Immune response induced by multi-wall carbon nanotubes: in vivo and in vitro studies on the medicinal leech. Risposta immunitatia indotta da nanotubi di carbonio a parete multipla: studi in vivo e in vitro sulla sanguisuga medicinale.

Since the production and use of engineered nanomaterials (NMs) is steadily increasing, the development of new reliable methods to analyze NMs effects are critical. Here we propose the medicinal leech as alternative animal model to study multi-walled carbon nanotubes (MWCNTs) effects by means of in vivo (environmental dispersion and MWCNT supplemented Matrigel injection) and in vitro studies on leeches macrophages. Our results show that water dispersed MWCNTs evoke a massive migration of CD45+/CD68+ monocyte-macrophages cells and the production of molecules involved in innate immune response, such as the pro-inflammatory cytokine IL-18 and amyloid fibrils. Ultrastructural analysis of MWCNTs-supplemented biomatrix revealed that in leech macrophages MWCNTs are internalized both actively (phagocytosis) and passively (membrane piercing). Finally, MWCNT in vitro treatment cause the decrease of cell proliferation rate and the increase of both ROS production and apoptotic rate. Moreover, in this study we identified and characterized in Hirudo two inflammatory markers striking similar to vertebrate\u2019s MIF and GMFG, showing that these molecules are involved in macrophage response to MWCNTs. Our combined experimental approaches, not only attest the ability of MWCNTs in inducing a potent inflammatory response, but also confirm the medicinal leech 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

Amyloidogenesis and Responses to Stress

Amyloidogenesis is a primitive, physiological response that seems to be an ancient process widely distributed in different cell types of evolutionary distant organisms. The amyloid fibril synthesis is part of a more general inflammatory response to stressful conditions all entailing overproduction of reactive oxygen species (ROS). Interestingly, this event has been integrated into additional physiological functions: (i) the formation of a scaffold promoting the activation and packaging of melanin; (ii) the formation of a scaffold to compartmentalize hormones in the cytoplasm; (iii) the ability to reversibly link different types of molecules to drive close to the nonself; (iv) the construction of a framework to close body lesions. Amyloid fibril formation is a cellular response harmonically integrated with the stress response but for a deregulation in assembling/dismantling, dangerous depots, as in a lot of pathologies, can occur

Butyrate and taurine exert a mitigating effect on the inflamed distal intestine of European sea bass fed with a high percentage of soybean meal

Abstract Background Due to the paucity of oceanic resources utilized in the preparation of diets for cultured fish, commercial feed producers have been trying to replace fishmeal (FM) using alternative protein sources such as vegetable protein meals (VMs). One of the main drawbacks of using VMs in fish feed is related to the presence of a variety of anti-nutritional factors, which could trigger an inflammation process in the distal intestine. This reduces the capacity of the enterocytes to absorb nutrients leading to reduced fish growth performances. Methods We evaluated the mitigating effects of butyrate and taurine used as feed additives on the morphological abnormalities caused by a soybean meal (SBM)-based diet in the distal intestine of sea bass (Dicentrarchus labrax). We used three experimental diets, containing the same low percentage of FM and high percentage of SBM; two diets were supplemented with either 0.2% sodium butyrate or taurine. Histological changes in the intestine of fish were determined by light and transmission electron microscopy. Infiltration of CD45+ leucocytes in the lamina propria and in the submucosa was assessed by immunohistochemistry. We also quantified by One-Step Taqman® real-time RT-PCR the messenger RNA (mRNA) abundance of a panel of genes involved in the intestinal mucosa inflammatory response such as TNFα (tumor necrosis factor alpha) and interleukins: IL-8, IL-1β, IL-10, and IL-6. Results Fish that received for 2 months the diet with 30% soy protein (16.7% SBM and 12.8% full-fat soy) developed an inflammation in the distal intestine, as confirmed by histological and immunohistochemistry data. The expression of target genes in the intestine was deeply influenced by the type of fish diet. Fish fed with taurine-supplemented diet displayed the lowest number of mRNA copies of IL-1β, IL-8, and IL-10 genes in comparison to fish fed with control or butyrate-supplemented diets. Dietary butyrate caused an upregulation of the TNFα gene transcription. Among the quantified interleukins, IL-6 was the only one to be not influenced by the diet. Conclusions Histological and gene expression data suggest that butyrate and taurine could have a role in normalizing the intestinal abnormalities caused by the SBM, but the underling mechanisms of action seem different

The main actors involved in parasitization of Heliothis virescens larva

At the moment of parasitization by another insect, the host Heliothis larva is able to defend itself by the activation of humoral and cellular defenses characterized by unusual reactions of hemocytes in response to external stimuli. Here, we have combined light and electron microscopy, staining reactions, and immunocytochemical characterization to analyze the activation and deactivation of one of the most important immune responses involved in invertebrates defense, i.e., melanin production and deposition. The insect host/parasitoid system is a good model to study these events. The activated granulocytes of the host insect are a major repository of amyloid fibrils forming a lattice in the cell. Subsequently, the exocytosed amyloid lattice constitutes the template for melanin deposition in the hemocel. Furthermore, cross-talk between immune and neuroendocrine systems mediated by hormones, cytokines, and neuromodulators with the activation of stress-sensoring circuits to produce and release molecules such as adrenocorticotropin hormone, alpha melanocyte-stimulating hormone, and neutral endopeptidase occurs. Thus, parasitization promotes massive morphological and physiological modifications in the host insect hemocytes and mimics general stress conditions in which phenomena such as amyloid fibril formation, melanin polymerization, pro-inflammatory cytokine production, and activation of the adrenocorticotropin hormone system occur. These events observed in invertebrates are also reported in the literature for vertebrates, suggesting that this network of mechanisms and responses is maintained throughout evolution

Cytokine Impregnated Biomatrix: A New Tool to Study Multi-Wall Carbon Nanotubes Effects on Invertebrate Immune Cells

The novel features of engineered nanoparticles, such as multi-wall carbon nanotubes (MWCNTs) are impressive and attractive for technology, however they dissolved in water and accumulate in soils through the application of sewage sludge, accidental spills, and deposition from the air, agrochemicals or soil remediation. Given that several studies have revealed that chronic exposure to these nanomaterials products through the ingestion of drinking water, inhalation and dermal contact may harbour potential risks to human health, risk assessment of this nanomaterials in the aquatic environment are becoming essential. Here we propose a freshwater invertebrate, the leech Hirudo medicinalis, as a model to assess the effects MWCNTs on the immune system by means of in vivo and in vitro experiments. For this study, we used a consolidated experimental approach based on injection in the body wall of the leech of the biomatrice Matrigel (MG), added with a specific macrophage chemoattractant, the cytokine Allograft inflammatory factor-1 (AIF-1) and/or with MWCNTs. MG sponges analysis show the presence of a larger number of cells positive for both CD68 and HmAIF-1, specific monocyte-macrophage markers. Ultrastructural analysis suggests that MWCNTs may be internalized by phagocytosis but they seem also to be able to pierce cell membranes during cells migration. Cells extracted from MG were also used for in vitro treatment with MWCNTs at different concentration (2.5, 5, 10, 25, 50 and 100 \ub5g/ml) for 24 h to study cell morphology changes and production of amyloid fibrils in order to encapsulate the foreign bodies. Our results, not only confirm the ability of MWCNTs in inducing a potent inflammatory response, but highlight rapid colorimetric assays that can be successfully used as sensitive tools for aquatic pollution biomonitoring

A new cellular type in invertebrates: first evidence of telocytes in leech Hirudo medicinalis

Abstract Telocytes, a peculiar cell type, were recently found in vertebrates. Hence this cell system has been reported as ubiquitous in the bodies of mammals and interpreted as an important player in innate immunity and tissue regeneration, it is reasonable to look for it also in invertebrates, that rely their integrity solely by innate immunity. Here we describe, at morphological and functional level, invertebrate telocytes from the body of leech Hirudo medicinalis (Annelida), suggesting how these cells, forming a resident stromal 3D network, can influence or participate in different events. These findings support the concepts that leech telocytes: i) are organized in a cellular dynamic and versatile 3D network likewise the vertebrate counterpart; ii) are an evolutionarily conserved immune-neuroendocrine system; iii) form an immuno-surveillance system of resident cells responding faster than migrating immunocytes recruited in stimulated area; iv) communicate with neighbouring cells directly and indirectly, via cell-cell contacts and soluble molecules secreted by multivesicular bodies; v) present within neo-vessels, share with immunocytes the mesodermal lineage; vi) are involved in regenerative processes. In conclusion, we propose that HmTCs, integrating so different functions, might explain the innate immune memory and can be associated with several aged related diseases

Acid phosphatase reaction (A-F) and anti CD68 immunofluorescence staining (G-L).

<p>Resident in untreated (A, G) and migrating macrophages-like cells in treated leeches (B-E) and (H-L), located under the epithelium (e) and among the muscle fibers (m), are positive for acid phosphatase reaction (arrowheads in A-F) and for anti-CD68 (G-L). (F) Quantitative evaluation of cell numbers. Column 1: number of cells in untreated sample, columns 2–10: number of cells in MWCNTs treated sample from 1h up to 5 weeks. *p<0.01. (J) Combined transmission and fluorescence images showing CD68⁺ cells (in red) in spatial association with MWCNTs aggregates (circled). Bars in A-E, G-I, K-L: 100μm; Bar in F, J: 10μm.</p

Morphological analysis (A-L).

<p>Morphological at optical (A-E, C, I) and transmission electron microscopes (F, H, J-L) analyses of sections from <i>H</i>. <i>medicinalis</i> body wall. The body wall of untreated leeches results practically avascular (A) and the botryoidal tissue (b) appears as a solid chord of clustered cells (B). After 1 hour from MWCNTs treatment, numerous neo-vessels (v) are found among muscles (m) and under the epithelium (e) (C). Within the new cavities (c) lined by the botryoidal tissue (b), immunocompetent circulating precursors cells (arrowhead) are clearly distinguishable (D). After 3 hours from treatment, numerous fibroblasts are visible immersed in an abundant extracellular matrix ECM (E, F). After 1 (G, H) up to 5 weeks (I) from MWCNTs treatment, numerous vessels (v) and fibroblasts (arrowheads) are still visible in the body wall. (J-L) Detail of MWCNTs (arrowheads) freely dispersed in the cytoplasm of macrophage-like cells. Bars in A, C, G: 100μm; Bars in B, D-E, I: 10μm; Bar in F: 5μm; Bar in H: 2μm; Bar in J: 500nm; Bars in K-L: 200nm.</p