Davide Malagoli

Autophagy and its physiological relevance in arthropods: Current knowledge and perspectives

Autophagic process is one of the best examples of a conserved mechanism of survival in eukaryotes. At the molecular level there are impressive similarities between unicellular and multicellular organisms, but there is increasing evidence that the same process may be used for different ends, i.e., survival or death, at least at cellular levels. Arthropods encompass a wide variety of invertebrates such as insects, crustaceans and spiders, and thus represent the taxon in which most of the investigations on autophagy in non-mammalian models are performed. The present review is focused on the genetic basis and the physiological meaning of the autophagic process on key models of arthropods. The involvement of autophagy in programmed cell death, especially during oogenesis and development, is also discussed.

Immunomodulation by recombinant human interleukin-8 and its signal transduction pathways in invertebrate hemocytes

Abstract. We report the presence of interleukin (IL)-8-immunoreactive molecules in hemocytes from the mollusc Mytilus galloprovincialis. Functional studies demonstrate that recombinant human (rh)IL-8 provokes conformational changes, induces chemotaxis, and increases bacterial phagocytic activity in hemocytes. rhIL-8 induces cell shape changes via protein kinase A and C pathways. These morphological changes are followed by reorganization of the actin microfilaments. The findings suggest that, as previously reported for other cytokines, IL-8 is well conserved and deeply involved in immune functions from invertebrates to mammals.

Invertebrate Humoral Factors: Cytokines as Mediators of Cell Survival

The presence and the different functional aspects of cytokine-related molecules in invertebrates are described. Cytokine-like factors affect immune functions, such as cell motility, chemotaxis, phagocytosis and cytotoxicity. In particular, cell migration shows a species-specific effect for IL-1alpha and TNF-alpha and a dose-correlated effect for IL-8, PDGF-AB and TGF-beta1. Apart from some exceptions, the phagocytic effect increases significantly at all the concentrations tested and with all the species used. PDGF-AB, TGF-beta1 and IL-8 provoke conformational changes in mollusk immunocytes, involving the signaling transduction pathways of phosphatidylinositol and cAMP. PDGF-AB and TGF-beta1 partially inhibit the induced programmed cell death in an insect cell line, and the survival effect is mediated by the activation of phosphatidylinositol 3-kinase, PKA and PKC. The exogenous administration of these growth factors in an invertebrate wound repair model showed that they are able to control the wound environment and promote the repair process by accelerating the coordinated activities involved. Moreover, IL-1alpha, IL-2 and TNF-alpha are able to induce nitric oxide synthase. PDGF-AB and TGF-beta1 provoke an increase in neutral endopeptidase-24.11 (NEP)-like activity in membrane preparations from mollusk immunocytes, while NEP deactivates the PDGF-AB- and TGF-beta1-induced cell shape changes. Cytokines are also involved in invertebrate stress response in a manner extremely similar to that in vertebrates. Several studies suggest the existence on the mollusk immunocyte membrane of an ancestral receptor capable of binding both IL-2 and CRH. Furthermore, the competition found between CRH and a large number of cytokines supports the idea that invertebrate cytokine receptors show a certain degree of promiscuity. The multiple functions of cytokines detected in invertebrates underline another characteristic of mammalian cytokines, i.e. their great pleiotropicity. Altogether, the studies on the function of the invertebrate humoral factors show a close overlapping with those found in vertebrates, and the hypothesized missing correlation between invertebrate and vertebrate cytokine genes that is emerging from the limited molecular biology data present in literature might represent a very peculiar strategy followed by Nature in the evolution of cytokines.

The evolution of the adipose tissue: A neglected enigma

The complexity of the anatomical distribution and functions of adipose tissue (AT) has been rarely analyzed in an evolutionary perspective. From yeast to man lipid droplets are stored mainly in the form of triglycerides in order to provide energy during periods when energy demands exceed caloric intake. This simple scenario is in agreement with the recent discovery of a highly conserved family of proteins for fat storage in both unicellular and multicellular organisms. However, the evolutionary history of organs such as the fat body in insects, playing a role in immunity and other functions besides energy storage and thermal insulation, and of differently distributed subtypes of AT in vertebrates is much less clear. These topics still await a systematic investigation using up-to-date technologies and approaches that would provide information useful for understanding the role of different AT subtypes in normal/physiological conditions or in metabolic pathologies of humans.

Targets and Effects of Yessotoxin, Okadaic Acid and Palytoxin: A Differential Review

In this review, we focus on processes, organs and systems targeted by the marine toxins yessotoxin (YTX), okadaic acid (OA) and palytoxin (PTX). The effects of YTX and their basis are analyzed from data collected in the mollusc Mytilus galloprovincialis, the annelid Enchytraeus crypticus, Swiss CD1 mice and invertebrate and vertebrate cell cultures. OA and PTX, two toxins with a better established mode of action, are analyzed with regard to their effects on development. The amphibian Xenopus laevis is used as a model, and the Frog Embryo Teratogenesis Assay-Xenopus (FETAX) as the experimental protocol.

Growth factors and chemokines: A comparative functional approach between invertebrates and vertebrates

Growth factors and cytokines control and coordinate a broad spectrum of fundamental cellular functions, and are evolutionarily conserved both in vertebrates and invertebrates. In this review, we focus our attention on the functional phylogenetic aspects of growth factors/cytokines like the Transforming Growth Factor-beta (TGF-beta), the Connective Tissue Growth Factor (CTGF), and the Vascular Endothelial Growth Factor (VEGF). We will also delve into the activites of two chemokine families, interleukin (IL)-8 (or CXCL8) and CC chemokine ligand 2/monocyte chemoattractant protein-1 (CCL2). These molecules have been selected for their involvement in immune responses and wound healing processes, where they mediate and finely regulate various regeneration processes like angiogenesis or fibroplasia, not only in vertebrates, but also in invertebrates.

Ecoimmunology: is there any room for the neuroendocrine system?

Ecological Immunology assumes that immunological defenses must be minimized in terms of cost (energy expenditure). To reach this goal, a complex and still largely unexplored strategy has evolved to assure survival. From invertebrates to vertebrates, an integrated immune-neuroendocrine response appears to be crucial for the hierarchical redistribution of resources within the body according to the specific ecological demands. Thus, on the basis of experimental data on the intimate relationship between stress and immune responses that has been maintained during evolution, we argue that a broader perspective based on the integration of immune and neuroendocrine responses should be adopted to describe the comprehensive strategy that the body utilizes to adapt to dynamic environmental conditions. We discuss the hypothesis that a bow-tie architecture might be suitable to describe the variety of immune-neuroendocrine inputs that continuously target cells and organs while, at the same time, fulfilling the basic requirement of minimizing the cost of immune-neuroendocrine responses. Bow-tie architectures are able to convert a variety of stimuli (fan in) into a wide range of fine-tuned responses (fan out) by passing through the integrating activity of a core (knot) constituted by a limited number of elements. Finally, we argue that the ecologically negotiated immune-neuroendocrine strategies may have deleterious effects in the post-reproductive period of life when, at least in humans, chronic, low-grade, systemic inflammation develops, in accordance with the antagonistic pleiotropy theory of aging.

Yessotoxin affects fMLP-induced cell shape changes inMytilus galloprovincialis immunocytes

Using computer‐assisted microscopic image analysis, we have found that algal yessotoxin (YTX) affects the immune response of Mytilus galloprovincialis. Indeed, YTX increases immunocyte cell motility through the involvement of both extracellular Ca2+and cAMP, but not through protein kinase A, protein kinase C or phosphoinositide 3‐kinase. Alone, however, the toxin does not induce any effect, as its action on cell motility is observed only after addition of the chemotactic substance N‐formyl‐Meth‐Leu‐Phe (fMLP). fMLP is known to induce cellular changes via both the phosphatidylinositol and cAMP pathways and, from this scenario, we can surmise that Ca2+and cAMP concentrations rise sufficiently in fMLP‐activated immunocytes to reveal YTX action. One possible explanation is that the toxin increases fMLP‐mediated cell activation by intervening in L‐type Ca2+‐channel opening through a cAMP‐dependent/PKA‐independent pathway.

Skin wound healing in different aged Xenopus laevis

Xenopus froglets can perfectly heal skin wounds without scarring. To explore whether this capacity is maintained as development proceeds, we examined the cellular responses during the repair of skin injury in 8‐ and 15‐month‐old Xenopus laevis. The morphology and sequence of healing phases (i.e., inflammation, new tissue formation, and remodeling) were independent of age, while the timing was delayed in older frogs. At the beginning of postinjury, wound re‐epithelialization occurred in form of a thin epithelium followed by a multilayered epidermis containing cells with apoptotic patterns and keratinocytes stained by anti‐inducible nitric oxide synthase (iNOS) antibody. The inflammatory response, early activated by recruitment of blood cells immunoreactive to anti‐tumor necrosis factor (TNF)‐α, iNOS, transforming growth factor (TGF)‐β1, and matrix metalloproteinase (MMP)‐9, persisted over time. The dermis repaired by a granulation tissue with extensive angiogenesis, inflammatory cells, fibroblasts, and anti‐α‐SMA positive myofibroblasts. As the healing progressed, wounded areas displayed vascular regression, decrease in cellularity, and rearrangement of provisional matrix. The epidermis restored to a prewound morphology while granulation tissue was replaced by a fibrous tissue in a scar‐like pattern. The quantitative PCR analysis demonstrated an up‐regulated expression of Xenopus suppressor of cytokine signaling 3 (XSOCS-3) and Xenopus transforming growth factor-β2 (XTGF-β2) soon after wounding and peak levels were detected when granulation tissue was well developed with a large number of inflammatory cells. The findings indicate that X. laevis skin wound healing occurred by a combination of regeneration (in epidermis) and repair (in dermis) and, in contrast to froglet scarless wound healing, the growth to a more mature adult stage is associated with a decrease in regenerative capacity with scar‐like tissue formation. J. Morphol. 274:956–964, 2013.

Purification and characterization of phenoloxidase from the hemocytes of Eurygaster integriceps (Hemiptera: Scutelleridae)

Insects protect themselves from microbial invaders by two main immune activities, namely the cellular and humoral reactions. Phenoloxidases are oxidative enzymes that have an important role in both cell-mediated and humoral immunity. In this study, the purification and biochemical characterization of a phenoloxidase from the hemocytes of the Sunn pest Eurygaster integriceps Puton (Hemiptera: Scutelleridae) were carried out. After the final purification step, the enzyme was purified 7.31-fold with a recovery of 3.94% and a specific activity of 4.95U/mg protein. Results of the biochemical characterization showed that the purified phenoloxidase has a maximum activity at pH 6 and at 30-35°C and is stable for 24-36h. Divalent cations such as Ca(2+) and Cu(2+) significantly increased the enzymatic activity and synthetic inhibitors such as phenylthiourea significantly decreased it. The purified phenoloxidase has a molecular mass of 22kDa. The current paper represents a further step towards the characterization of humoral immunity of E. integriceps in order to develop new strategies for the biological control of the Sunn pest.