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Freshwater ecosystems are among the most severely affected environments by species loss caused by climate change and intense anthropogenic pressure. To preserve biodiversity, biomonitoring plays a key role by providing reliable data on biological diversity and ecological status. Environmental DNA (eDNA) metabarcoding has emerged as a powerful and non-invasive alternative to traditional morphology-based sampling and identification methods. This study represents the first application of eDNA analysis to assess the invertebrate communities in three Sicilian Lakes: Poma, Piana degli Albanesi and Scanzano. Water samples were collected at two points in each lake and after filtration with nitrocellulose membranes, eDNA was extracted and metabarcoding analysis was performed. A total of 27 species were identified, belonging to Phyla of Annelida, Arthropoda and Rotifera. Notably, the analysis revealed the presence of alien species ( Daphnia parvula and Acanthocyclops americanus ), a dangerous species associated with the transmission of viral diseases ( Culex pipiens ), and potential new records for Sicily ( Stylaria lacustris , Platypalpus exilis , Pammene aurana , Limnephilus rhombicus ). These results provide a preliminary snapshot of invertebrate biodiversity at these sites, demonstrating how eDNA has the potential to complement, but not replace, traditional methods, contributing to the assessment of ecosystem status.

In recent years, the invasive Atlantic blue crab ( Callinectes sapidus ) has increased its spread throughout the Mediterranean Sea, threatening native biodiversity and local economies. This study aimed to valorize C. sapidus sampled in Sicily by utilizing its exoskeleton as a source of chitosan, astaxanthin, and bio-phenolic compounds. These biomolecules were evaluated for their reducing, radical scavenging, and antitumor activity. The ferric ion reducing antioxidant power (FRAP) and the free radical scavenging activity against radical 2,2-Diphenyl-1-picrylhydrazyl (DPPH) were significantly higher for chitosan (3.16 ± 0.10 mg AAE/g and 8.1 ± 0.10 µmol TE/g). No significant differences were observed among the tested biomolecules in their activity in scavenging the radical 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS). Both bio-phenolic compounds and astaxanthin exhibited dose-dependent cytotoxicity on CaCo-2 (IC 50 = 12.47 and 18 µg/mL) and HepG2 (IC 50 = 10.25 and 1.26 µg/mL) cell lines, while only bio-phenols showed no cytotoxic effect on differentiated CaCo-2 cells up to 20 µg/mL. These findings highlight the value of blue crab by-products in supporting a circular economy, offering a sustainable approach to managing this invasive species while providing bioactive compounds with promising medical and nutraceutical applications.

The intraspecies and interspecies Comparative Genomic Hybridization (CGH) between the closely related Cebidae species, capuchin monkeys ( Cebus capucinus , Sapajus apella ), and the tamarins ( Saguinus mystax, Leontocebus fuscicollis ) was performed to analyze their genomes. In particular, this approach determines balanced and unbalanced repetitive DNA sequence distribution and reveals dynamics during evolution. Capuchin monkeys are considered the most ancestral group with conserved syntenies compared to the hypothetical ancestral New World monkeys' karyotype. Also, more derived karyotypes of phylogenetically distant species from the Saguinus and Leontocebus genera are analyzed here. The distribution of repetitive sequences has been traditionally studied through classical staining methods of cytogenetics. It has been hypothesized that repeats are species-specific and their conservation across closely related species are also common; their role in the genome has been extensively studied even though its role in speciation is not well studied and understood. The CGH shows bright signals with balanced and imbalanced DNA involving different genome regions: such as predominantly repetitive DNA at centromeric positions, and interstitial distribution with extended blocks. Cross-species CGH demonstrated the origin of some heterochromatic regions and identified apomorphic heterochromatin expansion events. The uncovered distribution of repetitive sequences is analyzed from an evolutionary perspective to elucidate the genomic dynamics of the repetitive sequences at the level of chromosomal organization.

This study evaluates, for the first time, the reducing capacity, radical scavenger activity, and in vitro antitumor and anti-inflammatory effects of chitosan, astaxanthin, and bio-phenols extracted from the exoskeleton of Sicilian Procambarus clarkii , the most widespread species of invasive crayfish in the Mediterranean region. Among the extracted compounds, astaxanthin exhibited the highest antioxidant activity in all assays. Chitosan and polyphenols demonstrated reducing and radical scavenging activity; chitosan showed significant ferric ion reducing capacity in the FRAP test, while bio-phenolic compounds displayed notable radical scavenging activity in the DPPH and ABTS assays. Both astaxanthin and polyphenols showed dose-dependent cytotoxicity on two different cancer cell lines, with IC50 values of 1.45 µg/mL (phenolic extract) and 4.28 µg/mL (astaxanthin extract) for HepG2 cells and 2.45 µg/mL (phenolic extract) and 4.57 µg/mL (astaxanthin extract) for CaCo-2 cells. The bio-phenolic extract also showed potential anti-inflammatory effects in vitro by inhibiting nitric oxide production in inflamed RAW 264.7 macrophages, reducing the treated/control NO ratio to 77% and 74% at concentrations of 1.25 and 1.5 μg/mL, respectively. These results suggest that P. clarkii exoskeletons could be a valuable source of bioactive molecules for biomedical, pharmaceutical, and nutraceutical application while contributing to the sustainable management of this invasive species.

The cell-free aqueous extract from the coelomic fluid of Holothuria tubulosa was prepared and examined for its glucose-lowering effect on HepG2 cells in vitro. In particular, employing a combination of cytochemical, flow cytometric, PCR, and protein blot techniques, we evaluated its role on glucose internalization and storage and on the upregulation and surface translocation of the two glucose transporters GLUT-2 and -4. The changes in expression, synthesis, and/or activation of the GLUT2 -related transcription factor hepatocyte nuclear factor-1 alpha (HNF1α) and the GLUT-4-translocation regulatory factors insulin receptor substrate-1 (IRS-1) and AKT were also studied. Our results showed the improved glucose response by HepG2 cells, leading to an evident increase in glucose consumption/uptake and glycogen storage upon exposure. Moreover, the extract induced molecular reprogramming involving the upregulation of (i) IRS1 gene expression, (ii) the transcription and translation levels of HNF1α, AKT, and GLUT-4, (iii) the phosphorylation level of AKT, (iv) the synthesis of GLUT-2 protein, and (v) the translocation of GLUT-2 and -4 transporters onto the plasma membrane. Cumulatively, our results suggest that the coelomic fluid extract from H. tubulosa can be taken into consideration for the development of novel treatment agents against diabetes mellitus.

The Barcelona Tissue Bank was established from the merge of two previous multi-tissue banks. Potential donors are screened by Donor Center staff and multi-tissue retrieval is performed by specialized own teams. Tissue processing and preservation is performed in clean room facilities by specialised personnel. After quality control of both donor and all tissues results, the heart valves and vascular segments are stored until medical request. The aim of this report is to present the cardiovascular tissue activity and retrospectively evaluate the outcomes of the changes performed in last 20 years. Cardiovascular tissue from 4088 donors was received, specifically 3115 hearts and 2095 vascular segments were processed and evaluated. A total of 48% of the aortic valves, 68% of the pulmonary valves and 75% of the vascular segments were suitable for transplant. The main reason for discarding tissue was macroscopic morphology followed by microbiological results, for both valves and arteries. Altogether, 4360 tissues were distributed for transplantation: 2032 (47%) vascular segments, 1545 (35%) pulmonary valves and 781 (18%) aortic valves. The most common indication for aortic valve surgery was the treatment of endocarditis, while for pulmonary valves, it was congenital malformation reconstruction. Vascular segments were mainly used for reconstruction after ischemia. During this period, a number of changes were made with the goal of enhancing tissue quality, safety and efficacy. These improvements were achieved through the use of a new antibiotic cocktail, increasing of donor age criteria and changing the microbiological control strategy., (© 2023. The Author(s).)

The study of bioactive molecules of marine origin has created an important bridge between biological knowledge and its applications in biotechnology and biomedicine. Current studies in different research fields, such as biomedicine, aim to discover marine molecules characterized by biological activities that can be used to produce potential drugs for human use. In recent decades, increasing attention has been paid to a particular group of marine invertebrates, the Ascidians, as they are a source of bioactive products. We describe omics data and computational methods relevant to identifying the mechanisms and processes of innate immunity underlying the biosynthesis of bioactive molecules, focusing on innovative computational approaches based on Artificial Intelligence. Since there is increasing attention on finding new solutions for a sustainable supply of bioactive compounds, we propose that a possible improvement in the biodiscovery pipeline might also come from the study and utilization of marine invertebrates' innate immunity.

Freshwater ecosystems play a key role in global diversity and are subject to a series of anthropic impacts, often leading to biodiversity loss. The organisms inhabiting these sites continuously release DNA into the environment through cells, excrement, gametes and/or decomposing matter; thus, evaluation of this eDNA could revolutionize the monitoring of biodiversity. In this study, environmental DNA metabarcoding was used for the first time in three Sicilian lakes: Lake Poma, Piana degli Albanesi Lake and Lake Scanzano. Results obtained provide the first snapshot of vertebrate biodiversity in these three lakes, where little is known, to provide valuable information useful for creating a baseline of knowledge regarding the biodiversity in these three lakes. Another important result was the detection of marine species, most likely due to some kind of anthropogenic contamination. Environmental DNA is a useful tool to evaluate both biodiversity and the ecological status of the environment; it has the potential to complement traditional methods, and the use of both approaches may offer a more comprehensive understanding of the ecosystem.

The effects of lipopolysaccharide (LPS) on Mif (macrophage migration inhibitory factor) gene expression in the pharynx (haemapoetic tissue) of Ciona robusta were investigated using quantitative reverse-transcription PCR (qRT-PCR) and in situ hybridisation (ISH). To verify the induction of an inflammatory response in the pharynx, a qRT-PCR analysis was performed to evaluate the change in the expression of proinflammatory marker genes such as Mbl, Ptx-like, Tnf-α and Nf-kb, which were shown to be upregulated 1 h post LPS challenge. The change in the expression of the two Mif paralogs in the pharynx was assessed before and after stimulation, and qRT-PCR and ISH results showed that, although Mif2 and Mif2 were expressed in clusters of haemocytes in pharynx vessels, only Mif1 expression increased after LPS stimulation. This indicates that the Mif genes are differently regulated and respond to different ambient inputs that need further analysis., (© 2023 The Authors. Journal of Fish Biology published by John Wiley & Sons Ltd on behalf of Fisheries Society of the British Isles.)

The processing and initial testing of a new human tissue preparation is described. Full-thickness Acellular Dermal Matrix (ftADM) is the extracellular matrix (ECM) obtained by decellularization of full-thickness human skin from cadaveric donors. The safety, stability and usability of the graft are discussed with respect to the results of the residual cellular content, maintenance of ECM components, and biomechanical properties. Quantitative and qualitative analysis of the ECM demonstrated the absence of cell debris, while the native structure of human dermis was maintained. Biomechanical testing showed stiffness values comparable to other commercial products used for tendon reinforcement, suggesting that our ftADM could be successfully used not only in soft tissue regeneration surgeries, but also in tendon reinforcement. First case of ftADM in rotator cuff augmentation is described. Technical management of the patch during surgery and clinical outcomes are discussed., Competing Interests: Competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors., (Copyright © 2023. Published by Elsevier B.V.)

Aqueous extracts from Posidonia oceanica 's green and brown (beached) leaves and rhizomes were prepared, submitted to phenolic compound and proteomic analysis, and examined for their potential cytotoxic effect on HepG2 liver cancer cells in culture. The chosen endpoints related to survival and death were cell viability and locomotory behavior, cell-cycle analysis, apoptosis and autophagy, mitochondrial membrane polarization, and cell redox state. Here, we show that 24 h exposure to both green-leaf- and rhizome-derived extracts decreased tumor cell number in a dose-response manner, with a mean half maximal inhibitory concentration (IC 50 ) estimated at 83 and 11.5 μg of dry extract/mL, respectively. Exposure to the IC 50 of the extracts appeared to inhibit cell motility and long-term cell replicating capacity, with a more pronounced effect exerted by the rhizome-derived preparation. The underlying death-promoting mechanisms identified involved the down-regulation of autophagy, the onset of apoptosis, the decrease in the generation of reactive oxygen species, and the dissipation of mitochondrial transmembrane potential, although, at the molecular level, the two extracts appeared to elicit partially differentiating effects, conceivably due to their diverse composition. In conclusion, P. oceanica extracts merit further investigation to develop novel promising prevention and/or treatment agents, as well as beneficial supplements for the formulation of functional foods and food-packaging material with antioxidant and anticancer properties.

The Ascidian C. robusta is a powerful model for studying innate immunity. LPS induction activates inflammatory-like reactions in the pharynx and the expression of several innate immune genes in granulocyte hemocytes such as cytokines, for instance, macrophage migration inhibitory factors (CrMifs). This leads to intracellular signaling involving the Nf-kB signaling cascade that triggers downstream pro-inflammatory gene expression. In mammals, the COP9 (Constitutive photomorphogenesis 9) signalosome (CSN) complex also results in the activation of the NF-kB pathway. It is a highly conserved complex in vertebrates, mainly engaged in proteasome degradation which is essential for maintaining processes such as cell cycle, DNA repair, and differentiation. In the present study, we used bioinformatics and in-silico analyses combined with an in-vivo LPS exposure strategy, next-generation sequencing (NGS), and qRT-PCR to elucidate molecules and the temporal dynamics of Mif cytokines, Csn signaling components, and the Nf-κB signaling pathway in C. robusta . A qRT-PCR analysis of immune genes selected from transcriptome data revealed a biphasic activation of the inflammatory response. A phylogenetic and STRING analysis indicated an evolutionarily conserved functional link between the Mif-Csn-Nf-kB axis in ascidian C. robusta during LPS-mediated inflammation response, finely regulated by non-coding molecules such as microRNAs (miRNAs).

To date, drugs released into the aquatic environment are a real problem, and among antibiotics, sulfamethoxazole is the one most widely found in wastewater; thus, the evaluation of its toxicity on marine organisms is very important. This study, for the first time, investigates the in vitro effects of 4 concentrations of sulfamethoxazole (0.05 mg/L, 0.5 mg/L, 5 mg/L, 50 mg/L) on the fertilization and development of the sea urchin Arbacia lixula . The gametes were exposed to drugs in three different stages: simultaneously with, prior to, and post-fertilization. The results show a significant reduction in the percentage of fertilized oocytes at the highest drug concentrations. Moreover, an increase in anomalies and delays in embryo development following the treatment with the drug was demonstrated. Therefore, the data suggest that this antibiotic can alter the development of marine organisms, making it urgent to act to reduce their release and to determine the concentration range with the greatest impact.

Cytochromes P450 (CYP) are enzymes responsible for the biotransformation of most endogenous and exogenous agents. The expression of each CYP is influenced by a unique combination of mechanisms and factors including genetic polymorphisms, induction by xenobiotics, and regulation by cytokines and hormones. In recent years, Ciona robusta , one of the closest living relatives of vertebrates, has become a model in various fields of biology, in particular for studying inflammatory response. Using an in vivo LPS exposure strategy, next-generation sequencing (NGS) and qRT-PCR combined with bioinformatics and in silico analyses, compared whole pharynx transcripts from naïve and LPS-exposed C. robusta , and we provide the first view of cytochrome genes expression and miRNA regulation in the inflammatory response induced by LPS in a hematopoietic organ. In C. robusta , cytochromes belonging to 2B,2C, 2J, 2U, 4B and 4F subfamilies were deregulated and miRNA network interactions suggest that different conserved and species-specific miRNAs are involved in post-transcriptional regulation of cytochrome genes and that there could be an interplay between specific miRNAs regulating both inflammation and cytochrome molecules in the inflammatory response in C. robusta .

Background: Dermal scaffolds for tissue regeneration are nowadays an effective alternative in not only wound healing surgeries but also breast reconstruction, abdominal wall reconstruction and tendon reinforcement. The present study describes the development of a decellularization protocol applied to human split-thickness skin from cadaveric donors to obtain dermal matrix using an easy and quick procedure., Methods: Complete split-thickness donor was decellularized through the combination of hypertonic and enzymatic methods. To evaluate the absence of epidermis and dermal cells, and ensure the integrity of the extracellular matrix (ECM) structure, histological analysis was performed. Residual genetic content and ECM biomolecules (collagen, elastin, and glycosaminoglycan) were quantified and tensile strength was tested to measure the effect of the decellularization technique on the mechanical properties of the tissue., Results: Biomolecules quantification, residual genetic content (below 50 ng/mg dry tissue) and histological structure assessment showed the efficacy of the decellularization process and the preservation of the ECM. The biomechanical tests confirmed the preservation of native properties in the acellular tissue., Conclusions: The acellular dermal matrix obtained from whole split-thickness skin donor with the newly developed decellualrization protocol, maintains the desired biomechanical and structural properties and represents a viable treatment option for patients., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Pentraxins (PTXs) are a superfamily of conserved proteins which are components of the humoral arm of innate immunity. They are considered to be functional ancestors of antibodies and are classified into short and long types. In this study, we show that a pentraxin-like component (Ptx-like) with a C-terminal PTX domain, highly homologous to the short PTX of H. sapiens CRP, and a long N-terminal domain typical of long PTXs, is involved in the inflammatory response of Ciona robusta under LPS exposure in vivo. Analyses of protein domains as well as 3D modelling and phylogenetic tree supported the close relationship of Ptx-like with mammalian CRP, suggesting that C. robusta Ptx-like shares a common ancestor in the chordate lineages. qRT-PCR analysis showed that Ptx-like was transcriptionally upregulated during the inflammatory process induced by LPS inoculation and that it is involved in the initial phase as well as the secondary phase of the inflammatory response in which matrix remodelling and the achievement of homeostasis occur. In situ hybridisation assays revealed that gene transcription was upregulated in the pharynx post-LPS challenge in vivo, and that Ptx-like was expressed by clusters of haemocytes, mainly granulocytes, inside the pharynx vessels. We also found transcript-expressing granulocytes flowing in the musculature and in the lacunae of the circulatory system. These data supported that Ptx-like is a potential molecule of the acute-phase response in C. robusta immune defence systems against bacterial infection., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The transforming growth factor-β (TGF-β) family of cytokines performs a multifunctional signaling, which is integrated and coordinated in a signaling network that involves other pathways, such as Wintless, Forkhead box-O (FOXO) and Hedgehog and regulates pivotal functions related to cell fate in all tissues. In the hematopoietic system, TGF-β signaling controls a wide spectrum of biological processes, from immune system homeostasis to the quiescence and self-renewal of hematopoietic stem cells (HSCs). Recently an important role in post-transcription regulation has been attributed to two type of ncRNAs: microRNAs and pseudogenes. Ciona robusta , due to its philogenetic position close to vertebrates, is an excellent model to investigate mechanisms of post-transcriptional regulation evolutionarily highly conserved in immune homeostasis. The combined use of NGS and bioinformatic analyses suggests that in the pharynx, the hematopoietic organ of Ciona robusta , the Tgf-β, Wnt, Hedgehog and FoxO pathways are involved in tissue homeostasis, as they are in human. Furthermore, ceRNA network interactions and 3'UTR elements analyses of Tgf-β, Wnt, Hedgehog and FoxO pathways genes suggest that different miRNAs conserved (cin-let-7d, cin-mir-92c, cin-mir-153), species-specific (cin-mir-4187, cin-mir-4011a, cin-mir-4056, cin-mir-4150, cin-mir-4189, cin-mir-4053, cin-mir-4016, cin-mir-4075), pseudogenes (ENSCING00000011392, ENSCING00000018651, ENSCING00000007698) and mRNA 3'UTR elements are involved in post-transcriptional regulation in an integrated way in C. robusta .

The close phylogenetic relationship between Ciona robusta and vertebrates makes it a powerful model for studying innate immunity and the evolution of immune genes. To elucidate the nature and dynamics of the immune response, the molecular mechanisms by which bacterial infection is detected and translated into inflammation and how potential pattern recognition receptors (PRRs) are involved in pathogen recognition in tunicate C. robusta (formerly known as Ciona intestinalis), we applied an approach combining bacterial infections, next-generation sequencing, qRT-PCR, bioinformatics and in silico analyses (criteria of a p-value < 0.05 and FDR < 0.05). A STRING analysis indicated a functional link between components of the Tlr/MyD88-dependent signalling pathway (Tlr2, MyD88, and Irak4) and components of the Nf-κB signalling pathway (Nf-κB, IκBα, and Ikkα) (p-value < 0.05, FDR < 0.05). A qRT-PCR analysis of immune genes selected from transcriptome data revealed Mif as more frequently expressed in the inflammatory response than inflammation mediator or effector molecules (e.g., Il-17s, Tnf-α, Tgf-β, Mmp9, Tlrs, MyD88, Irak4, Nf-κB, and galectins), suggesting close interplay between Mif cytokines and Nf-κB signalling pathway components in the biphasic activation of the inflammatory response. An in silico analyses of the 3'-UTR of Tlr2, MyD88, IκBα, Ikk, and Nf-κB transcripts showed the presence of GAIT elements, which are known to play key roles in the regulation of immune gene-specific translation in humans. These findings provide a new level of understanding of the mechanisms involved in the regulation of the C. robusta inflammatory response induced by LPS and suggest that in C. robusta, as in humans, a complex transcriptional and post-transcriptional control mechanism is involved in the regulation of several inflammatory genes.

The 5' and 3' RACE is a method to obtain full-length 5' and 3' ends of cDNA using known cDNA sequences from expressed sequence tags (ESTs), subtracted cDNA, differential display, or library screening. Here is described the identification of full-length 5' and 3' ends of Ciona robusta Mif1 and Mif2 cDNA by using 5' and 3' RACE method.

Parathyroidectomy is a standard practice to treat recurrent or persistent hyperparathyroidism. However, this can lead to the onset of hypoparathyroidism, treatable with the autotransplantation of parathyroid tissue (PT). Tissue can be transplanted immediately after parathyroidectomy or cryopreserved and transplanted only in case of necessity. Since 2011, the Cord Blood Bank and Cardiovascular Tissue Bank of Emilia-Romagna has been storing PT for potential autologous transplantation. To date, there are highly variable data about the viability and function of PT after thawing. However, it is not clear if the PT quality is affected by different cryopreservation protocols and/or by the storage time. The aim of this study was to assess the ex vivo function and viability of the PTs of ten patients stored in the Bank. Tissue morphology was evaluated before and after cryopreservation through histological investigations. PT function was analyzed by assessing the ability of cryopreserved PT to synthesize and secrete parathyroid hormone (PTH) in response to different calcium concentrations. Moreover, viability and function were also investigated on tissue-isolated cells in culture. These data show that tested tissues appear to be viable and able to produce PTH even after 5 years of storage, and the histological architecture is well preserved.

Matrix metalloproteinases (MMPs) are a family of endopeptidases collectively able to degrade the components of the extracellular matrix (ECM), with important roles in many biological processes, such as embryogenesis, normal tissue remodelling, angiogenesis and wound healing. New views on the function of MMPs reveal that they regulate inflammatory response and therefore might represent an early step in the evolution of the immune system. MMPs can affect the activity of cytokines involved in inflammation including TGF-β and TNF-α. MMPs are widely distributed in all kingdoms of life and have likely evolved from a single-domain protein which underwent successive rounds of duplications. In this study, we focused on the Ciona robusta (formerly known as Ciona intestinalis) MMP gelatinase homologue. Gene organization, phylogenetic analysis and 3D modeling supported the closest correlation of C. robusta gelatinase with the human MMP-9. Real-time PCR analysis and zymographic assay showed a prompt expression induced by LPS inoculation and an upregulation of enzymatic activity. Furthermore, we showed that before of the well-known increase of TGF-β and TNF-α levels, a MMP-9like boost occurred, suggesting a possible involvement of MMP-9like in regulating inflammatory response in C. robusta., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Innate immune responses face infectious microorganisms by inducing inflammatory responses. Multiple genes within distinct functional categories are coordinately and temporally regulated by transcriptional 'on' and 'off' switches that account for the specificity of gene expression in response to external stimuli. Mechanisms that control transcriptional and post-transcriptional regulation are important in coordinating the initiation and resolution of inflammation. Macrophage migration inhibitory factor (MIF) is an important cytokine that, in Ciona robusta, is related to inflammatory response. It is well known that in C. robusta, formerly known as Ciona intestinalis, the pharynx is involved in the inflammatory reaction induced by lipopolysaccharide (LPS) injection in the body wall. Using this biological system, we describe the identification of two C. robusta MIFs (CrMIF1 and CrMIF2). The phylogenetic tree and modeling support a close relationship with vertebrate MIF family members. CrMIF1 and CrMIF2 possess two evolutionally conserved catalytic sites: a tautomerase and an oxidoreductase site with a conserved CXXC motif. Real-time PCR analysis shows a prompt expression induced by LPS inoculation in CrMIF1 and a late upregulation of CrMIF2 and in silico analyses of 3'UTR show a cis-acting GAIT element and a CPE element in 3'-UTR, which are not present in the 3'-UTR of CrMIF1, suggesting that different transcriptional and post-transcriptional control mechanisms are involved in the regulation of gene expression of MIF during inflammatory response in C. robusta., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The selective modulation of immunity is an emerging concept driven by the vast advances in our understanding of this crucial host defense system. Invertebrates have raised researchers' interest as potential sources of new bioactive molecules owing to their antibacterial, anticancer and immunomodulatory activities. A LipoPolySaccharide (LPS) challenge in the ascidian Ciona intestinalis generates the transcript, Ci8 short, with cis-regulatory elements in the 3' UTR region that are essential for shaping innate immune responses. The derived amino acidic sequence in silico analysis showed specific binding to human Major Histocompatibility Complex (MHC) Class I and Class II alleles. The role of Ci8 short peptide was investigated in a more evolved immune system using human Peripheral Blood Mononuclear Cells (PBMCs) as in vitro model. The biological activities of this molecule include the activation of 70kDa TCR ζ chain Associated Protein kinase (ZAP-70) and T Cell Receptor (TCR) Vβ oligo clonal selection on CD4 + T lymphocytes as well as increased proliferation and IFN-γ secretion. Furthermore Ci8 short affects CD4 + /CD25 high induced regulatory T cells (iTreg) subset selection which co-expressed the functional markers TGF-β1/Latency Associated Protein (LAP) and CD39/CD73. This paper describes a new molecule that modulates important responses of the human adaptive immune system., (Copyright © 2017 Elsevier GmbH. All rights reserved.)

Riboprobes (in situ hybridization) and antibodies (immunohistochemistry), previously used to show the upregulation of Ciona intestinalis (Type A) galectins (CiLgals-a, CiLgals-b) and phenoloxidase (CinPO2) immune-related genes, were tested on histological sections of the ovary. The ovarian follicles are composed of oocytes encased by follicular cells (FCs) and test cells (TCs). Results show the transcription upregulation of both CiLgals and CinPO2 genes in the vitellogenic FCs, conversely distinct cytolocalization of the proteins are shown. At vitellogenic stage, the CiLgals are localized in the FCs, in the oocyte cytoplasm, and close to the germinal vesicle (GV), whereas the CinPO2 was never identified in the FCs. In a presumptive advanced phase and at the post-vitellogenic stage the TCs appear to be labelled by the CinPO2 riboprobe, and the protein identified by the antibody suggesting an mRNA transcytosis process from FCs. At post-vitellogenic stage the CiLgals mainly enrich the GV nucleoplasm, whereas the CinPO2 is contained in TCs and in the ooplasm but never found in the GV. This finding sheds new light on a former paper in which TCs were reported to be the only CinPO2-producing cells in the ovarian follicle. Finally, CiLgals and CinPO2 genes transcription and proteins production seem to be associated with accessory cells during their differentiation from vitellogenic to post-vitellogenic stage. The present findings promote further research on the early upregulation of immune-related genes, and the potential multifunctional role of the produced proteins. In addition further insight on the accessory cells involvement in ascidian oogenesis are reported., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The transcription of two Ciona intestinalis galectin genes (CiLgals-a and CiLgals-b) is uparegulated by LPS in the pharynxis (hemocytes, vessel epithelium, endostilar zones) which is retained the main organ of the immunity. In this ascidian, for the first time we show, by immunohistochemistry and in situ hybridization methods, that these two immune-related genes are expressed in the gastric epithelium of naïve ascidians, whereas the galectins appear to be only contained in the intestine columnar epithelium. In addition, according to previous results on the pharynx, the genes are also expressed and galectins produced by hemocytes scattered in the connective tissue surrounding the gut. The genes expression and galectin localization in several tissues, including the previous findings on the transcription upregulation, the constitutive expression of these genes by endostylar zones and by the gastric epithelium suggest a potential multifunctional role of these galectins. In this respect, it is of interest to define where the CiLgals are normally found as related to the tissue functions. Such an approach should be a starting point for further investigations., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Although the Tumor necrosis factor gene superfamily seems to be very conserved in vertebrates, phylogeny, tissue expression, genomic and gene organization, protein domains and polymorphism analyses showed that a strong change has happened mostly in invertebrates in which protochordates were a constraint during the immune-molecules history and evolution. RT PCR was used to investigate differential gene expression in different tissues. The expression shown was greater in the pharynx. Single-nucleotide polymorphism has been investigated in Ciona intestinalis Tumor necrosis factor alpha (CiTNFα) mRNA isolated from the pharynx of 30 ascidians collected from Licata, Sicily (Italy), by denaturing gradient gel electrophoresis (DGGE). For this analysis, CiTNFα nucleotide sequence was separated into two fragments, TNF-1 and -2, respectively, of 630 and 540 bp. We defined 23 individual DGGE patterns (named 1 to 10 for TNF-1 and 1 to 13 for TNF-2). Five patterns for TNF-1 accounted for <10% of the individuals, whereas the pattern 13 of TNF-2 accounted for >20% of the individuals. All the patterns were verified by direct sequencing. Single base-pair mutations were observed mainly within COOH-terminus, leading to 30 nucleotide sequence variants and 30 different coding sequences segregating in two main different clusters. Although most of the base mutations were silent, four propeptide variants were detected and six amino acid replacements occurred within COOH-terminus. Statistical tests for neutrality indicated negative selection pressure on signal and mature peptide domains, but possible positive selection pressure on COOH-terminus domain. Lastly we displayed the in silico 3D structure analysis including the CiTNFα variable region., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Lysozyme is an important defense molecule of the innate immune system. Known for its bactericidal properties, lysozyme catalyzes the hydrolysis of b-(1,4)-glycosidic bonds between the N-acetyl glucosamine and N-acetyl muramic acid in the peptidoglycan layer of bacterial cell walls. In this study, the complete coding sequence of four g-type lysozymes were identified in Ciona intestinalis. Phylogenetic analysis and modelling supported the hypothesis of a close relationship with the vertebrate g-type lysozymes suggesting that the C. intestinalis g-type lysozyme genes (CiLys-g1, Cilys-g2, CiLys-g3, CiLys-g4) share a common ancestor in the chordate lineage. Protein motif searches indicated that C. intestinalis g-type lysozymes contain a GEWL domain with a GXXQ signature, typical of goose lysozymes. Quantitative Real-Time PCR analysis results showed that transcripts are expressed in various tissues from C. intestinalis. In order to determine the involvement of C. intestinalis g-type lysozymes in immunity, their expression was analyzed in the pharynx, showing that transcripts were significantly up-regulated in response to a challenge with lipopolysaccharide (LPS). These data support the view that CiLys g-type are molecules with potential for immune defense system against bacterial infection., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The diversification of cellular functions is one of the major characteristics of multicellular organisms which allow cells to modulate their gene expression, leading to the formation of transcripts and proteins with different functions and concentrations in response to different stimuli. CAP genes represent a widespread family of proteins belonging to the cysteine-rich secretory protein, antigen 5 and pathogenesis-related 1 superfamily which, it has been proposed, play key roles in the infection process and the modulation of immune responses in host animals. The ascidian Ciona intestinalis represents a group of proto-chordates with an exclusively innate immune system that has been widely studied in the field of comparative and developmental immunology. Using this biological system, we describe the identification of a novel APA mechanism by which an intronic polyadenylation signal is activated by LPS injection, leading to the formation of a shorter CAP mRNA capable of expressing the first CAP exon plus 19 amino acid residues whose sequence is contained within the first intron of the annotated gene. Furthermore, such an APA event causes the expression of a translational controlling cis-acting GAIT element which is not present in the previously isolated CAP isoform and identified in the 3'-UTR of other immune-related genes, suggesting an intriguing scenario in which both transcriptional and post-transcriptional control mechanisms are involved in the activation of the CAP gene during inflammatory response in C. intestinalis., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Transforming growth factor (TGF-β) is a well-known component of a regulatory cytokines superfamily that has pleiotropic functions in a broad range of cell types and is involved, in vertebrates, in numerous physiological and pathological processes. In the current study, we report on Ciona intestinalis molecular characterisation and expression of a transforming growth factor β homologue (CiTGF-β). The gene organisation, phylogenetic tree and modelling supported the close relationship with the mammalian TGF suggesting that the C. intestinalis TGF-β gene shares a common ancestor in the chordate lineages. Functionally, real-time PCR analysis showed that CiTGF-β was transcriptionally upregulated in the inflammatory process induced by LPS inoculation, suggesting that is involved in the first phase and significant in the secondary phase of the inflammatory response in which cell differentiation occurs. In situ hybridisation assays revealed that the genes transcription was upregulated in the pharynx, the main organ of the ascidian immune system, and expressed by cluster of hemocytes inside the pharynx vessels. These data supported the view that CiTGF-β is a potential molecule in immune defence systems against bacterial infection., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

ML superfamily represents a group of proteins playing important roles in lipid metabolism and innate immune response. In this study, we report the identification of the first component of the ML superfamily in the invertebrate Ciona intestinalis by means of a subtractive hybridization strategy. Sequence homology and phylogenetic analysis showed that this protein forms a specific clade with vertebrate components of the Niemann-Pick type C2 protein and, for this reason, it has been named Ci-NPC2. The putative Ci-NPC2 is a 150 amino acids long protein with a short signal peptide, seven cysteine residues, three putative lipid binding site and a three-dimensional model showing a characteristic β-strand structure. Gene expression analysis demonstrated that the Ci-NPC2 protein is positively upregulated after LPS inoculum with a peak of expression 1 h after challenge. Finally, in-situ hybridization demonstrated that the Ci-NPC2 protein is preferentially expressed in hemocytes inside the vessel lumen., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

High-definition optical coherence tomography (HD-OCT) permits real-time 3D imaging of the impact of selected agents on human skin allografts. The real-time 3D HD-OCT assessment of (i) the impact on morphological and cellular characteristics of the processing of human acellular dermal matrices (HADMs) and (ii) repopulation of HADMs in vitro by human fibroblasts and remodelling of the extracellular matrix by these cells. Four different skin decellularization methods, Dispase II/Triton X-100, Dispase II/SDS (sodium dodecyl sulphate), NaCl/Triton X-100 and NaCl/SDS, were analysed by HD-OCT. HD-OCT features of epidermal removal, dermo-epidermal junction (DEJ) integrity, cellularity and dermal architecture were correlated with reflectance confocal microscopy (RCM), histopathology and immunohistochemistry. Human adult dermal fibroblasts were in vitro seeded on the NaCl/Triton X-100 processed HADMs, cultured up to 19 days and evaluated by HD-OCT in comparison with MTT proliferation test and histology. Epidermis was effectively removed by all treatments. DEJ was best preserved after NaCl/Triton X-100 treatment. Dispase II/SDS treatment seemed to remove all cellular debris in comparison with NaCl/Triton X-100 but disturbed the DEJ severely. The dermal micro-architectural structure and vascular spaces of (sub)papillary dermis were best preserved with the NaCl/Triton X-100. The impact on the 3D structure and vascular holes was detrimental with Dispase II/SDS. Elastic fibre fragmentation was only observed after Dispase II incubation. HD-OCT showed that NaCl/Triton X-100 processed matrices permitted in vitro repopulation by human dermal fibroblasts (confirmed by MTT test and histology) and underwent remodelling upon increasing incubation time. Care must be taken in choosing the appropriate processing steps to maintain selected properties of the extracellular matrix in HADMs. Processing HADMs with NaCl/Triton X-100 permits in vitro the proliferation and remodelling activity of human dermal fibroblasts. HD-OCT provides unique real-time and non-invasive 3D imaging of tissue-engineered skin constructs and complementary morphological and cytological information., (© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Two distinct Ciona intestinalis phenoloxidases (CinPO1, 2) had previously been cloned and sequenced. The CinPO2 is involved in innate immunity and is expressed by inflammatory hemocytes that populate the tunic and pharynx vessels as a response to LPS inoculation. In situ hybridization and immunohistochemistry assays on histological section, showed that the expression of this gene and the produced protein are shared with oogenesis, embryogenesis and larval morphogenesis. Intriguingly, upregulation of gene transcription was found in the test cell layer that envelopes the ovary follicle, ovulated egg, and gastrula, as well as it was modulated in the zygotic nucleus of outer balstomers of 32-cell embryo, neurula presumptive epidermis tissue and larval mesenchyme. The anti-CinPO2 antibodies, specific for adult inflammatory cells, recognize epitopes in the cytoplasm of ovarian oocytes, ovulated eggs, development stages and larval mesenchyme. The overall findings disclose the precocious activation of the CinPO2 immunity-related gene, and show a developmentally programmed expression of this phenoloxidase. Furthermore, these findings support the multifunctional roles of immunity-related genes and allows us to explore new perspectives on ascidian development and immunity., (© 2015 Wiley Periodicals, Inc.)

We investigated the role of phenoloxidases (POs) in ascidians inflammatory reaction, a components of a copper-containing protein family involved in invertebrate immune system. In Ciona intestinalis two phenoloxidases (CinPO-1, CinPO-2) have been sequenced. In the present study, real time PCR analysis showed that both CinPO-1 and CinPO-2 genes were modulated by LPS inoculation suggesting that they are inducible and highly expressed in the inflamed pharynx. In situ hybridization disclosed CinPO-1 and CinPO-2 transcripts in pharynx hemocytes (granulocytes) and, mainly, in unilocular refractile granulocytes (URG) which mainly populated the inflamed tunic matrix. Interestingly, the genes are also upregulated by LPS in the endostyle (zones 7, 8 and 9) that is considered homolog to the vertebrate thyroid., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Immunohistochemical and in situ hybridization assays were performed to answer the question whether the endostyle, that is the initial gastro-intestinal trait of Ciona intestinalis pharynx, is involved in galectin (CiLgals-a and CiLgals-b) production during the pharynx inflammatory response to LPS inoculation. Specific anti-CiLgal-a and anti-CiLgals-b antibodies, and oligonucleotide probes, that mark inflammatory hemocytes inside the pharynx vessels and vessel epithelium as shown by a previous paper, were assayed on endostyle histological sections. For the first time, we show that galectins are produced by endostyle zones, and both CiLgals-a and -b genes are upregulated by LPS. CiLgals-a and CiLgals-b are constitutively expressed in the endostyle zone 2 and 3, respectively, both genes are upregulated by LPS in the zone 2, and CiLgals-b in the zone 3 and 4. The antibody-reacting material contained in intracellular and extracellular large vesicles suggest an unexpected vesicle-dependent transporting mechanism of galectins not provided with signal peptide. Differential expression and gene upregulation in not-treated and LPS-treated specimens, support the role of endostyle galectins both in filter feeding and defense responses., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

In humans, IL-17 is a proinflammatory cytokine that plays a key role in the clearance of extracellular bacteria promoting cell infiltration and production of several cytokines and chemokines. Here, we report on three Ciona intestinalis IL-17 homologues (CiIL17-1, CiIL17-2, CiIL17-3). The gene organization, phylogenetic tree and modeling supported the close relationship with the mammalian IL-17A and IL-17F suggesting that the C. intestinalis IL-17 genes share a common ancestor in the chordate lineages. Real time PCR analysis showed a prompt expression induced by LPS inoculation suggesting that they are involved in the first phase of inflammatory response. In situ hybridization assays disclosed that the genes transcription was upregulated in the pharynx, the main organ of the ascidian immune system, and expressed by hemocytes (granulocytes and univacuolar refractile granulocyte) inside the pharynx vessels., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Peroxinectins function as hemoperoxidase and cell adhesion factor involved in invertebrate immune reaction. In this study, the ascidian (Ciona intestinalis) peroxinectin gene (CiPxt) and its expression during the inflammatory response have been examined. CiPxt is a new member of the peroxidase-cyclooxygenase gene superfamily that contains both the peroxidase domain and the integrin KGD (Lys-Gly-Asp) binding motif. A phylogenetic tree showed that CiPxt is very close to the chordate group and appears to be the outgroup of mammalian MPO, EPO and TPO clades. The CiPxt molecular structure model resulted superimposable to the human myeloperoxidase. The CiPxt mRNA expression is upregulated by LPS inoculation suggesting it is involved in C. intestinalis inflammatory response. The CiPxt was expressed in hemocytes (compartment/morula cells), vessel epithelium, and unilocular refractile granulocytes populating the inflamed tunic matrix and in the zones 7, 8 and 9 of the endostyle, a special pharynx organs homolog to the vertebrate thyroid gland., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

A subtractive hybridization strategy for the identification of differentially expressed genes was performed between LPS-challenged and naive Ciona intestinalis. This strategy allowed the characterization of two transcripts (Ci8short and Ci8long) generated by the use of two Alternative Polyadenylation sites. The Ci8long transcript contains a protein domain with relevant homology to several components of the Receptor Transporting Protein (RTP) family not present in the Ci8short mRNA. By means of Real Time PCR and Northern Blot, the Ci8short and Ci8long transcripts showed a different pattern of gene expression with the Ci8short mRNA being strongly activated after LPS injection in the pharynx. In situ hybridization analysis demonstrated that the activation of the APA site also influenced the tissue localization of the Ci8short transcript. This analysis showed that the Ci8long mRNA was expressed in hemocytes meanwhile the Ci8short mRNA was highly transcribed also in vessel endothelial cells and in the epithelium of pharynx. These findings demonstrated that regulation of gene expression based on different polyadenylation sites is an ancestral powerful strategy influencing both the level of expression and tissue distribution of alternative transcripts.

Interest is increasing in biological scaffolds for tissue regeneration such as extracellular matrix membranes, developed through soft tissue decellularization. Extracellular matrix membranes were developed to heal different tendon and soft tissue lesions that are very frequent in the general population with high health-care costs and patient morbidity. The aim of this research was to evaluate a human dermal matrix (HDM) decellularized by a chemico-physical method. A primary culture of rat tenocytes was performed: tenocytes were seeded on HDM samples and on polystyrene wells as controls (CTR). Cell viability and synthetic activity were evaluated at 3 and 7 days. An in vitro microwound model was used to evaluate HDM bioactivity: after tenocyte expansion, artificial wounds were created, HDM extracts were added, and closure time and decorin synthesis were monitored histomorphometrically at 1, 4, 24, and 72 hr. A significant higher amount of collagen I was observed when cells were cultured on HDM in comparison with that on CTR (3 days: p < 0.0001; 7 days: p < 0.05). In HDM group, fibronectin synthesis was significantly higher at both experimental times (p < 0.0001). At 3 days, proteoglycans and transforming growth factor-β1 releases were significantly higher on HDM (p < 0.0001 and p < 0.005, respectively). The artificial microwound closure time and decorin expression were significantly enhanced by the addition of 50% HDM extract (p < 0.05). In vitro data showed that the decellularization technique enabled the development of a matrix with adequate biological and biomechanical properties.

Although ascidians belong to a key group in chordate phylogenesis, amino acid sequences of Ciona intestinalis galectin-CRDs (CiLgals-a and -b) have been retained too divergent from vertebrate galectins. In the present paper, to contribute in disclosing Bi-CRD galectin evolution a novel attempt was carried out on CiLgals-a and -b CRDs phylogenetic analysis, and their involvement in ascidian inflammatory responses was shown. CiLgals resulted aligned with Bi-CRD galectins from vertebrates (Xenopus tropicalis, Gallus gallus, Mus musculus, Homo sapiens), cephalochordates (Branchiostoma floridae), echinoderms (Strongylocentrotus purpuratus) and a mono-CRD galectin from the ascidian Clavelina picta. The CiLgals-a N-terminal and C-terminal CRDs contain the signature sequence involved in carbohydrate binding, whereas the CiLgals-b C-CRD presents only three out of seven key aminoacids and it could not be suitable as sugar binding motif. Sequence similarity between clusters suggests an evolutionary model based on CRD domain gene duplication and sequence diversification. In particular CiLgals-b N-CRD and C-CRD were similar to each other and both grouped with the ascidian C. picta mono-CRD. Homology modeling process shows a CiLgals molecular structure superimposed to chicken and mouse galectins. The CiLgals-a and CiLgals-b genes were upregulated by LPS inoculation suggesting that they are inducible and expressed in the inflamed pharynx as revealed by real-time PCR analysis. Finally, in situ hybridization and immunohistochemical assays showed their localization in the inflamed tissues, while immunoblotting analysis indicated that CiLgals can form oligomers., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The CAP superfamily is a group of proteins that have been linked to several biological functions such as reproduction, cancer, and immune defense. A differential screening between lipopolysaccharide (LPS)-challenged and naive Ciona intestinalis has been performed to identify LPS-induced genes. This strategy has allowed the isolation of a full-length 1471-bp cDNA encoding for a 413-amino-acid protein (CiCAP). In silico analysis has shown that this polypeptide displays a modular structure with similarities to vertebrate CAP-superfamily proteins and to a collagen-binding adhesin of Streptococcus mutans. Domain organization analysis and alignment of CiCAP to other vertebrate CAP proteins have revealed a novel structure suggesting that this protein originated from a common ancestor gene that gave rise to many subfamilies of mosaic proteins with novel functions. Quantitative mRNA expression performed by real-time polymerase chain reaction analysis has demonstrated that this gene is rapidly activated in the pharynx of C. intestinalis a few hours after LPS injection. Moreover, in situ hybridization has shown that CiCAP mRNA is highly expressed by hemocytes with large granules contained inside the pharynx vessels. Thus, CiCAP represents a protein with novel structural domains involved in ascidian immune responses.

Stressful conditions include a prompt release of corticosteroid hormones which can mediate gene expression through glucocorticoid receptors (GR). Since two seabass (Dicentrarchus labrax) GRs have been cloned and sequenced from peritoneal cavity cells (DlGR1) and liver (DlGR2), a comparative amino acid sequence analysis that included Haplochromis burtoni HbGRs, was carried out and homologies disclosed. The DlGR1 and DlGR2 deduced aminoacid sequences showed 61% identity (I) and 70% similarity (S). Moreover, DlGR2 was similar to HbGR2b (69% I, 73% S), and the DlGR1 to HbGR1 (72% I, 78% S). In addition, we examined the expression of the DlGRs after exogeneous cortisol inoculation into the peritoneal cavity, mimicking stress effects. At various times after the administration (3 h, 24 h, 1 week), gene expressions was evaluated in head kidney by real-time PCR. In addition, immunoblotting and densitometry analyses were performed with anti-DlGR1 antibodies. Although sea bass head kidney expressed both DlGR1 and DlGR2 they were differentially modulated by intraperitoneal implant of exogeneous cortisol., (Copyright (c) 2010 Elsevier Inc. All rights reserved.)

The purification, cloning, sequencing, molecular properties and expression of a fucose-binding lectin from the serum of Dicentrarchus labrax (DlFBL) have been previously reported. We now describe the distribution and expression of DlFBL during fish ontogeny. Immunohistochemistry and in situ hybridization assays were carried out at various developmental stages (from 10 days post-hatching larvae to juveniles). Another fucose-binding lectin, similar to DlFBL in biochemical, immunochemical and agglutinating properties, was extracted and purified from eggs and appeared to be localized in the embryo yolk sack residual. DlFBL was found in columnar and goblet cells of the intestinal epithelium of larvae (from 20 days post-hatching) and juveniles and in parenchymal tissue of juveniles. DlFBL mRNA and protein were detected in the intestinal epithelium and in hepatocytes. An amplification product from degenerate primers indicates that lectin isotypes with DlFBL epitopes are expressed in eggs and embryos. Whether the lectin fraction isolated from eggs and embryos includes DlFBL of maternal origin remains unclear.

In situ hybridisation and immunohistochemistry analyses have shown that the Ciona intestinalis tumour necrosis factor alpha gene (CiTNFalpha), which has been previously cloned and sequenced, is expressed either during the inflammatory pharynx response to lipopolysaccharide (LPS) or during the swimming larval phase of development. Granulocytes with large granules and compartment/morula cells are CiTNFalpha-producing cells in both inflamed pharynx and larvae. Pharynx vessel endothelium also takes part in the inflammatory response. Haemocyte nodules in the vessel lumen or associated with the endothelium suggest the involvement of CiTNFalpha in recruiting lymphocyte-like cells and promoting the differentiation of inflammatory haemocytes. Specific antibodies against a CiTNFalpha peptide have identified a 43-kDa cell-bound form of the protein. Observations of pharynx histological sections (at 4 and 8 h post-LPS inoculation) from naive and medium-inoculated ascidians have confirmed the CiTNFalpha-positive tissue response. Larval histological sections and whole-mount preparations have revealed that CiTNFalpha is expressed by trunk mesenchyme, preoral lobe and tunic cells, indicating CiTNFalpha-expressing cell immigration events and an ontogenetic role.

Collectins are a family of calcium-dependent lectins that are characterized by their collagen-like domains. Considerable interest has been focused on this class of proteins because of their ability to interact with components of the complement system activating a cascade of events responsible for the activation of the innate immune system. A differential screening between LPS-challenged and naïve Ciona intestinalis has been performed allowing the isolation of a full length cDNA encoding for a 221 AA protein. In silico analysis has shown that this polypeptide displays protein domains with similarities to mannose-binding lectins. A phylogenetic analysis suggested that C. intestinalis MBL has evolved early as a prototype of vertebrate MBL. Real-time PCR assay demonstrated that this gene is strongly activated after LPS injection in the tunica. In situ hybridization performed in LPS-induced animals has shown that this gene is expressed in granular amoebocytes and large granules hemocytes in the inflamed body wall tissue. Finally, an antimicrobial activity of the C. intestinalis MBL has been demonstrated.

A tumor necrosis factor-alpha (TNFalpha)-like gene from Ciona intestinalis (CiTNF alpha-like) body wall challenged with bacterial lipopolysaccharide (LPS) was cloned and sequenced 4 h after LPS inoculation. An open reading frame of 936 bp encoding a propeptide of 312 amino acids (35.4 kDa) displaying a transmembrane domain from positions 7 to 29, a TACE cleavage site, and a mature peptide domain of 185 amino acids (20.9 kDa), was determined with a predicted isoelectric point of 9.4. The phylogenetic tree based on deduced amino acid sequences of invertebrate TNF-like protein and vertebrate TNFs supported the divergence between the ascidian and vertebrate TNF families, whereas D. melanogaster Eiger A and B TNF-like sequences were distinctly separated from the chordate TNFs. Thus, the ascidian TNFalpha-like cytokine was upregulated by in vivo LPS challenge supporting its pro-inflammatory role. In the pharynx, increased expression levels were found following analysis by real-time polymerase chain reaction, whereas in situ hybridization assay showed positive hemocytes both in the tissue and in circulating hemocytes. Finally, Western blot with monoclonal antibodies disclosed human TNFalpha epitopes in a 15-kDa protein component of the hemolymph serum and in a 43-kDa protein contained in the hemocyte lysate supernatant prepared in the presence of detergents. Both soluble and hemocyte-bound CiTNF alpha-like protein therefore appeared to be modulated by the LPS challenge.

Based on previous cloning and sequencing study, real-time PCR and in situ hybridization assays of the inflamed body wall of LPS-injected Ciona intestinalis showed the enhanced gene expression of a collagen with FACIT structural features (Ci-type IX-Col 1alpha-chain). By using specific antibodies raised against an opportunely chosen Ci-type IX-Col synthetic peptide, the fibroblast property of hemocytes challenged in vitro with LPS (at 4h) was displayed by flow cytometry, while immunocytochemistry identified hemocytes with large granules (morula cells) as collagen-producing cells. Hemocyte lysate supernatant analyzed in immunoblotting contained a 60 kDa band identifiable as 1alpha-chain-Ci-type IX-Col. Observations of body wall sections (immunohistochemistry method) supported the role of hemocytes and showed that epidermis expressed Ci-type IX-Col 1alpha-chain in the time course of the inflammatory reaction (within 24h). Transcript and protein were mainly found in the epidermis that outlined the proximal side of the tunic matrix (at 24h after LPS injection), in cells associated with the epidermis at 4 and 192 h. In conclusion, the C. intestinalis inflammatory response to LPS challenge appeared to be composed of a complex reaction set, and for the first time we showed in ascidians a granulation tissue with FACIT-collagen production that could participate in inflammation and wound healing. Like in vertebrates, C. intestinalis acute inflammatory reactions result in a regulated pattern of tissue repair with collagen expression during remodelling. Ci-type IX-Col could be involved in a network of non-fibril-forming collagens that participates in the organization of extracellular matrix and defense responses.

Studies on inducible ascidian lectins may shed light on the evolutionary emergence of cytokine functions. Here, we show that the levels of opsonins, with IL1alpha-epitopes, increase in Ciona intestinalis hemolymph as a response to an inflammatory stimulus and, in particular, to intratunic injection of lipopolysaccharide (LPS). The inflammatory agent promptly (within 4 h) enhances Ca(2+)-independent serum hemagglutinating and opsonizing activities, which are both inhibited by D-galactose and D-galactosides (alpha-lactose, N-acetyl-D-lactosamine, thio-digalactoside), suggesting that anti-rabbit erythrocyte lectins with galectin properties are involved as opsonins. Inducible galectin molecules contain interleukin-1alpha (IL1alpha) epitopes, and their activities are specifically inhibited by anti-human recombinant IL1alpha antibody. Analysis by SDS-polyacrylamide gel electrophoresis has revealed that the density of the bands of several serum proteins increases within 4 h after LPS injection, correlated with the enhanced serum activity. Moreover, Western blot patterns demonstrate that several serum proteins (59, 37, 30, 23, 15 kDa) cross-react with the antibody as early as 4 h post-injection. Although we have not been able to establish whether, in adition to galectins, various types of D-galactose-specific lectins are contained in the serum, we show, for the first time in invertebrates, that galectin molecules with opsonic properties can be enhanced in response to a non-specific inflammatory stimulus, and that their release can be further stimulated by LPS. Finally, we reveal that multiple galectins share human IL1alpha epitopes, probably because of steric configuration and the oligomerization process.

To gain further insight into the role of cortisol in fish innate immune responses, we cloned and sequenced a 2592bp cDNA from sea bass (Dicentrarchus labrax) peritoneal leukocytes (PCLs) encoding a glucocorticoid receptor (DlGR1). The deduced aminoacid sequence displayed that DlGR1 belong to a multigenic family of steroid hormone receptors, and exhibited high homology (80%) to the Burton's mouth breeder (Haplochromis burtoni) HbGR1. The DlGR1 functional domains presented homologies with those of several vertebrate species. In situ hybridization assay revealed that DlGR1 was expressed in macrophages and neutrophils from the peritoneal cavity. Since in a previous paper, sea bass PCL chemiluminescence response (CL) has been related to increased respiratory burst of phagocytes stimulated with zymosan, PCLs, pre-incubated in vitro with cortisol at various concentrations, were assayed for their CL response. Dose-dependent cortisol inhibitory effects, and significant competitive activity of a low concentration of mifepristone (RU486), a glucocorticoid-receptor blocker, supported that cortisol-GR interaction was involved in modulating CL response via a genomic pathway. Results also indicated that cortisol could be effective through an additional not-genomic way, and showed that high doses of RU486 exerted an inhibitory effect on PCL chemiluminescence activity.

A novel fucose-binding lectin, designated SauFBP32, was purified by affinity chromatography on fucose-agarose, from the serum of the gilt head bream Sparus aurata. Electrophoretic mobility of the subunit revealed apparent molecular weights of 35 and 30 kDa under reducing and non-reducing conditions, respectively. Size exclusion analysis suggests that the native lectin is a monomer under the selected experimental conditions. Agglutinating activity towards rabbit erythrocytes was not significantly modified by addition of calcium or EDTA; activity was optimal at 37 degrees C, retained partial activity by treatment at 70 degrees C, and was fully inactivated at 90 degrees C. On western blot analysis, SauFBP showed intense cross-reactivity with antibodies specific for a sea bass (Dicentrarchus labrax) fucose-binding lectin. In addition, the similarity of the N-terminal sequence and a partial coding domain to teleost F-type lectins suggests that SauFBP32 is a member of this emerging family of lectins.

Collagens are highly preserved proteins in invertebrates and vertebrates. To identify the collagens in urochordates, the total RNA extracted from the pharynx of the ascidian Ciona intestinalis was hybridized with a heterologous probe specific for the echinoderm Paracentrotus lividus fibrillar type I-like larval collagen. Using this probe, two main bands (i.e. 6 and 2.8 kb mRNA) were observed on Northern blot hybridization. The cDNA library prepared from poly(A)+RNA extracted from pharyngeal tissue was screened and a cDNA that specifies a type IX-like collagen was identified. This molecule presents a conceptual open reading frame for a protein containing 734 amino acids. In particular, we showed a 1 alpha chain type IX-like collagen characterized by three short triple-helical domains interspersed with four non-triple-helical sequences, with structural features of fibril-associated collagens with interrupted triple-helices (FACIT) collagens. Northern blot hybridizations indicate a 2.8 kb transcript size. Sequence comparison indicated homology (47.64%, 48.95%) between the type IX-like collagen of C. intestinalis and mouse and human type IX collagen. In situ hybridization of tunic and pharynx tissues shows the presence of transcripts in connective tissue cells.