Gianluca Tettamanti

Programmed cell death and stem cell differentiation are responsible for midgut replacement in Heliothis virescens during prepupal instar

We have analyzed midgut development during the fifth larval instar in the tobacco budworm Heliothis virescens. In prepupae, the midgut formed during larval instars undergoes a complete renewal process. This drastic remodeling of the alimentary canal involves the destruction of the old cells by programmed cell-death mechanisms (autophagy and apoptosis). Massive proliferation and differentiation of regenerative stem cells take place at the end of the fifth instar and give rise to a new fully functioning epithelium that is capable of digesting and absorbing nutrients and that is maintained throughout the subsequent pupal stage. Midgut replacement in H. virescens is achieved by a balance between this active proliferation process and cell-death mechanisms and is different from similar processes characterized in other insects.

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.

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

Summry— 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.

Autophagy in invertebrates: insights into development, regeneration and body remodeling.

Autophagy is a process in which eukaryotic cells sequester and degrade cytoplasm and organelles via the lysosomal pathway. This process allows turnover of intracellular organelles, participates in the maintenance of cellular homeostasis and prevents accumulation of defective cellular structures. Increased autophagy is normally induced by environmental cues such as starvation and hormones, while excessive levels of autophagy can lead to autophagic programmed cell death (PCD), with features that differ from those of the apoptotic PCD process. Since autophagic PCD plays a key role in development, morphogenesis and regeneration in several animal taxa, identification of evolutionarily conserved components of the autophagic machinery is a basic starting point in order to unravel the role of autophagy under both physiological and pathological conditions. Here we summarize recent findings on the role of autophagy in two different invertebrate taxa, Platyhelminthes and Insects, focusing attention on two complex events occurring in those systems, namely planarian regeneration and insect metamorphosis. Both represent good models in which to investigate the process of autophagy and its relationship with other PCD mechanisms.

Hirudo medicinalis: A new model for testing activators and inhibitors of angiogenesis

An increasing body of evidence indicates that in the leech Hirudo medicinalis the angiogenic process is finely regulated and coordinated by the botryoidal tissue. In this paper we provide evidence on the involvement of botryoidal tissue cells in angiogenesis induced in H. medicinalis by a variety of stimuli including surgical wounds or the administration of modulators of neovascularization. Interestingly, we show that either human activators of vascular cell growth, or anti-angiogenic peptides like angiostatin and endostatin, or the drug mitomycin, can induce a prompt biological response in H. medicinalis. We show as well that angiogenesis in this invertebrate shares a surprising degree of similarity with neovascularization in vertebrates, both at the biochemical and cellular levels, because it involves similar growth factors/growth factor receptors, and relies on analogous cell–cell or cell–matrix interactions. For these reasons we suggest that H. medicinalis can be used as a reproducible model for testing activators or inhibitors of angiogenesis, and for investigating the biochemical, ultrastructural and cellular processes involved in new vessel formation.

Cell Lines Derived from Human Parthenogenetic Embryos Can Display Aberrant Centriole Distribution and Altered Expression Levels of Mitotic Spindle Check-point Transcripts

Human parthenogenetic embryos have recently been proposed as an alternative, less controversial source of embryonic stem cell (ESC) lines; however many aspects related to the biology of parthenogenetic embryos and parthenogenetic derived cell lines still need to be elucidated. We present here results on human cell lines (HP1 and HP3) derived from blastocysts obtained by oocyte parthenogenetic activation. Cell lines showed typical ESC morphology, expressed Oct-4, Nanog, Sox-2, Rex-1, alkaline phosphatase, SSEA-4, TRA 1-81 and had high telomerase activity. Expression of genes specific for different embryonic germ layers was detected from HP cells differentiated upon embryoid body (EBs) formation. Furthermore, when cultured in appropriate conditions, HP cell lines were able to differentiate into mature cell types of the neural and hematopoietic lineages. However, the injection of undifferentiated HP cells in immunodeficient mice resulted either in poor differentiation or in tumour formation with the morphological characteristics of myofibrosarcomas. Further analysis of HP cells indicated aberrant levels of molecules related to spindle formation as well as the presence of an abnormal number of centrioles and autophagic activity. Our results confirm and extend the notion that human parthenogenetic stem cells can be derived and can differentiate in mature cell types, but also highlight the possibility that, alteration of the proliferation mechanisms may occur in these cells, suggesting great caution if a therapeutic use of this kind of stem cells is considered.

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.

Functional amyloids in insect immune response.

The innate immune system of insects consists of humoural and cellular responses that provide protection against invading pathogens and parasites. Defence reactions against these latter include encapsulation by immune cells and targeted melanin deposition, which is usually restricted to the surface of the foreign invader, to prevent systemic damage. Here we show that a protein produced by haemocytes of Heliothis virescens (Lepidoptera, Noctuidae) larvae, belonging to XendoU family, generates amyloid fibrils, which accumulate in large cisternae of the rough endoplasmic reticulum and are released upon immune challenge, to form a layer coating non-self objects entering the haemocoel. This amyloid layer acts as a molecular scaffold that promotes localised melanin synthesis and the adhesion of immune cells around the non-self intruder during encapsulation response. Our results demonstrate a new functional role for these protein aggregates that are commonly associated with severe human diseases. We predict that insects will offer new powerful experimental systems for studying inducible amyloidogenesis, which will likely provide fresh perspectives for its prevention.

Molecular cloning, characterization and expression analysis of ATG1 in the silkworm, Bombyx mori

Atg1 is a Serine/Threonine protein kinase that plays a pivotal role in autophagy. A complete coding sequence of ATG1 is not available for the silkworm, Bombyx mori which is a good model for studying the autophagic process. In the present study we isolated two full-length cDNAs of 2175 (transcript variant A) and 2271 (transcript variant B) bases representing ATG1 in the silkworm. Phylogenetic analysis indicated that BmATG1 was closely related to orthologs of other insects. The encoded BmAtg1 proteins shared extensive homology with orthologs from yeast to mammals, showing high conservation at the N-terminal region where the catalytic domain and ATP- and Mg-binding sites are located. A de novo prediction of the three-dimensional structure for each protein is presented. We used real-time RT-PCR to quantify dynamic changes in mRNA copy number of BmATG1 in the midgut and fat body of fifth instar larvae undergoing starvation, as well as in other tissues of silkworm at the end of last larval instar. Our qPCR results revealed that BmATG1 expression levels at the end of larval life were comparable in the midgut, fat body and Malpighian tubules, while these were higher in the gonads; moreover, the mRNA copy number of ATG1 was very different among the anterior, middle and posterior silk glands. Real-time PCR analysis also showed that starvation significantly influenced BmATG1 mRNA copy number in the fat body of silkworm, inducing an upregulation 24h after food withdrawal, with only a slight effect in the midgut. Low expression levels of BmATG1 were observed in both tissues of control animals up to the second day of spinning phase.

Identification of Enterococcus mundtii as a pathogenic agent involved in the “flacherie” disease in Bombyx mori L. larvae reared on artificial diet

Enterococcus mundtii was shown to be directly correlated with flacherie disease of the silkworm larvae reared on artificial diet supplemented with chloramphenicol. Its identification was carried out by means of light and electron microscopy and nucleotide sequencing of 16S gene. The bacterium is capable of rapidly multiplying in the silkworm gut and of invading other body tissues, as demonstrated by deliberate infection of germfree larvae and by subsequent TEM observations. E. mundtii can endure alkaline pH of the silkworm gut and it has been proved to adapt in vitro to commonly applied doses of chloramphenicol, whose use can further contribute to reduce competition by other bacteria in Bombyx mori alimentary canal. The modality of transmission of the infection to the larvae was among the objectives of the present research. Since contamination of the progeny by mother moths can be avoided through routine egg shell disinfection, a trans-ovarian vertical transmission can be ruled out. On the other hand the bacterium was for the first time identified on mulberry leaves, and therefore artificial diet based on leaf powder could be a source of infection. We showed that while microwaved diet could contain live E. mundtii cells, the autoclaved diet is safe in this respect. Being E. mundtii also part of the human-associated microbiota, and since B. mori is totally domestic species, a possible role of man in its epidemiology can be postulated.