Earthworm immunity.

Earthworms belonging to oligochaete annelids became a model for comparative immunologists in the early sixties with the publication of results from transplantation experiments that proved the existence of self/nonself recognition in earthworms. This initiated extensive studies on the earthworm immune mechanisms that evolved to prevent the invasion of pathogens. In the last four decades important cellular and humoral pathways were described and numerous biologically active compounds were characterized and often cloned.

Identification and cloning of an invertebrate-type lysozyme from Eisenia andrei.

Lysozyme is a widely distributed antimicrobial protein having specificity for cleaving the beta-(1,4)-glycosidic bond between N-acetylmuramic acid (NAM) and N-acetylglucosamine (GlcNAc) of peptidoglycan of the bacterial cell walls and thus efficiently contributes to protection against infections caused mainly by Gram-positive bacteria. In the present study, we assembled a full-length cDNA of a novel invertebrate-type lysozyme from Eisenia andrei earthworm (EALys) by RT-PCR and RACE system. The primary structure of EALys shares high homology with other invertebrate lysozymes; however the highest, 72% identity, was shown for the destabilase I isolated from medicinal leech. Recombinant EALys expressed in Escherichia coli exhibited the lysozyme and isopeptidase activity. Moreover, real-time PCR revealed increased levels of lysozyme mRNA in coelomocytes of E. andrei after the challenge with both Gram-positive and Gram-negative bacteria.

Overcoming immunoescape mechanisms of BCL1 leukemia and induction of CD8+ T-cell-mediated BCL1-specific resistance in mice cured by targeted polymer-bound doxorubicin.

BALB/c mice bearing syngeneic BCL1 leukemia, a mouse model of human chronic lymphocytic leukemia, were treated with polymer-bound doxorubicin conjugate targeted with BCL1-specific monoclonal antibody. Such treatment can cure up to 100% of mice and the cured mice show long-lasting resistance to BCL1 leukemia. We show that both CD4+ and CD8+ T cells are required for establishment of the resistance, but only CD8+ T cells are necessary for its maintenance. BCL1 cells express MHC class I and II and also costimulatory molecules CD80 and CD86, which can aid eliciting of antitumor response. On the other hand, BCL1 cells also use several immunoescape mechanisms, such as expression of PD-L1, PD-L2, and interleukin-10. BCL1 cells thus can be recognized by BCL1-specific T cells, but instead of effective priming, such T cells are anergized or deleted by apoptosis. Moreover, BCL1 leukemia progression is accompanied by robust expansion of CD4+CD25+Foxp3+ regulatory T (Treg) cells. Although it has been shown that depletion of Treg cells in tumor-bearing mice can retard tumor growth, direct evidence that expansion of Treg cells can promote tumor growth was lacking. In this study, we provide first direct evidence that expanded Treg cells can indeed promote tumor progression by using mice with selectively expanded Treg cells before inoculation of BCL1 leukemia. Finally, we have also shown that elimination of some immunoescape mechanism (e.g., deletion of Treg) can significantly improve the therapeutic outcome of chemotherapy.

Characterization, molecular cloning and localization of calreticulin in Eisenia fetida earthworms.

Calreticulin is a highly conserved calcium-binding protein affecting many cellular processes inside and outside of the endoplasmic reticulum (ER). It participates in the regulation of Ca(2+) homeostasis, acts as a chaperone and modulates gene transcription, integrin-mediated cell signalling as well as cell adhesion. Here we report on the sequence characterization of a calreticulin-coding cDNA of Eisenia fetida earthworms. The neighbor-joining phylogeny tree constructed based on the deduced amino acid sequence indicates a common origin of the E. fetida calreticulin molecule and that of mollusks. A polyclonal anti-calreticulin antibody used for immunocytochemistry and immunohistochemistry localized the protein in the mesenchymal lining of the coelomic cavity and in coelomocytes of E. fetida. In situ hybridization revealed high expression of E. fetida calreticulin in various cells and tissues, namely epidermis, neurons of the ventral nerve cord, intestine, sperms, body wall muscles and some coelomocytes. Real-time PCR confirmed the strong expression of calreticulin in the nervous system, particularly in cerebral ganglia, in body wall muscles and in seminal vesicles. Moreover, a high calreticulin expression was measured in the muscular pharynx.

An invertebrate TNF functional analogue activates macrophages via lectin-saccharide interaction with ion channels.

The invertebrate pattern-recognition protein named coelomic cytolytic factor (CCF) and the mammalian cytokine tumor necrosis factor (TNF) share functional analogies that are based on a similar saccharide recognition specificity. In particular, CCF and TNF have been shown to interact with ion channels on the surface of vertebrate cells via N,N'-diacetylchitobiose lectin-like activity. In the present study, we show that CCF-induced membrane depolarization results in the release of TNF, IL-6 and nitric oxide (NO) by macrophages via nuclear factor-kappaB signaling. Interestingly, our data suggest that TNF contributes, through lectin-saccharide interaction, to the secretion of IL-6 and NO induced by CCF. This experimental non-physiological setting based on the interaction of an invertebrate defense lectin with vertebrate cells involved in the innate immune response may have highlighted an evolutionarily ancient mechanism of macrophage activation in vertebrates.

Evidence for proteins involved in prophenoloxidase cascade Eisenia fetida earthworms.

The prophenoloxidase cascade represents one of the most important defense mechanisms in many invertebrates. Following the recognition of microbial saccharides by pattern recognition molecules, proteinases cleave inactive prophenoloxidase to its active form, phenoloxidase. Phenoloxidase is a key enzyme responsible for the catalysis of the melanization reaction. Final product melanin is involved in wound healing and immune responses. Prophenoloxidase cascade has been widely described in arthropods; data in other invertebrate groups are less frequent. Here we show detectable phenoloxidase activity in 90-kDa fraction of the coelomic fluid of earthworms Eisenia fetida. Amino acid sequencing of peptides from the active fraction revealed a partial homology with invertebrate phenoloxidases and hemocyanins. Moreover, the level of phenoloxidase activity is lower and the activation slower as compared to other invertebrates.

Comparative study of the CCF-like pattern recognition protein in different Lumbricid species.

Coelomic fluid of the Lumbricid Eisenia fetida contains a 42-kDa pattern recognition protein named coelomic cytolytic factor (CCF) that binds microbial cell wall components and triggers the activation of the prophenoloxidase cascade, an important invertebrate defense pathway. Here we report on the sequence characterization of CCF-like molecules of other Lumbricids: Aporrectodea caliginosa, Aporrectodea icterica, Aporrectodea longa, Aporrectodea rosea, Dendrobaena veneta, Lumbricus rubellus and Lumbricus terrestris, and show that CCF from E. fetida has a broader saccharide-binding specificity, being the only one recognizing N,N'-diacetylchitobiose. We suggest that the broad recognition repertoire of E. fetida CCF reflects a particular microbial environment this species lives in.

Relationship between hemolytic molecules in Eisenia fetida earthworms.

The coelomic fluid of the earthworm Eisenia fetida has been reported to contain a variety of proteins causing the lysis of red blood cells-EFAF (Eisenia fetida andrei factor), fetidin, lysenin, eiseniapore, and hemolysins isolated either from coelomic fluid (H1, H2, H3) or from cell lysate (CL(39) and CL(41)). We document the presence of two distinct genes with a high level of homology. These genes encode fetidin and lysenin but their level of expression differs in individual E. fetida andrei animals.