Homolog of allograft inflammatory factor-1 induces macrophage migration during innate immune response in leech.

Allograft inflammatory factor-1 (AIF-1) is a 17-kDa cytokine-inducible calcium-binding protein that, in vertebrates, plays an important role in the allograft immune response. Its expression is mostly limited to the monocyte/macrophage lineage. Until recently, AIF-1 was assumed to be a novel molecule involved in inflammatory responses. To clarify this aspect, we have investigated the expression of AIF-1 after bacterial challenge and its potential role in regulating the innate immune response in an invertebrate model, the medicinal leech (Hirudo medicinalis). Analysis of an expressed sequence tag library from the central nervous system of Hirudo revealed the presence of the gene Hmaif-1/alias Hmiba1, showing high homology with vertebrate aif-1. Immunohistochemistry with an anti-HmAIF-1 polyclonal antibody revealed the constitutive presence of this protein in spread CD68(+) macrophage-like cells. A few hours after pathogen (bacterial) injection into the body wall, the amount of these immunopositive cells co-expressing HmAIF-1 and the common leucocyte marker CD45 increased at the injected site. Moreover, the recombinant protein HmAIF-1 induced massive angiogenesis and was a potent chemoattractant for macrophages. Following rHmAIF-1 stimulation, macrophage-like cells co-expressed the macrophage marker CD68 and the surface glycoprotein CD45, which, in vertebrates, seems to have a role in the integrin-mediated adhesion of macrophages and in the regulation of the functional responsiveness of cells to chemoattractants. CD45 is therefore probably involved in leech macrophage-like cell activation and migration towards an inflammation site. We have also examined its potential effect on HmAIF-1-induced signalling.

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.

Effects of Carbon Nanotube Environmental Dispersion on an Aquatic Invertebrate, Hirudo medicinalis.

The recent widespread applications of nanomaterials, because of their properties, opens new scenarios that affect their dispersal in the environment. In particular multiwall carbon nanotubes (MWCNTs), despite their qualities, seem to be harmful for animals and humans. To evaluate possible toxic effects caused by carbon nanotube environmental dispersion, with regard to aquatic compartment, we proposed as experimental model a freshwater invertebrate: Hirudo medicinalis. In the present study we analyse acute and chronic immune responses over a short (1, 3, 6 and 12 hours) and long time (from 1 to 5 weeks) exposure to MWCNTs by optical, electron and immunohistochemical approaches. In the exposed leeches angiogenesis and fibroplasia accompanied by massive cellular migration occur. Immunocytochemical characterization using specific markers shows that in these inflammatory processes the monocyte-macrophages (CD45+, CD68+) are the most involved cells. These immunocompetent cells are characterized by sequence of events starting from the expression of pro-inflammatory cytokines (in particular IL-18), and amyloidogenensis. Our combined experimental approaches, basing on high sensitive inflammatory response can highlight adverse effects of nanomaterials on aquatic organisms and could be useful to assess the MWCNTs impact on aquatic, terrestrial animal and human health.

Hirudo medicinalis: avascular tissues for clear-cut angiogenesis studies?

Basic studies on angiogenesis in normal and pathologic conditions, as well as research on drugs or genes/proteins that stimulate or regulate the angiogenic process, can rely on an increasing number of experimental models. Among non-mammalian models, Zebrafish is adopted by an increasing number of research groups. Moreover, angiogenesis and vasculogenesis in invertebrates like the leech Hirudo medicinalis share a high degree of similarity with the same processes occurring in humans, both under the structural/functional and biochemical points of view. Interestingly, Hirudo angiogenic growth factor receptors respond to corresponding human/mammalian recombinant growth factors and cytokines; in addition, Hirudo endogenous angiogenic growth factors and receptors react with antibodies against their human/mammalian counterparts. Furthermore, as it will be shown in this review, Hirudo has the unique advantage of having a virtually avascular muscular body wall, whereas the reliability of such a peculiar feature as a model for physiologically vascularised mammalian tissues has to be thoroughly investigated. Hirudo has proven so far to allow unambiguous, clear-cut studies on the angiogenic potential of gene-products or drugs, as well as on the anti-angiogenic compounds. This article will review the biology of angiogenesis in Hirudo and the data so far collected on angiogenesis stimulation/modulation in this model; an example describing a study on the biological activity of a naked DNA vector for angiogenesis gene therapy will also be provided.

Leeches: immune response, angiogenesis and biomedical applications.

The innate immune response is the first line of defence strategies in invertebrates against attack of infectious agents. A detailed analysis of the immune mechanisms involved in annelids has been performed in oligochaets, but few data are available in polichaets and hirudineans. The aim of this review is to describe the responses of leeches to different kinds of stimuli (infections following non-self agent attacks, surgical lesions, grafts). Furthermore, the use of this invertebrate as a novel experimental model to be used to screen drugs and genes, which are responsible for positive and negative modulation of angiogenesis, is discussed.

Assessment of the biological activity of an improved naked-DNA vector for angiogenesis gene therapy on a novel non-mammalian model.

From the basic expression vector p/hVEGF165, containing a cDNA sequence encoding the 165-amino-acid isoform of human vascular endothelial growth factor (VEGF165), we generated an improved construct (p163/hVEGF165) by subcloning at the 5' exact end of the VEGF165 cDNA a 163-bp IRES element belonging to the 1,014-bp, 5'-untranslated region of the murine VEGF gene. This IRES structure caused enhanced synthesis and increased secretion of the mature protein both in HEK-293 and in COS-7 cells, when compared to the basic construct. Both p/hVEGF165 and p163/hVEGF165 vectors were tested for in vivo angiogenic activity on a novel hirudinean model. As expected, the p/hVEGF165 vector injected as naked DNA was able to induce angiogenesis in the non-vascularized muscular tissue of Hirudo medicinalis. This model also allowed us to monitor intracellular synthesis of VEGF165 and subsequent interstitial secretion from muscle cells. Interestingly, significantly larger muscle tissue areas underwent marked angiogenesis when the improved vector p163/hVEGF165 was injected in H. medicinalis. It thus appears that the p163/hVEGF165 construct allows enhanced expression of the human VEGF165 gene, which in turn is responsible for increased secretion of biologically active growth factor by transduced cells. Since a naked-DNA vector very similar to p/hVEGF165 was recently found to be very active in humans for treatment of heart and limb ischemia, we suggest that our improved construct might be further tested in view of potential therapeutic applications.

Leech responses to tissue transplantation.

The aim of the present work is to describe histologically, histochemically and immunocytochemically, the sequence of events that lead to first and second set rejection of allo- or xenograft in leeches. Graft responses of leeches are comparable and are described following specific steps: inflammatory phase, rejection phase and granulation tissue formation (including re-epithelialisation, angiogenesis and fibroplasia).The responses to first and second graft in first set graft rejection as well as to the first transplant in second set graft experiments are identical and in the time span of a week all grafts are destroyed and disappear. In the second set graft rejection experiments the responses against the second transplant are markedly accelerated. The second graft shows massive structural alterations and it is rapidly rejected, within 3-4 days.Our results permit to highlight that in leeches there is a specific responsiveness of immune system similar to those described in highly divergent phyla.

The leech: a novel invertebrate model for studying muscle regeneration and diseases.

We focused our studies on the leech, Hirudo medicinalis. This invertebrate 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. Hirudo is also a good invertebrate model to test the actions of drugs and gene products, since the responses evoked by the different stimuli are clear and easily detectable due to their small size and anatomical simplicity. Here we review the use of this invertebrate model to investigate muscle regeneration and the role of hematopoietic stem cells in this process. Our recent data, summarized in this review, demonstrate that the injection of an appropriate combination of the matrigel biopolymer supplemented with Vascular Endothelial Growth factor (VEGF) in the leech Hirudo medicinalis is a remarkably effective tool for isolating a specific population of hematopoietic/endothelial precursor cells, which in turn can differentiate in muscle cells. Thus leeches can be considered as a new emerging model for studying endothelial and hematopoietic precursors cells involved in muscle post-natal growth and regeneration processes.

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.

A hedgehog homolog is involved in muscle formation and organization of Sepia officinalis (mollusca) mantle.

Our study focuses on the possible involvement of the Hedgehog (Hh) pathway in the differentiation of striated muscle fibres in cuttlefish (Sepia officinalis) mantle. We show here that both an hh-homolog signalling molecule and its receptor Patched (Ptc) are expressed in a specific population of myoblasts which differentiates into the radial fast fibres. To evaluate the functional significance of hh expression in developing cuttlefish, we inhibited the Hedgehog signalling pathway by means of cyclopamine treatment in cuttlefish embryos. In treated embryos, the gross anatomy was considerably compromised, displaying an extremely reduced mantle with a high degree of morphological abnormalities. TUNEL and BrdU assays showed that the absence of an hh signalling induces apoptosis and reduces the proliferation rate of muscle precursors. We therefore hypothesize a possible involvement of Hh and its receptor Ptc in the formation of striated muscle fibres in cuttlefish.

Collagen reorganization in leech wound healing.

BACKGROUND INFORMATION: Leeches respond to surgical lesions with the same sequence of events as that described for wound healing in vertebrates, where collagen is important for the development of tensions in healing wounds, functioning as an extracellular scaffold for accurate regeneration of the structures disrupted by surgical or traumatic actions. RESULTS: In surgically lesioned leeches, newly synthesized collagen is arranged in hierarchical structures. Fibrils can be packed and shaped to form cords or tubular structures, thus acting as an extracellular scaffold that directs and organizes the outgrowth of new vessels and the migration of immune cells towards lesioned tissues. In these animals, the general architecture of collagen fibrils, generated during tissue regeneration, shows similarities to both the structural pattern of collagen bundles and assembly processes observed in several vertebrate systems (fish scales, amphibian skin and human cornea). CONCLUSIONS: The production of extracellular matrix during wound healing in leeches is a surprising example of conservation of an extremely close relationship between the structure and function of molecular structures. It could be hypothesized that collagen structures, characterized not only by a striking structural complexity, but also by multifunctional purposes, are anatomical systems highly conserved throughout evolution.

Active supercontraction in rolling-up muscles of Glomeris marginata (myriapoda, diplopoda).

The lateroventral muscles of Glomeris marginata keep the animal rolled up and are able to develop and maintain great tension. Their fibers are not equipped with a particularly strong contractile apparatus but can super-contract. The sarcomere shortens its resting length by up 60% and in a typical supercontraction the thick filaments pass through the Z-line into adjacent sarcomeres. The Z-line structure changes according to the contraction state: It passes from a homogeneous, dense zig-zag line in decontracted fibers to a rarified, vaguely outlined Z-band in supercontracted fibers, in which it is possible to see actin and myosin filaments. The Z-line is thus involved in an active expanding process and is functionally very different from the fragmented and discontinuous Z-line of "classical" supercontracting muscles. The different meaning of the two cases of supercontraction is discussed.

Differentiation of slow and fast fibers in tentacles of Sepia officinalis (Mollusca).

The tentacles of Sepia officinalis are cylindrical muscular structures that can be quickly everted and elongated to capture prey. The combination of both velocity and extensive elongation of the tentacles is due to the presence of both cross-striated and helical muscles. The complex organization and differentiation of different fibers has been studied in cuttlefish extracted from egg gel coats at different developmental stages, and in completely developed animals. Tentacle muscles start to differentiate centrifugally from the area close to the axial nervous system, where two types of myocytes can be recognized. These populations of myocytes, which may be distinguished morphologically and which express different myosin isoforms, give rise to fast and slow muscles. The presence in molluscs of slow and fast muscles arising from different populations of myocytes, as in vertebrate muscle development, could be considered as an example of evolutionary conservation.

The multifunctional role of fibroblasts during wound healing in Hirudo medicinalis (Annelida, Hirudinea).

Extracellular matrix components play a key role during the angiogenic process for a correct development of blood vessels: fibroblasts are the main cell type involved in the regulation of ECM protein production. In this study we characterize H. medicinalis fibroblasts and demonstrate that they take part to the regulation of angiogenesis that occurs during wound healing process. Massive proliferation and phenotypic modification are two distinctive markers of fibroblast activation. These cells, that are usually responsible for collagen production and function as an energy reservoir, are recruited during wound healing to form a collagen scaffold through a direct mechanic action and through secretion of specific proteoglycans. In addition we show that the activity of fibroblasts is modulated by EGF, a growth factor involved in wound healing in vertebrates. The formation of bundles of collagen fibrils by fibroblasts is fundamental for the development and migration of new blood vessels in lesioned areas during wound repair: administration of lovastatin in explanted leeches affects fibroblasts, damages collagen "scaffold" and indirectly causes the reduction of neo-capillary formation.