as prognostic marker of stage II CRC (ColoLipidGene signature). In order to explore in depth the transcriptomic and genomic scenarios of ABCA1, ACSL1, AGPAT1 and SCD genes, we performed a transcriptomic meta-analysis in more than one thousand CRC individuals. Additionally we analyzed their genomic coding sequence in 95 patients, to find variants that could orchestrate CRC prognosis. We found that genetic variant rs3071, located on SCD gene, defines a 9.77% of stage II CRC patients with high risk of death. Moreover, individuals with upregulation of ABCA1 and AGPAT1 expression have an increased risk of CRC recurrence, independently of tumor stage. ABCA1 emerges as one of the main contributors to signature's prognostic effect. Indeed, both high ABCA1 expression and presence of tumoral genetic variants located in ABCA1 coding region, seem to be associated with CRC risk of death. In addition the non-synonymous polymorphism rs2230808, located on ABCA1, is associated with gene expression. Patients carrying at least one copy of minor allele showed higher levels of ABCA1 expression than patients carrying homozygous major allele. This study broaden the prognostic value of ABCA1, ACSL1, AGPAT1 and SCD genes, independently of CRC tumor stage, leading to future precision medicine approaches and "omics"-guided therapies., Ministerio de Economía y Competitividad del Gobierno de España (MINECO, Plan Nacional I+D+i AGL2016-76736-C3), Gobierno regional de la Comunidad de Madrid (P2013/ABI-2728, ALIBIRD-CM) and EU Structural Funds.

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.

In jawed vertebrates, the crosstalk between immune and endocrine system as well as many fundamental mechanisms of T cell development are evolutionary conserved. Oestrogens affect mammalian thymic function and plasticity, but the mechanisms of action and the oestrogen receptors involved remain unclear. To corroborate the oestrogenic regulation of thymic function in teleosts and to identify the implicated oestrogen receptor subtypes, we examined the distribution of nuclear and membrane oestrogen receptors within the thymus of the European Sea bass, Dicentrarchus labrax, in relation to its morpho-functional organisation. Immunohistological analysis specified thymus histology and organisation in teleosts and described, for the first time, Hassall's corpuscle like structures in the medulla of sea bass. All oestrogen receptors were expressed at the transcript and protein level, both in T cells and in stromal cells belonging to specific functional areas. These observations suggest complex regulatory actions of oestrogen on thymic function, notably through the stromal microenvironment, comprising both, genomic and non-genomic pathways that are likely to affect T cell maturation and trafficking processes. Comparison with birds, rodents and humans supports the thymic localization of oestrogen receptors and suggests that oestrogens modulate T cell maturation in all gnathostomes. (C) 2017 Elsevier Ltd. All rights reserved. ANR [ANR-15-CE32-0014] FR CNRS 3730 SCALE info:eu-repo/semantics/publishedVersion

Background: Immunoglobulins (Igs) are fundamental components of the adaptive immune system of vertebrates, with the IgT/IgZ isotype specific of Teleosts. In this paper we describe the identification of an IgT heavy chain from the European sea bass (Dicentrarchus labrax L.), its molecular characterization and tissue mRNA localization by in situ hybridization.Results: Sea bass IgT consists of 552 aa (Accession Number KM410929) and it contains a putative 19 amino acids long signal peptide and one potential N-glycosylation site. The C-region consists of four CH domains; each contains the cysteine and tryptophan residues required for their correct folding. Based on the recent sequencing of sea bass genome, we have identified five different genomic contigs bearing exons unequivocally pertaining to IgT (CH2, CH3 and CH4), but none corresponded to a complete IgH locus as IgT sequences were found in the highly fragmented assembled genomic regions which could not be assigned to any major scaffold. The 3D structure of sea bass IgT has been modelled using the crystal structure of a mouse Ig gamma as a template, thus showing that the amino acid sequence is suitable for the expected topology referred to an immunoglobulin-like architecture. The basal expression of sea bass IgT and IgM in different organs has been analysed: gut and gills, important mucosal organs, showed high IgT transcripts levels and this was the first indication of the possible involvement of sea bass IgT in mucosal immune responses. Moreover, sea bass IgT expression increased in gills and spleen after infection with nodavirus, highlighting the importance of IgT in sea bass immune responses. In situ hybridization confirmed the presence of IgT transcripts in the gut and it revealed a differential expression along the intestinal tract, with a major expression in the posterior intestine, suggesting the hindgut as a site for the recruitment of IgT+ cells in this species. IgT transcripts were also found in gill filaments and parallel lamellae and, for the first time, we identified scattered IgT positive cells in the liver, with a strong signal in the hepatic parenchyma.Conclusions: In conclusion, we performed a full molecular characterization of IgT in sea bass that points out its possible involvement in mucosal immune responses of this species.

Cellular and molecular data have evidenced a gut-associated lymphoid tissue in a variety of teleost species, abundantly containing T cells, whose origin, selection and functions are still unclear. This study reports CD4, CD8-α, MHCI-α, MHCII-β, rag-1 and TCR-β gene transcription along the intestine (anterior, middle and posterior segments) and in the thymus of one year-old Dicentrarchus labrax (L.). Real-time PCR findings depicted a main role of the thymus in T cell development, but also rag-1 and CD8-α transcripts are detected in the intestine, having significant expression in the posterior segment. In the whole intestine TCR-β and CD8-α exceeded CD4 transcripts. RNA ISH confirmed these data and detailed that mucosal CD8-α+ cells were especially numerous in the epithelium and in aggregates in the lamina propria. Regional differences in T cell-specific gene expressions are first described in the intestine of a bony fish. High non-specific cytotoxic activity against xenogeneic and allogeneic cells was found in lymphocytes purified from the intestinal mucosa, providing further insight into their local defence roles. L'articolo è disponibile sul sito dell'editore http://www.sciencedirect.com/

The crop of adult Phormia regina consists of a duct that diverges from the esophagus, just in front of the cardia, and extends ventrally and posteriorly into the thorax and abdomen where it forms a bilobed sac. Flattened epithelial cells produce the cuticular lining of the crop. When empty, or partially full, the epithelial cells and cuticular lining form folds extending into the lumen, thus providing for expansion as the crop sac fills. Covering the sac on the hemolymph side is a layer of anastomosed, intrinsic muscles connected to one another by intercellular cytoplasmic bridges. Mitochondria are located at the periphery of the sarcomere. Also inside the sarcomere are glycogen, sarcoplasmic reticula, and transverse tubular systems (T-system). I, A, and Z-bands are present and the Z-bands are not in register making the muscle-type supercontractile. Important structures, not previously researched and associated with the crop muscles, are the crop nerves. Coming off the corpora cardiaca, and running down each side of the crop duct, is a pair of nerves, each housing several axons. These nerves extend to and branch over the crop sac. Here they penetrate the muscle mass and form neuromuscular junctions where electron-dense droplets of neurosecretion are released. Based on the literature, and research in our laboratory, it has now been shown that these nerves carry adipokinetic hormone, Drosophila insulin-like peptide, and a dromyosuppressin-like neuropeptide.

Using a serotonin antibody and confocal microscopy, this study reports for the first time direct serotonergic innervation of the muscle sheath covering the secretory region of the salivary glands of adult tsetse fly, Glossina pallidipes Austen. Reports to date, however, note that up until this finding, dipteran species previously studied lack a muscle sheath covering of the secretory region of the salivary glands. Direct innervation of the salivary gland muscle sheath of tsetse would facilitate rapid deployment of saliva into the host, thus delaying a host response. Our results also suggest that the neuronal and abnormal pattern seen in viral infected glands by the Glossina pallidipes salivary gland hypertrophy virus (GpSGHV) is due to a compensatory increased branching of the neurons of the salivary glands, which is associated with the increased size of the salivary glands in viral infected flies. This study shows for the first time serotonin in the cell bodies of the brain and thoracico-abdominal ganglion in adult tsetse, G. pallidipes Austen (Diptera: Glossinidae). A hypothesis is proposed as to whether innervation of the muscle sheath covering of the secretory region of the salivary glands is present in brachyceran compared with nematoceran dipterans; and, a plea is made that more research is needed to develop a blood feeding model, similar to that in the blow flies, for elucidating the various mechanisms involved in production and deployment of saliva.

Climacostol, a compound produced by the ciliated protozoan Climacostomum virens, displayed cytotoxic properties in vitro. This study demonstrates that it has anti-tumour potential. Climacostol caused a reduction of viability/proliferation of B16-F10 mouse melanoma cells, a rapidly occurring DNA damage, and induced the intrinsic apoptotic pathway characterised by the dissipation of the mitochondrial membrane potential, the translocation of Bax to the mitochondria, the release of Cytochrome c from the mitochondria, and the activation of Caspase 9-dependent cleavage of Caspase 3. The apoptotic mechanism of climacostol was found to rely on the up-regulation of p53 and its targets Noxa and Puma. In vivo analysis of B16-F10 allografts revealed a persistent inhibition of tumour growth rate when melanomas were treated with intra-tumoural injections of climacostol. In addition, it significantly improved the survival of transplanted mice, decreased tumour weight, induced a remarkable reduction of viable cells inside the tumour, activated apoptosis and up-regulated the p53 signalling network. Importantly, climacostol toxicity was more selective against tumour than non-tumour cells. The anti-tumour properties of climacostol and the molecular events associated with its action indicate that it is a powerful agent that may be considered for the design of pro-apoptotic drugs for melanoma therapy.

MHC II-β chain gene transcripts were quantified by real-time PCR and localised by in situ hybridization in the developing thymus of the teleost Dicentrarchus labrax, regarding the specialization of the thymic compartments. MHC II-β expression significantly rose when the first lymphoid colonization of the thymus occurred, thereafter increased further when the organ progressively developed cortex and medulla regions. The evolving patterns of MHC II-β expression provided anatomical insights into some mechanisms of thymocyte selection. Among the stromal cells transcribing MHC II-β, scattered cortical epithelial cells appeared likely involved in the positive selection, while those abundant in the cortico-medullary border and medulla in the negative selection. These latter most represent dendritic cells, based on typical localization and phenotype. These findings provide further proofs that efficient mechanisms leading to maturation of naïve T cells are operative in teleosts, strongly reminiscent of the models conserved in more evolved gnathostomes.

To investigate mucosal immunity, from a whole transcriptome of sea bass gills we identified and successively cloned complete cDNA sequences of IgT and IgD heavy chains. These sequences have been employed to prepare riboprobes for ISH, and to identify conserved regions of sea bass IgT/IgD in order to select sequences from which synthetic peptides have been prepared. The peptides have been used as immunogens in rabbits to obtain antisera against IgT (RaIgT1) and IgD (RaIgD1) and by employing either riboprobes and antisera, the pattern of IgT/IgD expressing tissues and the presence of IgT/IgD immunoreactive cells has been investigated. The results by qPCR showed that the IgT gene is mainly expressed in muscle, intestine and gills, and the IgD gene in head kidney. By ISH, the presence of IgT-expressing cells has been detected in the intestine, with their number increasing toward the posterior segment, and in the gills. These data have been confirmed by IHC employing the rabbit antiserum RaIgT1, and extended by revealing the presence of IgTimmunoreactive cells in head kidney and spleen, as well as intense reactivity in intestinal mucus. As expected, in mucosal tissues IgT-immunoreactive cells outnumbered IgM-immunoreactive cells. By IIF and flow cytometry with RaIgT1 we determined the number of IgT-positive cells in organs and tissues, being around 32 ± 12 % in head kidney leukocytes, 14 ± 6 % in GIALT and 23 ± 10 % in spleen leukocytes. At the microscope the IIF-positive cells had the morphology of lymphocytes. By western blotting of head kidney lysates, RaIgT1 stained a 180 kDa band in non-reducing gels and a 74 kDa band in reducing gels and, in addition, RaIgT1 showed cross-reaction with partially purified rainbow trout IgT. Leukocytes from the head kidney were enriched in RaIgT1-positive cells by magnetic sorting, and PCR analysis showed an enrichment of IgT and depletion of IgM expression, respectively, in RaIgT1-enriched cells. These data suggest we obtained an antibody against IgT cells of sea bass. Preliminary data employing the RaIgD1 showed by IHC the presence of immunoreactive cells in gills, spleen, head kidney, intestine, and work is in progress to highlights the characteristics of sea bass IgD expressing cells. Acknowledgements. The work was financially supported by EU-6FP project Targetfish, n°311993.

Light, scanning electron, and transmission electron microscopy analyses were conducted to examine the morphology and ultrastructure of the salivary glands of Glossina pallidipes. Three distinct regions, each with a characteristic composition and organization of tissues and cells, were identified: secretory, reabsorptive and proximal. When infected with the salivary gland hypertrophy (SGH) virus, glands showed a severe hypertrophy, accompanied by profound changes in their morphology and ultrastructure. In addition, the muscular fibers surrounding the secretory region of the glands were disrupted. The morphological alterations in the muscular tissue, caused by viral infection, could be an important aspect of the pathology and may shed light on the mode of action of the SGH virus. Results were discussed with regard to the potential effect of viral infection on normal salivation and on the ability of infected tsetse flies to transmit a trypanosome parasite.

CD4 is a transmembrane glycoprotein fundamental for cell-mediated immunity. Its action as a T cell coreceptor increases the avidity of association between a T cell and an antigen-presenting cell by interacting with portions of the complex between MHC class II and TR molecules. In this paper we report the cDNA cloning, expression and structural analysis of a CD4 homologue from sea bass (Dicentrarchus labrax). The sea bass CD4 cDNA consists of 2071 bp that translates in one reading frame to give the entire molecule containing 480 amino acids. The analysis of the sequence shows the presence of four putative Ig-like domains and that some fundamental structural features, like a disulphide bond in domain D2 and the CXC signalling motif in the cytoplasmic tail, are conserved from sea bass to mammals. Real-time PCR analysis showed that very high levels of CD4 mRNA transcripts are present in thymus, followed by gut and gills. In vitro stimulation of head kidney leukocytes with LPS and PHA-L gave an increase of CD4 mRNA levels after 4 h and a decrease after 24 h. Homology modelling has been applied to create a 3D model of sea bass CD4 and to investigate its interaction with sea bass MHC-II. The analysis of the 3D complex between sea bass CD4 and sea bass MHC-II suggests that the absence of a disulfide bond in the CD4 D1 domain could make this molecule more flexible, inducing a different conformation and affecting the binding and the way of interaction between CD4 and MHC-II. Our results will add new insights into the sea bass T cell immune responses and will help in the identification of T cell subsets in teleost fishes to better understand the evolution of cell-mediated immunity from fish to mammals. L'articolo è disponibile sul sito dell'editore http://www.sciencedirect.com/

Leptoconops spp. are small midges, members of the family Ceratopogonidae, that are relatively widespread in wetlands with sandy or silty-clay soils, including many tourist sites. Although very few of the species are proven vectors of pathogens, the blood-feeding females attack mammals, including humans, in large swarms and their painful bites may cause severe reactions, especially in children. Although Leptoconops spp. may limit the socio-economic development of an area, there are currently no effective methods for the control of their natural populations, and the repellents and netting used against mosquitoes are ineffective against these midges. The diurnal control of the adults with pesticides may have unacceptable impacts on non-target species, including humans, and be ineffective because the adult females can easily be carried, from untreated areas to treated, on the wind. In the present study, the use of a diflubenzuron-based insecticide against the larvae of Leptoconops (Holoconops) kerteszi Kieffer, 1908 - a sand-reproducing species that is widespread in certain coastal areas of the Italian province of Grosseto - was explored. In Grosseto, in summer, attacks by swarms of adult L. kerteszi create problems for the local people and the many tourists. The encouraging results of preliminary tests are discussed in relation to the potential use of diflubenzuron for the integrated control of L. kerteszi populations.

Contrary to what was assumed regarding the presence of respiratory proteins in insects, a functional hemocyanin was recently found in larvae and adults of the stoneflies species Perla marginata, whereas in the close species Perla grandis, hemocyanin functionality was deduced from sequence data. In order to verify if the presence of this ancient trait is widespread within the order and to investigate why stoneflies have maintained it, we have extended the search for hemocyanin to species of other Plecoptera families. In particular, we assessed the presence of hemocyanin in the larval stage of nine Plecoptera species, belonging to six of the seven families of the European stonefly-fauna, and analyzed its potential functionality as deduced by sequence data. We cloned and sequenced the corresponding cDNAs and studied their expression with RT-PCR technique. Moreover, we performed homology studies using the deduced amino acid sequences. On the basis of our analysis, we hypothesized a functional role of the hemocyanin only for two species: Dinocras cephalotes and Isoperla grammatica (Perloidea). In all the investigated Nemouroidea and in Siphonoperla torrentium (Perloidea), this protein may have been lost. Larval size, life-cycle length, trophic role and environmental induction are discussed as possible explanations of these different physiological requirements.

Different developmental stages (from eggs to 1-year-old juveniles) of the teleost fish Dicentrarchus labrax (L.) were assayed for CD4 gene expression. RT-PCR revealed the appearance of CD4 transcripts in postlarvae from 51 days post-hatching (dph). This finding overlaps the first detection of CD8-a mRNA. Real-time PCR with specific primers quantified CD4, CD8-a and TCR-b transcripts in larvae and postlarvae (25, 51, 75 and 92 dph) and 1-year-old thymus. At 92 dph, TcR-b and CD8-a transcripts were significantly higher (P < 0.001) than in previous stages, as CD4 transcripts compared with 51 dph (P < 0.01). High levels of TCR-b and CD8-a transcripts were found in the thymus, while CD4 transcripts were lower (P < 0.05 vs. TCR-b). In situ hybridization identified CD4 mRNAs at 51 dph, localized in thymocytes of the outer and lateral zones of the thymic glands. From 75 dph on the signal was mainly detected in the outer region, drawing a cortex–medulla demarcation. Developmental expression of CD4 and CD8-a almost coincided. In each adult thymic lobe CD4þ and CD8-aþ thymocytes filled the cortex. The expression patterns of CD4 and CD8-a largely overlap, except in the medulla, where CD4þ thymocytes were isolated, while CD8-aþ ones mainly arranged in cords. These results provide new information about the thymic compartmentalization and lymphocyte differentiation pathways in a teleost, almost demonstrating that double negative thymocytes fill the cortex giving rise to further selection in the medulla. L'articolo è disponibile sul sito dell'editore http://www.sciencedirect.com/

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 b and CD8a. RT-PCR of RNA extracted from larvae revealed TCRb transcripts from day 25 posthatching (ph) and CD8a transcripts from 26 days later. At day 51 ph, CD8a and TCRb 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, CD8a+ and TCRb+ thymocytes almost filled the cortex and extended in large cords in the medulla. A CD8a TCRb+ subcapsular lymphoid zone was evident near the septa coming from the inner connective capsule that delimited the thymus. The localisation of CD8a and TCRb transcripts demonstrated a compartmentalisation of the juvenile thymus due to distinct localisation of thymocytes at different developmental stages. L'articolo è disponibile sul sito dell'editore http://www.sciencedirect.com/

In female phlebotomine sandflies, little is known about the reproductive accessory glands that presumably contribute to egg production and/or oviposition. The main protein secreted in the accessory glands of female Phlebotomus papatasi was recently characterised as a lipase-like protein, the first to be found in the female accessory glands of any insect. This protein, named PhpaLIP (for Phlebotomus papatasi lipase), has now been detected and localized in the reproductive tissues of female P. papatasi, at different stages of the gonotrophic cycle, using a polyclonal anti-PhpaLIP serum and both confocal scanning laser and immuno-electron microscopy. PhpaLIP appears to be always present in the accessory glands (with a secretory peak shortly before oviposition) but was also detected in the follicle cells of the ovarioles, within the developing vitelline envelope, and in the oviducts. The results are discussed in relation to the functions that PhpaLIP could have during the gonotrophic cycle, in the various reproductive structures of female P. papatasi.

The reproductive accessory glands of the adult female have a functional versatility in insects, contributing to reproduction in various ways. The major protein secreted by the accessory glands of female Phlebotomus papatasi (Diptera, Psychodidae) has already been characterised and named PhpaLIP (for P. papatasi lipase) because, in terms of its amino-acid sequence, it is very similar to a wide range of vertebrate lipases. The gene coding for PhpaLIP has now been cloned into a pQE30 vector and expressed in Escherichia coli. When the recombinant PhpaLIP was tested in vitro, it was found to have not only lipase-like activity (when p-nitrophenyl caprylate was used as the substrate) but also specific antibacterial activity against some Gram-positive and Gram-negative bacteria. The possible physiological roles of PhpaLIP in P. papatasi are discussed, in the light of these results.

The hyperparathyroidism-jaw tumor (HPT-JT) syndrome is an autosomal dominant disorder characterized by the occurrence of parathyroid tumors and ossifying jaw fibromas. The gene causing HPT-JT, HRPT2, is located on chromosome 1q31.2 and consists of 17 exons that encode a 531-amino acid protein, designated parafibromin. We recently identified six Roma families in Portugal with 56 members (11 affected and 45 asymptomatic), who had the HPT-JT syndrome. We postulated that they may have a common ancestor and that the HPT-JT syndrome may be due to a mutation of the HRPT2 gene. Haplotype analysis using 14 chromosome 1q24-q32 polymorphic markers showed that the 11 affected individuals shared a common haplotype defined by seven markers that spanned an approximately 12.5-cM region, flanked centromerically by D1S202 and telomerically by D1S306. DNA sequence analysis identified a 2-bp (TG or GT) frameshift deletion in exon 8, which predicts a truncated parafibromin protein, in all 11 affected individuals. This mutation was also found in 19 unaffected individuals (age range, 12-74 yr) who shared the affected haplotype, suggesting a low age-related penetrance for HPT-JT in these families. Thus, the HPT-JT syndrome in six Roma families from Portugal is due to a novel founder mutation in the HRPT2 gene.

Background Due to optimal safety of sublingual immunotherapy (SLIT), it was suggested that a slow up-dosing phase maybe not necessary, and therefore the treatment will be more patient-friendly, avoiding dosing mistakes. Patients Twenty adult patients suffering allergic rhinitis due to Parietaria, were enrolled. Half of them received the traditional schedule and the other half immediately started with 200 STU. Results No difference was observed between the traditional up-dosing treatment schedule and no-updosing treatment schedule in terms of side effects, even mild local side effects was grater with traditional regimen.

The essential oils from Magnolia grandiflora and Chrysactinia mexicana leaves, and from Schinus molle leaves and fruit, were characterized by gas chromatography/flame-ionization detection and gas chromatography/mass spectrometry. Twenty-eight compounds from M. grandiflora leaves were identified (representing 93.6% of the total area of the gas chromatogram), with the major component being bornyl acetate (20.9%). Colorless and yellow oils were obtained from the C. mexicana leaves with 18 (86.7%) and 11 (100%) compounds identified, respectively. In both fractions, the principal component was sylvestrene (36.8% and 41.1%, respectively). The essential oils of S. molle leaves and fruit were each separated into colorless and yellow fractions, in which 14 (98.2) and 20 (99.8%) compounds were identified. The main component was α-phellandrene in all fractions (between 32.8% and 45.0%). The M. grandiflora oil displayed antifungal activity against five dermatophyte strains. The oils from S. molle and M. grandiflora leaves had antimicrobial activity against Staphylococcus aureus and Streptococcus pyogenes, which cause skin infections that potentially may lead to sepsis. However, the antioxidant activities of all oils were small (half maximal effective concentration values >250 μg/mL).