Motility of Mytilus galloprovincialis hemocytes: Sensitivity to paracetamol in vitro exposure.

Pharmaceuticals released into the environment (PiEs) represent an environmental problem of growing concern for the health of ecosystems and humans. An increasing number of studies show that PiEs pose a risk to aquatic organisms. The aim of the present work was to contribute to increasing the knowledge of the effects of PiE on marine biota focusing on the effect of paracetamol on the motility of hemocytes in Mytilus galloprovincialis, a bivalve mollusk species widely utilized as bioindicator organism. Hemocytes are the immunocompetent cells of bivalve mollusks. An early and key stage of mollusk immune response is represented by the recruitment and migration of these cells to the site of infection. Therefore, motility is an intrinsic characteristic of these cells. Here, we first characterized the spontaneous cell movement of M. galloprovincialis hemocytes when plated in a TC-treated polystyrene 96-well microplate. Two different cellular morphotypes were distinguished based on their appearance and motility behavior: spread cells and round-star-shaped cells. The two motility morphotypes were characterized by different velocities as well as movement directness, which were significantly lower in round-star-shaped cells with respect to spread cells. The sensitivity of the motility of M. galloprovincialis hemocytes to paracetamol at different concentrations (0.02, 0.2 and 2 mg/L) was investigated in vitro after 1h and 24h exposure. Paracetamol induced alterations in the motility behavior (both velocity and trajectories) of the hemocytes and the effects were cell-type specific. The study of hemocyte movements at the single cell level by cell tracking and velocimetric parameters analysis provides new sensitive tools for assessing the effects of emerging pollutants at the cellular levels in non-target organisms.

In Vitro Anti-Inflammatory and Vasculoprotective Effects of Red Cell Extract from the Black Sea Urchin Arbacia lixula.

Sea urchins have emerged as an important source of bioactive compounds with anti-inflammatory and antioxidant properties relevant to human health. Since inflammation is a crucial pathogenic process in the development and progression of atherosclerosis, we here assessed the potential anti-inflammatory and vasculoprotective effects of coelomic red-cell methanolic extract of the black sea urchin Arbacia lixula in an in vitro model of endothelial cell dysfunction. Human microvascular endothelial cells (HMEC-1) were pretreated with A. lixula red-cell extract (10 and 100 µg/mL) before exposure to the pro-inflammatory cytokine tumor necrosis factor (TNF)-α. The extract was non-toxic after 24 h cell treatment and was characterized by antioxidant power and phenol content. The TNF-α-stimulated expression of adhesion molecules (VCAM-1, ICAM-1) and cytokines/chemokines (MCP-1, CCL-5, IL-6, IL-8, M-CSF) was significantly attenuated by A. lixula red-cell extract. This was functionally accompanied by a reduction in monocyte adhesion and chemotaxis towards activated endothelial cells. At the molecular level, the tested extract significantly counteracted the TNF-α-stimulated activation of the pro-inflammatory transcription factor NF-κB. These results provide evidence of potential anti-atherosclerotic properties of A. lixula red-cell extract, and open avenues in the discovery and development of dietary supplements and/or drugs for the prevention or treatment of cardiovascular diseases.

Microalgae: A Promising Future.

Microalgae are photosynthetic unicellular microorganisms that represent an extremely important component of the aquatic ecosystem productivity, diversity, and functioning [...].

Cytoskeletal Responses and Aif-1 Expression in Caco-2 Monolayers Exposed to Phorbol-12-Myristate-13-Acetate and Carnosine.

The dis(re)organization of the cytoskeletal actin in enterocytes mediates epithelial barrier dys(re)function, playing a key role in modulating epithelial monolayer's integrity and remodeling under transition from physiological to pathological states. Here, by fluorescence-based morphological and morphometric analyses, we detected differential responses of cytoskeletal actin in intestinal epithelial Caco-2 cell monolayers at two different stages of their spontaneous differentiation, i.e., undifferentiated cells at 7 days post-seeding (dps) and differentiated enterocyte-like cells at 21 dps, upon challenge in vitro with the inflammation-mimicking stimulus of phorbol-12-myristate-13-acetate (PMA). In addition, specific responses were found in the presence of the natural dipeptide carnosine detecting its potential counteraction against PMA-induced cytoskeletal alterations and remodeling in differentiated Caco-2 monolayers. In such an experimental context, by both immunocytochemistry and Western blot assays in Caco-2 monolayers, we identified the expression of the allograft inflammatory factor 1 (AIF-1) as protein functionally related to both inflammatory and cytoskeletal pathways. In 21 dps monolayers, particularly, we detected variations of its intracellular localization associated with the inflammatory stimulus and its mRNA/protein increase associated with the differentiated 21 dps enterocyte-like monolayer compared to the undifferentiated cells.

Bioactive Potential of Two Marine Picocyanobacteria Belonging to Cyanobium and Synechococcus Genera.

Coccoid cyanobacteria produce a great variety of secondary metabolites, which may have useful properties, such as antibacterial, antiviral, anticoagulant or anticancer activities. These cyanobacterial metabolites have high ecological significance, and they could be considered responsible for the widespread occurrence of these microorganisms. Considering the great benefit derived from the identification of competent cyanobacteria for the extraction of bioactive compounds, two strains of picocyanobacteria (coccoid cyanobacteria < 3 µm) (Cyanobium sp. ITAC108 and Synechococcus sp. ITAC107) isolated from the Mediterranean sponge Petrosia ficiformis were analyzed. The biological effects of organic and aqueous extracts from these picocyanobacteria toward the nauplii of Artemia salina, sea urchin embryos and human cancer lines (HeLa cells) were evaluated. Methanolic and aqueous extracts from the two strains strongly inhibited larval development; on the contrary, in ethyl acetate and hexane extracts, the percentage of anomalous embryos was low. Moreover, all the extracts of the two strains inhibited HeLa cell proliferation, but methanol extracts exerted the highest activity. Gas chromatography-mass spectrometry analysis evidenced for the first time the presence of ß-N-methylamino-l-alanine and microcystin in these picocyanobacteria. The strong cytotoxic activity observed for aqueous and methanolic extracts of these two cyanobacteria laid the foundation for the production of bioactive compounds of pharmacological interest.

Chemical characterization of red cells from the black sea urchin Arbacia lixula by X-ray photoelectron spectroscopy.

Red spherula cells (RSC) from sea urchin coelomic fluid have attracted great interest for their specific and intriguing properties, such as for example antimicrobial activities and immune response, that probably tie in with their red characteristic pigments. Although to date different studies have been reported aimed to chemically characterize their pigments extracted from the cells, few data are available about the chemical characterization of the cell surface. In this work, a systematic chemical characterization of the RSC surface by X-ray photoelectron spectroscopy (XPS) analysis is described. The results were compared with data on colorless cells from the same coelomic fluid sample. Our observations evidenced that the two cell types were characterized by the presence of different chemical functional groups. In particular, the colorless cells are dominated by the presence of alkyl, alcohol, amide, and carboxyl groups in accordance with other similar cell types, enriched in Na+ and Cl- ions. Traces of elements like S (sulphonates) and P (phosphates) are also present. On the other hand, the RSC in addition to the alkyl groups show a reduction in the content of amide groups, accompanied by the anomalous presence of keto-enolic groups that probably can be associated with the presence of quinones/hydro-quinones from red pigments. A chemical enrichment in elements such as Cl- and Mg2+ and sulphate groups (-R-O-SO3 -), as well as the presence of sulphides and phosphates traces, is evident. The absence of carbonate groups is also observed in both cell populations, confirming the absence of sodium and magnesium carbonate salts. No traces of toxic elements (i.e., heavy metals) have been revealed.

Allograft Inflammatory Factor-1 in Metazoans: Focus on Invertebrates.

Allograft inflammatory factor-1 (AIF-1) is a calcium-binding scaffold/adaptor protein often associated with inflammatory diseases. Originally cloned from active macrophages in humans and rats, this gene has also been identified in other vertebrates and in several invertebrate species. Among metazoans, AIF-1 protein sequences remain relatively highly conserved. Generally, the highest expression levels of AIF-1 are observed in immunocytes, suggesting that it plays a key role in immunity. In mammals, the expression of AIF-1 has been reported in different cell types such as activated macrophages, microglial cells, and dendritic cells. Its main immunomodulatory role during the inflammatory response has been highlighted. Among invertebrates, AIF-1 is involved in innate immunity, being in many cases upregulated in response to biotic and physical challenges. AIF-1 transcripts result ubiquitously expressed in all examined tissues from invertebrates, suggesting its participation in a variety of biological processes, but its role remains largely unknown. This review aims to present current knowledge on the role and modulation of AIF-1 and to highlight its function along the evolutionary scale.

Screening of Three Echinoderm Species as New Opportunity for Drug Discovery: Their Bioactivities and Antimicrobial Properties.

Echinoderms are a renewable resource with an economic value due to their increasing demand as food and/or source of bioactive molecules exerting antitumor, antiviral, anticoagulant, antioxidant, and antimicrobial activities. In this framework, the present study is aimed at investigating the antibacterial, antioxidant, and hemolytic activities in the three Echinoderm species Echinaster sepositus, Arbacia lixula, and Sphaerechinus granularis. The sea star E. sepositus showed lysozyme-like activity (mean diameter of lysis of 13.4 ± 0.2 mm), an antimicrobial activity against the human emerging pathogens Staphylococcus aureus, Pseudomonas aeruginosa, and Candida famata, and a strong lytic activity (100 ± 0.05%) towards the human red blood cells. Furthermore A. lixula and E. sepositus had the highest antioxidant activity (1792.75 ± 233.7 and 1765.65 ± 484.58 nmolTE/mL, resp.). From toxicological assays, it was shown that E. sepositus was not toxic towards HeLa cells and Vibrio fischeri, encouraging the exploitation of this species in the pharmaceutical field. Therefore, our findings have implications due to the ongoing explosion of antibiotic-resistant infections because of the new opportunistic pathogens and the need to discover antibacterial agents with new modes of action. Also the recorded antioxidant activity taking into account the need to find natural antioxidants useful for human health is intriguing.

Molecular and expression analysis of the Allograft inflammatory factor 1 (AIF-1) in the coelomocytes of the common sea urchin Paracentrotus lividus.

Allograft inflammatory factor 1 (AIF-1) is a highly conserved gene involved in inflammation, cloned and characterized in several evolutionary distant animal species. Here, we report the molecular identification, characterization and expression of AIF-1 from the common sea urchin Paracentrotus lividus. In this species, AIF-1 encodes a predicted 151 amino acid protein with high similarity to vertebrate AIF-1 proteins. Immunocytochemical analyses on coelomocytes reveal localization of the AIF-1 protein in amoebocytes (perinuclear cytoplasmic zone) and red sphaerulocytes (inside granules), but not in vibratile cells and colorless sphaerula cells. The significant increase of AIF-1 expression (mRNA and protein) found in the coelomocytes of the sea urchin after Gram + bacterial challenge suggests the involvement of AIF-1 in the inflammatory response. Our analysis on P. lividus AIF-1 contributes to elucidate AIF-1 function along the evolutionary scale and consolidate the key evolutionary position of echinoderms throughout metazoans with respect to the common immune paths.

The sea urchin Paracentrotus lividus immunological response to chemical pollution exposure: The case of lindane.

In the marine environment organochlorine insecticides can be broadly detected in water, sediments, and biota. These pollutants may have major ecological consequences since they may affect marine organisms and endanger organismal growth, reproduction or survival. In this study we investigated the modification of some sea urchin immunological parameters in response to subchronic lindane (γ-HCH) exposure. Adult specimens of the sea urchin Paracentrotus lividus were exposed to two different concentrations (0.1 and 0.5 mg L(-1)) of lindane. After 24 and 48h of treatment, we examined the lindane influence on coelomocytes vitality and enumeration as well on some humoral parameters. Our results showed that the presence of the pesticide affected both cellular and humoral components of the immune system. In particular, P. lividus coelomocytes vitality did not change but a decrease of the total cell number and an increase of the red cells was recorded. Haemolytic and lysozyme-like activities as well as antibacterial activity on Vibrio alginolyticus of treated animals decreased. Sea urchin immunological competence modifications might represent a tool for monitoring disease susceptibility thus providing biological criteria for the implementation of water quality standards to protect marine organisms.

Toxicity assessment of Amphidinium carterae, Coolia cfr. monotis and Ostreopsis cfr. ovata (Dinophyta) isolated from the northern Ionian Sea (Mediterranean Sea).

In many coastal areas the abundant proliferation of microalgae producing biotoxins determines the occurrence of Harmful Algal Blooms (HABs). Their presence in temperate waters is well documented and often associated with marine toxin-derived disease. The occurrence and toxicity of three harmful microalgae (Amphidinium carterae, Coolia cfr. monotis and Ostreopsis cfr. ovata) from the northern Ionian Sea (Mediterranean Sea) is hereby reported. The three dinoflagellates were sampled both on macroalgae and water and their morphology and occurrence were compared to those of other Mediterranean sites. The toxicity of the three cultured strains was tested by Artemia salina and hemolysis tests and their effects on the first stages of the sea urchin development was also evaluated. The contemporary presence of the three species inhibited the in vitro sea urchin embryonic development. But this action could be ascribed to the sole Ostreopsis as the addition of the single species to the sea urchins embryos evidenced no effects in presence of Amphidinium or Coolia cells, and an irregular segmentation in presence of Ostreopsis. In particular, this latter species exerted a cytotoxic effect in a dose-dependent manner, with a production of deformed embryos even at very low cell concentration (42 cells mL⁻¹). Nevertheless, when algal cell lysate was added, some effects on the sea urchin development was detected for each dinoflagellate, and also in this case Ostreopsis has proved to be the most toxic species. However, the lysate of Amphidinium and Ostreopsis strongly affects the A. salina nauplii vitality, while the hemolytic activity was very low for Amphidinium and Coolia lysate and very strong for Ostreopsis. Our results highlight the importance to monitoring the presence of these dinoflagellates whose effects may also be reflected on the early life stages of marine organisms, especially those species that are important from both an ecological and economic point of view, as the sea urchins are.

Zinc effect on the sea urchin Paracentrotus lividus immunological competence.

Pollution by heavy metals has become one of the most important problems in marine coastal areas as a consequence of anthropogenic inputs. Among metal contaminants, zinc, being considered not very toxic, is sometimes released into the sea in appreciable quantities and its concentration is loosely regulated. In this work we analyzed the effects of a high zinc concentration on the sea urchin Paracentrotus lividus immune system. In particular, after 24 h of zinc treatment, we evaluated coelomocytes morphology and composition as well as the zinc influence on some humoral parameters such as hemolysis, lysozyme-like activity and antibacterial activity on Vibrio alginolyticus. Our results evidenced that the presence of zinc affected both cellular and acellular components of the sea urchin immune system. The P. lividus coelomocytes changed in morphology and number; moreover, the amebocytes changed from a petaloid to a filipodial-like shape and the red spherula cells increased in number. Among the considered humoral effectors lysozyme-like activity and antibacterial activity on V. alginolyticus decreased in short-term to zinc treatment. The modifications in the sea urchin immunological competence might give an early indication of disease susceptibility thus suggesting to consider the examined defence mechanisms as potential biological indicators of metal pollution.

Cytotoxic and antimitotic activities in aqueous extracts of eight cyanobacterial strains isolated from the marine sponge Petrosia ficiformis.

Marine cyanobacteria are photosynthetic prokaryotes of significant ecological interest, living free or in association with invertebrates. They are also considered as excellent sources of antineoplastic, antibacterial, antiviral and antifungal compounds. In this work, aqueous extracts from eight cyanobacterial strains isolated from the Mediterranean sponge Petrosia ficiformis have been investigated for their bioactive properties. Bioassays with human erythrocytes, Artemia salina nauplii, and Paracentrotus lividus gametes and embryos were performed. Some aqueous extracts exhibited citolytic effect on human erythrocytes and toxic activity against A. salina nauplii. Furthermore antimitotic activity was evidenced during sea urchin embryos development and disorganization of blastomeres with altered cell-cell contact was also induced. Some of the isolated cyanobacterial strains, belonging to Leptolyngbya and Synechococcus genera with an high citotoxic activity, should be further investigated to better characterize their bioactive molecules. Our data confirm cyanobacteria as an interesting source of novel bioactive compounds with potential applications in pharmaceutics.

Kupffer cells promote lead nitrate-induced hepatocyte apoptosis via oxidative stress.

BACKGROUND: Apoptosis and its modulation are crucial factors for the maintenance of liver health, allowing hepatocytes to die without provoking a potential harmful inflammatory response through a tightly controlled and regulated process. Since Kupffer cells play a key role in the maintenance of liver function, the aim of this study was to verify whether Kupffer cells are involved in the induction of liver apoptosis after i.v. injection of Pb(NO3)2 likely by secretion mechanisms. RESULTS: The in vivo hepatic apoptosis, induced by Pb(NO3)2 was prevented by a pre-treatment with gadolinium chloride (GdCl3), a Kupffer cells toxicant, that suppresses Kupffer cell activity and reduces to a half the apoptotic rate. In addition, in vivo Pb(NO3)2 administration deprives hepatocytes of reduced glutathione, whereas the loss of this important oxidation-preventing agent is considerably mitigated or abolished by pre-treatment with GdCl3. However, incubation of isolated hepatocytes and Kupffer cells and HepG2 cells with Pb(NO3)2 for 24 hours induced necrotic but not apoptotic cells. Apoptosis of hepatocytes and HepG2 cells was observed only after the addition of conditioned medium obtained from Kupffer cells cultured for 24 hours with Pb(NO3)2, thus indicating the secretion of soluble mediators of apoptosis by Kupffer cells. Apoptosis in the HepG2 cells was observed upon 24-hours incubation of HepG2 cells with 1 mM buthionine sulfoximine, a glutathione depleting agent, thus showing that there is an oxidative apoptogenic pathway in HepG2 cells. CONCLUSION: Pb(NO3)2 has, at most, a direct necrotic (but not apoptogenic) effect on hepatocytes and HepG2 cells, giving a clue about the regulatory role of Kupffer cells in the induction of liver apoptosis after a single Pb(NO3)2 injection without pre-treatment with GdCl3, probably via secreting soluble factors that trigger oxidative stress in target cells.

Phagocytosis of apoptotic cells by liver: a morphological study.

The present review deals with the morphological features of the removal of apoptotic cells by liver. The engulfment of cells undergoing apoptosis can be considered a specialized form of phagocytosis, playing a major role in the general tissue homeostasis in physiological and pathological conditions. In fact, defects of phagocytosis of apoptotic cells might have deleterious consequences for neighboring healthy cells, i.e., pathogenesis of inflammatory disease or dysregulation of the immune system. Phagocytosis of apoptotic cells by liver is a complex phenomenon, involving multiple molecular mechanisms of recognition (i.e., lectin-like receptors and receptors for externalized phosphatydilserine) of both parenchymal (hepatocytes) and nonparenchymal (Kupffer and endothelial cells) liver cells, often operating in cooperation. The data discussed in the present review are drawn from studies of phagocytosis of apoptotic cells in the liver, carried out with in vivo and in situ adhesion experiments as well as in vitro assays. Our results indicate that the three main liver cell types (hepatocytes, Kupffer, and endothelial cells) are able to recognize and internalize apoptotic cells by means of specific receptors (galactose and mannose-specific receptor; receptor for phosphatydilserine) and by cytoskeletal reorganization that favors the engulfment of the apoptotic cells. The "flags" for the identification of apoptotic cells by the liver are modifications of the surface of dead cells, i.e., sugar residues and phosphatydilserine exposition. Vitronectin receptor is not involved in such a recognition. The adhesions between modified cell surfaces of apoptotic cells and phagocytes generate cytoplasmatic signaling pathways that drive apoptotic cells to their final fate within the phagocytes (i.e., lysosomal digestion).