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

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

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

Dopaminergic inhibition of human neutrophils is exerted through D1-like receptors and affected by bacterial infection

Dopamine (DA) affects immune functions in healthy subjects (HS) and during disease by acting on D1-like (D1 and D5) and D2-like (D2, D3 and D4) dopaminergic receptors (DR); however, its effects on human polymorphonuclear leukocytes (PMN) are still poorly defined. We investigated DR expression in human PMN and the ability of DA to affect cell migration and reactive oxygen species (ROS) production. Experiments were performed on cells from HS and from patients (Pts) with bacterial infections as well, during the acute phase and after recovery. Some experiments were also performed in mice knockout (KO) for the DRD5 gene. PMN from HS express both D1-like and D2-like DR, and exposure to DA results in inhibition of activation-induced morphological changes, migration and ROS production which depend on the activation of D1-like DR. In agreement with these findings, DA inhibited migration of PMN obtained from wild-type mice, but not from DRD5KO mice. In Pts with bacterial infections, during the febrile phase D1-like DRD5 on PMN were downregulated and DA failed to affect PMN migration. Both D1-like DRD5 expression and DA-induced inhibition of PMN migration were however restored after recovery. Dopaminergic inhibition of human PMN is a novel mechanism which is likely to play a key role in the regulation of innate immunity. Evidence obtained in Pts with bacterial infections provides novel clues for the therapeutic modulation of PMN during infectious disease

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

Cellular responses induced by multi-walled carbon nanotubes: in vitro study on the medicinal leech macrophages

The intrinsic characteristics of engineered nanoparticles, such as multi-wall carbon nanotubes (MWCNTs) are impressive and attractive for technology, however their environmental dispersion could be potentially hazardous to animal and human health. Since the production and use of MWCNTs is steadily increasing, intentional or unintentional environmental discharges may occur. For this reason, the development of new methods and the identification of reliable models to completely understand MWCNTs effects are critical. Here we propose a freshwater invertebrate, the medicinal leech, as alternative model to assess the effects of MWCNTs on immune system. Our previous studies have already demonstrated that in vivo MWCNT treatment induces the activation of leech\u2019s macrophages. In this study, we focus on the direct effects of MWCNTs on these cells. We used the consolidated experimental approach, based on the injection in the leech body wall of the biomatrice Matrigel (MG) added with the cytokine Allograft inflammatory factor-1 (HmAIF-1), to specifically chemoattract macrophages. Cells extracted from MG were cultured and characterized with the specific markers CD45 and CD68, confirming their belonging to the monocyte-macrophage lineage. Primary macrophage cultures were then subjected to an in vitro treatment with MWCNTs at different concentrations (2.5, 5, 10, 25, 50 and 100 \u3bcg/ml). Our results indicate that leech macrophages, once in close contact with MWCNTs, actively produce amyloid material to encapsulate the foreign bodies. We also demonstrated that MWCNTs in vitro treatment cause the decrease of cell proliferation rate and the increase of the apoptotic rate. Furthermore, since oxidative stress is linked with inflammation and amyloid production, reactive oxygen species has been evaluated, confirming that their production rate increases after MWCNT treatment. 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 improved and successfully used to study the possible harmful effects of any nanomaterial. Moreover, since autophagic cell death pathway activation is emerging as a possible consequence of MWCNT treatment, in the future we will attempt to clarify this aspect in order to completely understand MWCNT-induces toxicity