Morpho-structural adaptations of the integument in different aquatic organisms.

The integument acts as a barrier to protect the body from harmful pathogenic infectious agents, parasites, UV rays, trauma, and germs. The integument of invertebrates and vertebrates are structurally different: while invertebrates usually have a simple monolayer epidermis frequently covered by mucus, cuticles, or mineralized structures, vertebrates possess a multilayered epidermis with several specialized cells. This study aims to describe by morphological, histological, and immunohistochemical analyses, the morpho-structural adaptations throughout evolution of the integument of gastropod Aplysia depilans (Gmelin, 1791), ascidian Styela plicata (Lesuer, 1823), myxine hagfish Eptatretus cirrhatus (Forster, 1801) and teleost Heteropneustes fossilis (Bloch, 1794) for the first time, with special reference to sensory epidermal cells. Different types of cells could be identified that varied according to the species; including mucous cells, serous glandular cells, clavate cells, club cells, thread cells, and support cells. In all integuments of the specimens analyzed, sensory solitary cells were identified in the epidermis, immunoreactive to serotonin and calbindin. Our study provided an essential comparison of integuments, adding new information about sensory epidermal cells phylogenetic conservation and on the structural changes that invertebrates and vertebrates have undergone during evolution.

Spirulina promotes macrophages aggregation in zebrafish (Danio rerio) liver.

The immune system of teleosts offers many ideas to deepen the immune mechanisms and cells in general. The use of zebrafish as an experimental model is increased in recent years, thanks to its genetic and anatomical characteristics. It is known that several natural compounds exert an action on the immune system, boosting it. Spirulina, a non-toxic blue-green alga, has been declared a superfood for its peculiar biological activities. In this study, we test the immunostimulant effect of spirulina on zebrafish liver macrophages by immunohistochemical analysis using optical and confocal microscopy. Our results have shown an increase in the number of macrophages after feeding with spirulina, furthermore, this natural 'superfood' can induce macrophages aggregation. These data not only provide information on the possible effect of this alga as a complementary feed on the immune cells of teleost, but also improve the knowledge of the immune mechanisms of vertebrates.

Confocal Identification of Immune Molecules in Skin Club Cells of Zebrafish (Danio rerio, Hamilton 1882) and Their Possible Role in Immunity.

The immune system of a fish has cellular and molecular defense mechanisms that are substantially retained throughout the evolution of vertebrates. The innate immune system provides biological processes, such as phagocytosis and mechanical barriers, to implement an efficient defensive response after exposure to chemical or biological contaminants, pollutants, and contact with parasites, germs, and pathogens. Club cells (CCs) are widespread in the skin of Ostariophysi. After a predator attack or exposure to toxins and parasites, these cells can produce alarming substances. Given their effectiveness against viruses, parasites, and common skin lesions, recent studies have suggested that CCs are a component of the immune system. This study aims to immunohistochemically characterize the CCs for the first time in the skin of zebrafish, using mitogen-activated protein kinase (MAPK) p38, Toll-like receptor (TLR)2, Piscidin1, and inducible nitric oxide synthase (iNOS) peptides involved in the function of all types of vertebrate immune cells. According to our analysis, the intermediate layer of the epidermis exhibited rounded, oval, and elongated CCs, with central acidophilic cytoplasm and a spherical basophilic nucleus, that are positive to the antibodies tested. Our results may confirm that CCs could be involved in the immune function, increasing our knowledge of the immune system of teleosts.

Expression of Antimicrobic Peptide Piscidin1 in Gills Mast Cells of Giant Mudskipper Periophthalmodon schlosseri (Pallas, 1770).

The amphibious teleost Giant mudskipper (Periophthalmodon schlosseri, Pallas 1770) inhabit muddy plains and Asian mangrove forests. It spends more than 90% of its life outside of the water, using its skin, gills, and buccal-pharyngeal cavity mucosa to breathe in oxygen from the surrounding air. All vertebrates have been found to have mast cells (MCs), which are part of the innate immune system. These cells are mostly found in the mucous membranes of the organs that come in contact with the outside environment. According to their morphology, MCs have distinctive cytoplasmic granules that are released during the degranulation process. Additionally, these cells have antimicrobial peptides (AMPs) that fight a variety of infections. Piscidins, hepcidins, defensins, cathelicidins, and histonic peptides are examples of fish AMPs. Confocal microscopy was used in this study to assess Piscidin1 expression in Giant Mudskipper branchial MCs. Our results demonstrated the presence of MCs in the gills is highly positive for Piscidin1. Additionally, colocalized MCs labeled with TLR2/5-HT and Piscidin1/5-HT supported our data. The expression of Piscidin1 in giant mudskipper MCs highlights the involvement of this peptide in the orchestration of teleost immunity, advancing the knowledge of the defense system of this fish.

Confocal Characterization of Intestinal Dendritic Cells from Myxines to Teleosts.

Dendritic cells (DCs) are antigen-presenting cells (APCs) that regulate the beginning of adaptive immune responses. The mechanisms of tolerance to antigens moving through the digestive tract are known to be regulated by intestinal DCs. Agnatha and Gnathostoma are descendants of a common ancestor. The Ostracoderms gave rise to Cyclostomes, whereas the Placoderms gave rise to Chondrichthyes. Sarcopterygii and Actinopterygii are two evolutionary lines of bony fishes. Brachiopterygii and Neopterygii descend from the Actinopterygii. From Neopterygii, Holostei and Teleostei evolved. Using immunohistochemistry with TLR-2, Langerin/CD207, and MHC II, this study aimed to characterize intestinal DCs, from myxines to teleosts. The findings reveal that DCs are positive for the antibodies tested, highlighting the presence of DCs and DC-like cells phylogenetically from myxines, for the first time, to teleosts. These findings may aid in improving the level of knowledge about the immune system's evolution and these sentinel cells, which are crucial to the body's defense.

Detecting Intestinal Goblet Cells of the Broadgilled Hagfish Eptatretus cirrhatus (Forster, 1801): A Confocal Microscopy Evaluation.

The fish intestine operates as a complicated interface between the organism and the environment, providing biological and mechanical protections as a result of a viscous layer of mucus released by goblet cells, which serves as a barrier against bacteria, viruses, and other pathogens, and contributes to the functions of the immune system. Therefore, goblet cells have a role in preserving the health of the body by secreting mucus and acting as sentinels. The ancient jawless fish broadgilled hagfish (Eptatretus cirrhatus, Forster, 1801) has a very basic digestive system because it lacks a stomach. By examining the presence, localization, and co-localization of 5-HT, TLR2, iNOS, and Piscidin1, this study intends to provide insight into the potential immune system contributions arranged by the gut goblet cells of broadgilled hagfish. Our results characterize intestinal goblet cells of broadgilled hagfish, for the first time, with the former antibodies, suggesting the hypothesis of conservation of the roles played by these cells also in primitive vertebrates. Moreover, this study deepens the knowledge about the still little-known immune system of hagfish.

Immunogenicity, effectiveness, safety and psychological impact of COVID-19 mRNA vaccines.

In December 2019, a new single-stranded RNA coronavirus, SARS-CoV-2, appeared in China and quickly spread around the world leading to a pandemic. Infection with SARS-CoV-2 generates symptoms ranging from asymptomatic to severe, occasionally requiring hospitalization in intensive care units, and, in more severe cases, leading to death. Scientists and researchers around the world have made a real race against time to develop various vaccines to slow down and stop the spread of the virus. In addition to conventional viral vector vaccines, new generation mRNA vaccines, BNT152b2 (Comirnaty) and mRNA-1273 (Spikevax), have been developed respectively by Pfizer/BioNTech and Moderna. These vaccines act on immune cells to induce an immune response with the production of specific antibodies against Spike protein of SARS-CoV-2, and to stimulate the differentiation of T and B memory cells. The objective of this review is to provide a detailed picture of the validity of these new vaccines and the safety of vaccination. Not only was the immunogenic effect of mRNA vaccines evaluated, but also the psychosocial impact they had on the population. The data collected show that this type of vaccine can also be an excellent candidate for future treatment and eradication of possible new pathologies with viral and non-viral etiology.

Histological and Chemical Analysis of Heavy Metals in Kidney and Gills of Boops boops: Melanomacrophages Centers and Rodlet Cells as Environmental Biomarkers.

Industrialization has resulted in a massive increase in garbage output, which is frequently discharged or stored in waterways like rivers and seas. Due to their toxicity, durability, bioaccumulation, and biomagnification, heavy metals (such as mercury, cadmium, and lead) have been identified as strong biological poisons. Their presence in the aquatic environment has the potential to affect water quality parameters and aquatic life in general. Teleosts' histopathology provides a sensitive indicator of pollutant-induced stress, because their organs have a central role in the transformation of different active chemical compounds in the aquatic environment. In particular, the gills, kidneys, and liver are placed at the center of toxicological studies. The purpose of this study is to examine the morphological changes caused by heavy metals in the kidney and gills of Boops boops, with a focus on melanomacrophages centers (MMCs) and rodlet cells (RCs) as environmental biomarkers, using histological and histochemical stainings (hematoxylin/eosin, Van Gieson trichrome, Periodic Acid Schiff reaction, and Alcian Blue/PAS 2.5), and immunoperoxidase methods. Our findings show an increase of MMCs and RCs linked to higher exposure to heavy metals, confirming the role of these aggregates and cells as reliable biomarkers of potential aquatic environmental changes reflected in fish fauna. The cytological study of RCs and MMCs could be important in gaining a better understanding of the complicated immune systems of teleosts.

Localization of vasoactive intestinal peptide and toll-like receptor 2 immunoreactive cells in endostyle of urochordate Styela plicata (Lesueur, 1823).

The endostyle is the first component of the ascidian digestive tract, it is shaped like a through and is located in the pharynx's ventral wall. This organ is divided longitudinally into nine zones that are parallel to each other. Each zone's cells are physically and functionally distinct. Support elements are found in zones 1, 3, and 5, while mucoproteins secreting elements related to the filtering function are found in zones 2, 4, and 6. Zones 7, 8, and 9, which are located in the lateral dorsal section of the endostyle, include cells with high iodine and peroxidase concentrations. Immunohistochemical technique using the following antibodies, Toll-like receptor 2 (TLR-2) and vasoactive intestinal peptide (VIP), and lectin histochemistry (WGA-wheat-germagglutinin), were used in this investigation to define immune cells in the endostyle of Styela plicata (Lesueur, 1823). Our results demonstrate the presence of immune cells in the endostyle of S. plicata, highlighting that innate immune mechanisms are highly conserved in the phylogeny of the chordates. RESEARCH HIGHLIGHTS: Immune cells positive to TLR-2 and VIP in the endostyle of Styela plicata. Expression of WGA in several zones of endostyle. Use of comparative biology to improve the knowledge about immunology in ascidians.

Rodlet cells in kidney of goldfish (Carassius auratus, Linnaeus 1758): A light and confocal microscopy study.

Rodlet cells (RCs) have always been an enigma for scientists. RCs have been given a variety of activities over the years, including ion transport, osmoregulation, and sensory function. These cells, presumably as members of the granulocyte line, are present only in teleosts and play a role in the innate immune response. RCs are migratory cells found in a variety of organs, including skin, vascular, digestive, uropoietic, reproductive, and respiratory systems, and present distinct physical properties that make them easily recognizable in tissues and organs. The development of RCs can be divided into four stages: granular, transitional, mature, and ruptured, having different morphological characteristics. Our study aims to characterize the different stages of these cells by histomorphological and histochemical techniques. Furthermore, we characterized these cells at all stages with peroxidase and fluorescence immunohistochemical techniques using different antibodies: S100, tubulin, α-SMA, piscidin, and for the first time TLR-2. From our results, the immunoreactivity of these cells to the antibodies performed may confirm that RCs play a role in fish defense mechanisms, helping to expand the state of the art on immunology and immune cells of teleosts.

Expression of the Antimicrobial Peptide Piscidin 1 and Neuropeptides in Fish Gill and Skin: A Potential Participation in Neuro-Immune Interaction.

Antimicrobial peptides (AMPs) are found widespread in nature and possess antimicrobial and immunomodulatory activities. Due to their multifunctional properties, these peptides are a focus of growing body of interest and have been characterized in several fish species. Due to their similarities in amino-acid composition and amphipathic design, it has been suggested that neuropeptides may be directly involved in the innate immune response against pathogen intruders. In this review, we report the molecular characterization of the fish-specific AMP piscidin1, the production of an antibody raised against this peptide and the immunohistochemical identification of this peptide and enkephalins in the neuroepithelial cells (NECs) in the gill of several teleost fish species living in different habitats. In spite of the abundant literature on Piscidin1, the biological role of this peptide in fish visceral organs remains poorly explored, as well as the role of the neuropeptides in neuroimmune interaction in fish. The NECs, by their role as sensors of hypoxia changes in the external environments, in combination with their endocrine nature and secretion of immunomodulatory substances would influence various types of immune cells that contain piscidin, such as mast cells and eosinophils, both showing interaction with the nervous system. The discovery of piscidins in the gill and skin, their diversity and their role in the regulation of immune response will lead to better selection of these immunomodulatory molecules as drug targets to retain antimicrobial barrier function and for aquaculture therapy in the future.

Immunostimulant and Antidepressant Effect of Natural Compounds in the Management of Covid-19 Symptoms.

In recent years, the use of natural compounds as adjuvant treatments and alternatives to traditional pharmacological therapies has become increasingly popular. These compounds have a wide range of biological effects, such as: antioxidant, anti-aging, hypocholesterolizing, hypoglycemic, antitumoral, antidepressant, anxiolytic activity, etc. Almost all of these compounds are easily available and are contained in different foods. At the end of 2019 the Coronavirus SARS-CoV-2 appeared in China and quickly spread throughout the world, causing a pandemic. The most common symptoms of this infection are dry cough, fever, dyspnea, and in severe cases bilateral interstitial pneumonia, with consequences that can lead to death. The nations, in trying to prevent the spread of infection, have imposed social distancing and lockdown measures on their citizens. This had a strong psychological-social impact, leading to phobic, anxious and depressive states. Pharmacological therapy could be accompanied by treatment with several natural compounds, such as vitamins, baicalein, zinc and essential oils. These compounds possess marked immunostimulant activity, strengthening the immune response and mitigating interactions between the virus and the host cell. They also have an antidepressant effect, acting on certain neurotransmitters.

Toll-like receptor 2 and α-Smooth Muscle Actin expressed in the tunica of a urochordate, Styela plicata.

The tunicate, Styela plicata (Lesueur, 1823) present an open circulator system with a tubular heart and blood flowing in lacunae among organs, bathing the tissues directly. Blood vascular lacunae are present in the tunica that is situated outside the epidermis and present a fibrous structure. The cells of the tunic are in straight contact with the blood vessels or are highly mobile. Ascidians are considered model organisms in comparative immunology of the chordate, and hold an important phylogenetic position as sister group of vertebrates. In recent years, numerous studies have reported the presence of Toll-like receptors (TLRs) in the genome of non-mammalian organisms including invertebrates. Two TLRs, designated Ci-TLR1 and Ci-TLR2 were expressed in the stomach, intestine and in numerous hemocytes of Ciona intestinalis, demonstrating that these key transmembrane proteins are evolutionarily conserved in ascidians. In this study for the first time, hemocytes aggregates were identified by confocal immunofluorescence techniques, using TLR2 antibody in the tunica of Styela plicata; furthermore, α-Smooth Muscle Actin (α-SMA) expression has been shown in the cells lining the vessels of the tunic. Our results support the view that the TLR-mediated innate immune functions are conserved in ascidian tissues.

Effects of spirulina diet on the oogenesis of zebrafish: morphological analysis and immunohistochemical determination of the vitellogenin.

Morphological analysis and immunohistochemical determination of vitellogenin (Vtg) were carried out in zebrafish ovaries (Danio rerio). The goal was to determine the nutraceutical effect of Spirulina feed on ovarian maturation stages of adult zebrafish. After 30 d, in the stages of advanced maturation a prevalence of oocytes was found in the experimental group. Immunohistochemical investigations revealed a significant increase in Vtg, precursor of vitelline (Vn) in egg yolk, whose expression increased in the liver in relation to the stages of maturation. In the zebrafish liver, the expression of Vtg was extremely low before vitellogenesis, and then increased and it was maintained at a high level until the final stage of ovarian maturation. The results showed that food additives such as Spirulina can improve fertility in laboratory farmed fish. The data obtained from this study may be a contribution to wider research aimed at improving reproduction in all vertebrates, including humans.

Immunohistochemical Characterization of PepT1 and Ghrelin in Gastrointestinal Tract of Zebrafish: Effects of Spirulina Vegetarian Diet on the Neuroendocrine System Cells After Alimentary Stress.

Gastrointestinal function in vertebrates is influenced by stressors, such as fasting and refeeding, different types of diet and hormonal factors. The aim of this paper was to analyze the effect of a Spirulina (Arthrospira platensis) diet, a microalga known for its nutraceutical properties, on the gastrointestinal tract of zebrafish (Danio rerio) regarding expression of oligopeptide transporter 1 (PepT1) and ghrelin (GHR). Food deprivation and refeeding was investigated to elucidate expression of PepT1 and GHR at a gastrointestinal level and the zebrafish compensatory mechanism. PepT1 is responsible for absorbing di- and tripeptides through a brush border membrane of intestinal mucosa. GHR is a brain-gut peptide in fish and mammals, stimulating growth hormone secretion and regulating appetite. Samples were taken after 2 and 5 days of specimen fasting, and 2 and 5 days of refeeding with Sera Spirulina tabs, in which the major constituent is Spirulina sp. (50.2% protein). Morphological and immunohistochemical analysis of PepT1 and GHR were carried out. Control specimen intestinal tract showed normal morphology of the digestive tract. Fasting caused fold structural changes and intestinal lumen constriction. Immunohistochemical analysis showed a PepT1 level reduction after fasting and an increase after refeeding, reaching very high levels after 5 days, compared to controls. GHR levels increased after food deprivation and gradually decreased after refeeding. Increased expression of PepT1 in refeeding fish suggests a compensatory physiological mechanism, as does the increase in GHR levels in fasting fish followed by a reduction after refeeding. A compensatory mechanism may be induced by fasting and refeeding and by a higher protein Spirulina diet. The microalga, for its nutraceutical properties, is an excellent candidate for animal breeding and human diet.

PCB-126 effects on aryl hydrocarbon receptor, ubiquitin and p53 expression levels in a fish product (Sparus aurata L.).

The aim of the present study is to determine if Ahr ligands as PCB-126, a dioxin-like, might contribute to inhibition of the tumour suppressor p53 by promoting its degradation through proteasome-ubiquitin system (UPS). The findings show, in the presence of PCB-126, a significant increase in p53 immunoreactivity in fish compared to the control. Subsequently, there is a decrease in p53 immunoreactivity at 24 h which is maintained even at 72 h. There is also a slight decrease in ubiquitin immunoreactivity to 12 h compared to the control and a marked decrease to 24 and 72 h. It's very important to underline as in this study we demonstrate a marked decrease in ubiquitin and p53 immunoreactivity at 24 and 72 h. Our result emphasise the need to deeply the role of this receptor in UPS regulation as potential therapeutic target for cancer treatment.

Effects of fasting and refeeding on the digestive tract of zebrafish (Danio rerio) fed with Spirulina (Arthrospira platensis), a high protein feed source.

In the present work, morphological and molecular effects of short-term feed deprivation and refeeding with Spirulina (Arthrospira platensis) on zebrafish digestive tract were determined. Once elucidated the proximate composition of Spirulina feed, immunohistochemical and western blot analyses of peptide transporter (PepT1) and cholecystokinin (CCK8) were carried out in the gastrointestinal tract of zebrafish, previously morphologically investigated. Two and five fasting days caused not only morphostructural alterations, but also the downregulation of PepT1 and CCK8 proteins. Conversely, the recovery of normal morphological conditions, along with an increased PepT1 and CCK8 expression, were observed after refeeding with Spirulina. The increase of PepT1 expression in zebrafish may be responsible for the enhanced CCK8 secretion, so that both proteins may contribute to an improved digestion process during refeeding. These observations could be supported not only by compensatory mechanisms induced by fasting and refeeding but also by an higher protein quality of Spirulina-based diet.

Role of AHR, AHRR and ARNT in response to dioxin-like PCBs in Spaurus aurata.

The aryl hydrocarbon receptor (AHR) mediates a variety of biological responses to ubiquitous dioxin and PCB dioxin-like. AHR together with ARNT, AHRR, represent a novel basic helix-loop-helix/PAS family of transcriptional regulators. Their interplay may affect the xenobiotic response. The aim of this study was to investigate, by histological, immunohistochemical investigations and western-blot analysis, the expression of AHR, ARNT and AHRR in liver of seabrem (Spaurus aurata) after exposure at different time to dioxin-like PCB126 in order to deep the knowledge about their specific role. The findings showed a significant increase of AHR and ARNT expression in juvenile fishes after 12 h than control group. The induction of AHR and ARNT is also significant at 24 and 72 hours compared to the control group. Furthemore, induction of AHRR expression has proved to increase both 12 h but this induction does not seem significant to 24 and 72 hours. The most important data of this work is that the induction of AHRR, when the action of the toxic persistence substances, as dioxin and PCB-126, it is not enough to reduce AHR signaling and thus its hyperactivation leads to toxic effects in seabrem (Spaurus aurata). All this confirms the importance of AHR ligands as new class of drugs that can be directed against severe disease such as cancer.

Relaxin improves multiple markers of wound healing and ameliorates the disturbed healing pattern of genetically diabetic mice.

Diabetic mice are characterized by a disrupted expression pattern of VEGF (vascular endothelial growth factor), and impaired vasculogenesis during healing. Experimental evidence suggests that RLX (relaxin) can improve several parameters associated with wound healing. Therefore we investigated the effects of porcine-derived RLX in diabetes-related wound-healing defects in genetically diabetic mice. An incisional wound model was produced on the back of female diabetic C57BL/KsJ-m+/+Lept(db) (db+/db+) mice and their normal littermates (db(+/+)m). Animals were treated daily with porcine RLX (25 µg/mouse per day, subcutaneously) or its vehicle. Mice were killed on 3, 6 and 12 days after skin injury for measurements of VEGF mRNA and protein synthesis, SDF-1α (stromal cell-derived factor-1α) mRNA and eNOS (endothelial NO synthase) expression. Furthermore, we evaluated wound-breaking strength, histological changes, angiogenesis and vasculogenesis at day 12. Diabetic animals showed a reduced expression of VEGF, eNOS and SDF-1α compared with non-diabetic animals. At day 6, RLX administration resulted in an increase in VEGF mRNA expression and protein wound content, in eNOS expression and in SDF-1α mRNA. Furthermore, the histological evaluation indicated that RLX improved the impaired wound healing, enhanced the staining of MMP-11 (matrix metalloproteinase-11) and increased wound-breaking strength at day 12 in diabetic mice. Immunohistochemistry showed that RLX in diabetic animals augmented new vessel formation by stimulating both angiogenesis and vasculogenesis. RLX significantly reduced the time to complete skin normalization and this effect was abrogated by a concomitant treatment with antibodies against VEGF and CXCR4 (CXC chemokine receptor 4), the SDF-1α receptor. These data strongly suggest that RLX may have a potential application in diabetes-related wound disorders.

Activation of the Ahr signalling pathway by polychlorobiphenyls causes a marked induction of cytochrome P450 only after depletion of vitellogenin in Sparus aurata.

The effects of polychlorinated biphenyl congeners 126 and 153 (PCB-126 and PCB-153) on vitellogenin (Vtg) and cytochrome (CYP1A1) expression were evaluated in 60 juvenile Sparus aurata. Fish were divided into four groups and the control group (Group A) was compared to fish exposed to PCB-126 (10-8M) (Group B), PCB-153 (10-6M) (Group C) singly and also in combination (Group D) for 12, 24 and 72 h. Hepatic expression of Vtg and CYP1A1 were analyzed using histological examinations and by immunochemical (Western blotting and immunohistochemistry) methods. Vtg increased in juvenile fishes of Groups B, C and D after 12h respect to Group A and decreased after 24 and 72 h respect to 12h in each group. CYP1A1 increased after 12 and 24h in all groups vs control group and increased in Group B only at 72 h vs in control group. The results showed that chemical interaction and endocrine disruption in fish might produce deleterious consequences not only for fish but also for human.