The immuneregulator role of neprilysin (NEP) in invertebrates

Neprilysin (NEP) represents an important enzyme in both vertebrates and invertebrates. In the present report we have focused our attention to invertebrates. In particular, a structure related to CD10/NEP as well as its activity in different tissues, such as immunocytes, nervous tissue and muscle of various species were detected. Moreover, the role played by the enzyme in the interactions between host and parasite has also been reported. The findings indicate that NEP immunoregulation is a well-balanced process that, with appropriate physiological and homeostatic responses to challenges, allows the survival and well-being of the species

Amyloid and allorecognition in the colonial ascidian Botryllus schlosseri.

Allorecognition, i.e., the ability of intraspecific nonself recognition is widely distributed among colonial, sessile marine organisms in the form of colony specificity. In the cosmopolitan compound ascidian Botryllus schlosseri, colony specificity is controlled by a highly polymorphic Fu/HC locus: two colonies sharing at least one alleleat the Fu/HC locus can fuse into a chimeric colony; if no alleles are shared, a typical inflammatory reaction occurs, with the recruitment of a specific hemocyte type, the cytotoxic morula cells (MCs), inside the tips of the ampullae (the blind termini ofthe tunic vasculature) extending towards the alien colony, their extravasation in the tunic and their final degranulation. As a consequence of allorecognition, necrotic, melanic spots (points of rejection; PORs) form along the contact border, due to the release, by MCs, of their granular content, mainly represented by quinones, polyphenols and the enzyme phenoloxidase (PO), upon the perception of the allogeneic humoral factors diffusing from the alien colony through the partially fused tunics. It is remarkable that the deposition of melanin and the cell death is confined to the immediate outside of the ampullar tips, suggesting that the diffusion of PO and the products of its activity are, in some way, prevented in order to limit cytotoxicity to the immediate neighbourhood of the contact region. In this context, we looked for factors released by MCs that could limit the spreading of cytotoxicity and melanisation. We found that MCs share with vertebrate melanocytes similar packaging of melanin precursors, entrapped in a 3Dscaffold of amyloid fibrils. They contribute to form the electron dense content of MC granules that, after stimulation, flake off and is released in the surrounding medium. Released amyloid fibrils limit the diffusion of the produced melanin. The search for genes and factor controlling both melanogenesis and amyloidogenesis, revealed an evolutionary conserved machinery involved in the processes and an unexpected cross talk between the two Botryllus immunocyte types, i.e., phagocytes and MCs. Furthermore, this work confirms the physiological role of amyloid in tunicate immunity

Ras activation in Hirudo medicinalis angiogenic process

In some leeches like Hirudo medicinalis, any kind of stimulation (surgical wound or growth factor injection) provokes the botryoidal tissue response. This peculiar tissue, localized in the loose connective tissue between gut and body wall, is formed by granular botryoidal cells and flattened endothelial-like cells. Under stimulation, the botryoidal tissue changes its shape to form new capillaries. In mammals, the molecular regulation of the angiogenic phenotype requires coordinated input from a number of signalling molecules: among them the GTPase Ras is one of the major actor. In our current study, we determine whether Ras activation alone would be sufficient to drive vessels formation from leech botryoidal tissue. Our findings indicate that assembly and disassembly of actin filaments regulated by Ras protein is involved in morphological modification of botryoidal tissue cells during leech angiogenic process

Hirudo medicinalis as alternative model for in vivo and in vitro studies on nanomaterials toxicity.

Due to the steady increase of production and use of engineered nanomaterials (NMs), intentional or unintentional discharges into the environment may occur. Since it is critical to develop new methods to fully understand NMs bioaccumulation and cytotoxicity, a reliable model in which analyze NMs effects both in vivo and in vitro is more than ever necessary. Here we propose the leech, Hirudo medicinalis, as alternative animal model to study multi-walled carbon nanotubes (MWCNTs) effects by means of an extended study that includes in vivo and in vitro treatments. First, MWCNTs were dispersed in leeches\u2019 water to mimic a possible environmental exposure. In a second assay, a MWCNTs-supplemented biomatrix was injected in leeches\u2019 body wall. Thirdly, leeches macrophages were isolated and cultured to analyze their responses after MWCNTs in vitro treatment. Our results show that water dispersed MWCNTs evoke in the leech body wall a strong inflammatory response, involving mainly monocyte-macrophages cells. Ultrastructural analysis of MWCNTs-supplemented biomatrix revealed that in leech macrophages MWCNTs are internalized both in an active (phagocytosis) and a passive manner (membrane piercing). Finally, MWCNTs in vitro treatment cause the decrease of cell proliferation rate and the increase of ROS production and of the apoptotic rate. Our combined experimental approaches, not only attest the ability of MWCNTs in inducing a potent inflammatory response, but also confirm Hirudo medicinalis as a good alternative model that can be successfully used to study, both in vivo and in vitro, the possible harmful effects of any nanomaterial

Functional amyloid formation in LPS activated cells from invertebrates to vertebrates

LPS stimulation provokes serious cellular stress with an increase of cytoplasmic reactive oxygen species (ROS). We have investigated, among the different cellular defenses, amyloidogenesis as common physiological response to attenuate oxidative stress. Optical and electron microscopic observations of the following LPS activated cell lines [insect (larval hemocytes, IPLB-LdFB and Drosophila Schneider\u2019s S2 cells); mouse (NIH3T3 embryonic fibroblasts); Human (Human Umbilical Vein Endothelial Cells (HUVEC), neutrophils, and mesenchymal stem cells] reveal that, all are characterized by irregular profiles, cytoplasmic empty vacuoles or by cisternae containing fibrillar material. The compartmentalized fibrillar material shows staining properties typical of amyloid fibrils. LPS activation leads to ROS generation, resulting in pH acidification. Stimulated cells show pink cytoplasm in May-Gr\ufcnwald Giemsa differential staining, giving a gross indication of a lower intracellular pH. Moreover the activation of amyloidogenesis is also linked with an extensive production of ACTH and \u3b1-MSH in all cultured cell types. We suggest that amyloidogenesis is a common, physiological cellular response to weak ROS, starting when other anti-stress cellular systems failed to restore homeostasis. The morphological evidence and/or functional characterization of synthesized amyloid fibrils could be an early indicator of oxidative stress that may lead to a general inflammatory process

Amyloid/Melanin distinctive mark in invertebrate immunity

Protostomes and Deuterostomes show the same nexus between melanin production, and amyloid fibril production, i.e., the presence of melanin is indissolubly linked to amyloid scaffold that, in turn, is conditioned by the redox status/cytoplasmic pH modification, pro-protein cleavage presence, adrenocorticotropin hormone (ACTH), melanocyte-stimulating hormone (\u3b1-MSH), and neutral endopeptidase (NEP) overexpressions. These events represent the crucial component of immune response in invertebrates, while in vertebrates these series of occurrences could be interpreted as a modest and very restricted innate immune response. On the whole, it emerges that the mechanisms involving amyloid fibrils/pigment synthesis in phylogenetically distant metazoan (viz, cnidaria, molluscs, annelids, insects, ascidians and vertebrates) are evolutionary conserved. Furthermore, our data show the relationship between immune and neuroendocrine systems in amyloid/melanin synthesis. Indeed the process is closely associated to ACTH-\u3b1-MSH production, and their role in stress responses leading to pigment production reflects and confirms again their ancient phylogeny

Identification, Isolation and Expansion of Myoendothelial Cells Involved in Leech Muscle Regeneration

Adult skeletal muscle in vertebrates contains myoendothelial cells that express both myogenic and endothelial markers, and which are able to differentiate into myogenic cells to contribute to muscle regeneration. In spite of intensive research efforts, numerous questions remain regarding the role of cytokine signalling on myoendothelial cell differentiation and muscle regeneration. Here we used Hirudo medicinalis (Annelid, leech) as an emerging new model to study myoendothelial cells and muscle regeneration. Although the leech has relative anatomical simplicity, it shows a striking similarity with vertebrate responses and is a reliable model for studying a variety of basic events, such as tissue repair. Double immunohistochemical analysis were used to characterize myoendothelial cells in leeches and, by injecting in vivo the matrigel biopolymer supplemented with the cytokine Vascular Endothelial Growth Factor (VEGF), we were able to isolate this specific cell population expressing myogenic and endothelial markers. We then evaluated the effect of VEGF on these cells in vitro. Our data indicate that, similar to that proposed for vertebrates, myoendothelial cells of the leech directly participate in myogenesis both in vivo and in vitro, and that VEGF secretion is involved in the recruitment and expansion of these muscle progenitor cells

In Vivo Isolation and Characterization of Stem Cells with Diverse Phenotypes Using Growth Factor Impregnated Biomatrices

BACKGROUND: The stimulation to differentiate into specific cell types for somatic stem cells is largely due to a series of internal and external signals coming from the microenvironment that surrounds the stem cell. Even though intensive research has been made, the basic mechanisms of plasticity and/or the molecules regulating stem cells proliferation and differentiation are not completely determined. Potential answers concerning the problems could be derived from the studies of stem cells in culture. METHODOLOGY/PRINCIPLE FINDINGS: We combine a new procedure (using the matrigel biopolymer supplemented with a selected cytokine/growth factor) with classic techniques such as light, confocal and electron microscopy, immunohistochemistry and cell culture, to perform an analysis on stem cells involved in the leech (Hirudo medicinalis) repair tissues. The leech has a relative anatomical simplicity and is a reliable model for studying a variety of basic events, such as tissue repair, which has a striking similarity with vertebrate responses. Our data demonstrate that the injection of an appropriate combination of the matrigel biopolymer supplemented with a selected cytokine/growth factor in the leech Hirudo medicinalis is a remarkably effective tool for isolating a specific cell population in vivo. A comparative analysis of biopolymer in vivo sorted stem cells indicates that VEGF recruited cells of a hematopoietic/endothelial phenotype whereas MCP-1/CCL2 isolated cells that were of an early myeloid lineage. CONCLUSION: Our paper describes, for the first time, a method allowing not only the isolation of a specific cell population in relation to the cytokine utilized but also the possibility to culture a precise cell type whose isolation is otherwise quite difficult. This approach could be broadly applied to isolate stem cells of diverse origins based on the recruitment stimuli employed

Blood circulating miR-28-5p and let-7d-5p associate with premature ageing in Down syndrome

Persons with Down syndrome (DS) undergo a premature ageing with early onset of age-related diseases. The main endpoint of this study was the identification of blood circulating microRNAs (c-miRs) signatures characterizing DS ageing process. A discovery phase based on array was performed in plasma samples obtained from 3 young (31 ± 2 years-old) and 3 elderly DS persons (66 ± 2 years-old). Then, a validation phase was carried out for relevant miRs by RT-qPCR in an enlarged cohort of 43 DS individuals (from 19 up to 68 years-old). A group of 30 non-trisomic subjects, as representative of physiological ageing, was compared. In particular miR-628-5p, miR-152-3p, miR-28-5p, and let-7d-5p showed a lower level in younger DS persons (age ≤ 50 years) respect to the age-matched controls. Among those, miR-28-5p and let-7d-5p were found significantly decreased in physiological ageing ( oldest group ), thus they emerged as possible biomarkers of premature ageing in DS. Moreover, measuring blood levels of beta amyloid peptides, Aβ-42 was assessed at the lowest levels in physiological ageing and correlated with miR-28-5p and let-7d-5p in DS, while Aβ-40 correlated with miR-628-5p in the same cohort. New perspectives in terms of biomarkers are discussed