Proteomic analysis of body wall and coelomic fluid in Sipunculus nudus.

In order to make clear the protein compositions of Sipunculus nudus and investigate its immune-related proteins, proteomic analysis was performed on body wall and coelomic fluid of Sipunculus nudus. A total of 1659 proteins were identified, and 539 proteins were differentially expressed in the coelomic fluid compared to those in the body wall, of which 415 proteins were up-regulated while 124 proteins were down-regulated. Gene Ontology (GO) analysis showed that the GO terms involved in the two parts of Sipunculus nudus were similar, with metabolic processes, catalytic activity and cell occupying the top categories of biological process, molecular function and cellular component, respectively. KEGG pathway analysis showed that 49 pathways in body wall and 48 in coelomic fluid were mapped respectively, and these pathways were mainly related to cellular processes, environmental information processing, genetic information processing and metabolism. The COG analysis showed that 757 proteins from body wall and 889 from coelomic fluid were classified into 26 COG categories, respectively. Pfam annotation revealed the mainly immune-related proteins contained in Sipunculus nudus, such as insulin-like growth factor binding protein, catalase, basement membrane proteoglycan, titin. Our research provides the first proteomic information of Sipunculus nudus, which contributes to the study of functional proteins in Sipunculus nudus and is of great significance for the application of Sipunculus nudus in functional foods and medicines.

Pattern recognition receptors in annelids.

The existence of pattern recognition receptors (PRRs) on immune cells was discussed in 1989 by Charles Janeway, Jr., who proposed a general concept of the ability of PRRs to recognize and bind conserved molecular structures of microorganisms known as pathogen-associated molecular patterns (PAMPs). Upon PAMP engagement, PRRs trigger intracellular signaling cascades resulting in the expression of various proinflammatory molecules. These recognition molecules represent an important and efficient innate immunity tool of all organisms. As invertebrates lack the instruments of the adaptive immune system, based on "true" lymphocytes and functional antibodies, the importance of PRRs are even more fundamental. In the present review, the structure, specificity, and expression profiles of PRRs characterized in annelids are discussed, and their role in innate defense is suggested.

Metagenomic sequencing suggests a diversity of RNA interference-like responses to viruses across multicellular eukaryotes.

RNA interference (RNAi)-related pathways target viruses and transposable element (TE) transcripts in plants, fungi, and ecdysozoans (nematodes and arthropods), giving protection against infection and transmission. In each case, this produces abundant TE and virus-derived 20-30nt small RNAs, which provide a characteristic signature of RNAi-mediated defence. The broad phylogenetic distribution of the Argonaute and Dicer-family genes that mediate these pathways suggests that defensive RNAi is ancient, and probably shared by most animal (metazoan) phyla. Indeed, while vertebrates had been thought an exception, it has recently been argued that mammals also possess an antiviral RNAi pathway, although its immunological relevance is currently uncertain and the viral small RNAs (viRNAs) are not easily detectable. Here we use a metagenomic approach to test for the presence of viRNAs in five species from divergent animal phyla (Porifera, Cnidaria, Echinodermata, Mollusca, and Annelida), and in a brown alga-which represents an independent origin of multicellularity from plants, fungi, and animals. We use metagenomic RNA sequencing to identify around 80 virus-like contigs in these lineages, and small RNA sequencing to identify viRNAs derived from those viruses. We identified 21U small RNAs derived from an RNA virus in the brown alga, reminiscent of plant and fungal viRNAs, despite the deep divergence between these lineages. However, contrary to our expectations, we were unable to identify canonical (i.e. Drosophila- or nematode-like) viRNAs in any of the animals, despite the widespread presence of abundant micro-RNAs, and somatic transposon-derived piwi-interacting RNAs. We did identify a distinctive group of small RNAs derived from RNA viruses in the mollusc. However, unlike ecdysozoan viRNAs, these had a piRNA-like length distribution but lacked key signatures of piRNA biogenesis. We also identified primary piRNAs derived from putatively endogenous copies of DNA viruses in the cnidarian and the echinoderm, and an endogenous RNA virus in the mollusc. The absence of canonical virus-derived small RNAs from our samples may suggest that the majority of animal phyla lack an antiviral RNAi response. Alternatively, these phyla could possess an antiviral RNAi response resembling that reported for vertebrates, with cryptic viRNAs not detectable through simple metagenomic sequencing of wild-type individuals. In either case, our findings show that the antiviral RNAi responses of arthropods and nematodes, which are highly divergent from each other and from that of plants and fungi, are also highly diverged from the most likely ancestral metazoan state.

Isolation of an invertebrate-type lysozyme from the nephridia of the echiura, Urechis unicinctus, and its recombinant production and activities.

Invertebrates, unlike vertebrates which have adaptive immune system, rely heavily on the innate immune system for the defense against pathogenic bacteria. Lysozymes, along with other immune effectors, are regarded as an important group in this defense. An invertebrate-type (i-type) lysozyme, designated Urechis unicinctus invertebrate-type lysozyme, Uu-ilys, has been isolated from nephridia of Urechis unicinctus using a series of high performance liquid chromatography (HPLC), and ultrasensitive radial diffusion assay (URDA) as a bioassay system. Analyses of the primary structure and cDNA cloning revealed that Uu-ilys was approximately 14 kDa and composed of 122 amino acids (AAs) of which the precursor had a total of 160 AAs containing a signal peptide of 18 AAs and a pro-sequence of 20 AAs encoded by the nucleotide sequence of 714 bp that comprises a 5' untranslated region (UTR) of 42 bp, an open reading frame (ORF) of 483 bp, and a 3' UTR of 189 bp. Multiple sequence alignment showed Uu-ilys has high homology to i-type lysozymes from several annelids. Relatively high transcriptional expression levels of Uu-ilys was detected in nephridia, anal vesicle, and intestine. The native Uu-ilys exhibited comparable lysozyme enzymatic and antibacterial activities to hen egg white lysozyme. Collectively, these data suggest that Uu-ilys, the isolated antibacterial protein, plays a role in the immune defense mechanism of U. unicinctus. Recombinant Uu-ilys (rUu-ilys) produced in a bacterial expression system showed significantly decreased lysozyme lytic activity from that of the native while its potency on radial diffusion assay detecting antibacterial activity was retained, which may indicate the non-enzymatic antibacterial capacity of Uu-ilys.

Evolutionary Origins of Toll-like Receptor Signaling.

Toll-like receptors (TLRs) are transmembrane pattern recognition receptors that are best known for their roles in innate immunity for the detection of and defense against microbial pathogens. However, TLRs also have roles in many nonimmune processes, most notably development. TLRs direct both immune and developmental programs by activation of downstream signaling pathways, often by activation of the NF-κB pathway. There are two primary TLR subtypes: 1) TLRs with multiple cysteine clusters in their ectodomain (mccTLRs) and 2) TLRs with a single cysteine cluster in their ectodomain (sccTLRs). For some time, it has been known that TLRs and the biological processes that they control are conserved in organisms from insects to mammals. However, genome and transcriptome sequencing has revealed that many basal metazoans also have TLRs and downstream NF-κB signaling components. In this review, we discuss what is known about the structure, biological function, and downstream signaling pathways of TLRs found in phyla from Porifera through Annelida. From these analyses, we hypothesize that mccTLRs emerged in the phylum Cnidaria, that sccTLRs evolved in the phylum Mollusca, and that TLRs have dual immune and developmental biological functions in organisms as ancient as cnidarians.

Neuro-immune lessons from an annelid: The medicinal leech.

An important question that remains unanswered is how the vertebrate neuroimmune system can be both friend and foe to the damaged nervous tissue. Some of the difficulty in obtaining responses in mammals probably lies in the conflation in the central nervous system (CNS), of the innate and adaptive immune responses, which makes the vertebrate neuroimmune response quite complex and difficult to dissect. An alternative strategy for understanding the relation between neural immunity and neural repair is to study an animal devoid of adaptive immunity and whose CNS is well described and regeneration competent. The medicinal leech offers such opportunity. If the nerve cord of this annelid is crushed or partially cut, axons grow across the lesion and conduction of signals through the damaged region is restored within a few days, even when the nerve cord is removed from the animal and maintained in culture. When the mammalian spinal cord is injured, regeneration of normal connections is more or less successful and implies multiple events that still remain difficult to resolve. Interestingly, the regenerative process of the leech lesioned nerve cord is even more successful under septic than under sterile conditions suggesting that a controlled initiation of an infectious response may be a critical event for the regeneration of normal CNS functions in the leech. Here are reviewed and discussed data explaining how the leech nerve cord sensu stricto (i.e. excluding microglia and infiltrated blood cells) recognizes and responds to microbes and mechanical damages.

Toll-like receptor genes (TLRs) from Capitella capitata and Helobdella robusta (Annelida).

Toll-like receptors (TLRs) are an important part of the innate immunity system and are found throughout the animal kingdom, but have not yet been reported in annelids. We searched shotgun reads of the genomes of the leech Helobdella and polychaete Capitella for TLR homologs. We found 105 TLR homologs in Capitella and 16 in Helobdella. The deduced phylogeny of these sequences, together with TLRs from other animal phyla, reveals three major clades. One clade consists of a mixture of both vertebrates and invertebrates, including sequences from Capitella and Helobdella, while the other two clades contain only invertebrate TLRs.

What characterizes house dust mite sensitive individuals in a house dust mite free community in Reykjavik, Iceland?

BACKGROUND: Previous studies show that 6-9% of young adults in Reykjavik are sensitised to the house dust mite (HDM) Dermatophagoides pteronyssinus (D. pteronyssinus). However, only negligible amounts of HDM and HDM allergens were detected in their homes. The study investigates what characterizes these individuals. METHODS: We investigated all participants in the European Community Respiratory Health Surveys I and II (ECRHS I and II) with D. pteronyssinus specific IgE, in the years 1991-92. A grass positive but D. pteronyssinus negative control group was recruited from the same cohort. A detailed questionnaire was administered and the specific IgE (Pharmacia CAP system) against six D. pteronyssinus cross-reactive allergens was measured. RESULTS: Of 601 ECRHS I participants with available IgE results, 88% returned for ECRHS II, 8.4 years later. Of 49 individuals with D. pteronyssinus specific IgE in ECRHS I, 24 had become negative in ECRHS II. Compared with controls, HDM sensitive subjects were more often men who had lived on farms or kept aquaria fish in childhood. Of those with specific IgE against D. pteronyssinus in ECRHS I and II, 75% had detectable IgE antibodies (>or=0.35 kU/l) to cross-reactive allergens compared with none in the control group (p<0.0001): Lepidoglyphus destructor (L. destructor) (67%), shrimp (58%), cockroach (33%), mosquito (17%), tropomyosin (17%) and blood worm (4%). CONCLUSIONS: Icelanders with specific IgE to D. pteronyssinus are more often men who spent time on farms in childhood and today have high prevalence of IgE antibodies cross-reactive to D. pteronyssinus.

Innate immunity in lophotrochozoans: the annelids.

Innate immunity plays a major role as a first defense against microbes. Effectors of the innate response include pattern recognition receptors (PRR), phagocytic cells, proteolytic cascades and peptides/proteins with antimicrobial properties. Each element of these events has been well studied in vertebrates and in some invertebrates such as annelids. From these different researches, it appears that mammalian innate immunity could be considered as a mosaic of invertebrate immune responses. Annelids belonging to the lophotrochozoans' group are primitive coelomates that possess specially developed cellular immunity against pathogens including phagocytosis, encapsulation and spontaneous cytotoxicity of coelomocytes against allogenic or xenogenic cells. They have also developed an important humoral immunity that is based on antimicrobial, hemolytic and clotting properties of their body fluid. In the present review, we will emphasize the different immunodefense strategies that adaptation has taken during the course of evolution of two classes of annelids i.e. oligochaetes and achaetes.

Antibacterial properties of hemerythrin of the sand worm Nereis diversicolor.

OBJECTIVES: To investigate the immune defense of the annelid Nereis diversicolor and the key role of a oxygen-binding protein, the metalloprotein MPII animals were subjected to bacteria infection. METHODS AND RESULTS: Using RACE-PCR, we have cloned the complete cDNA coding for the MPII related to the hemerythrin family in the sand worm Hediste diversicolor. This cDNA (883 pb) codes for a polypeptide of 119 amino acid residues with no signal peptide. Previous works have identified this protein as a cadmium scavenger. We here clearly demonstrated that this protein is also involved in the worm defence towards bacteria growth by its iron scavenger ability. This protein is expressed and produced in a haematopoietic center that floats freely in the coelomic fluid before stored in a particular hemocyte type: the granulocyte type 1. During bacterial challenge, this protein contained in these cells is discharged into the blood stream 3-4 hours after the infection and remains active for approximately 10 hours. This time period blocks progression of the pathogen and its attachment to tissues. CONCLUSION: These results reflect that MPII in conjunction with others partners like lysozyme act as defence molecule for the sand worm.

Annelid neuroimmune system.

Neuropeptides have been found in nervous central or immune systems of Annelids. Since these signaling molecules are found free in the hemolymph, they are considered as hormones. Hormonal processes along with enzymatic processing similar to that found in vertebrates occur in annelids. Furthermore, amino acid sequence determination of annelids precursor gene products reveals the presence of the respective peptides that exhibit high sequence identity to their mammalian counterparts. Nevertheless, specific neuropeptides to annelids or invertebrates have also been in these animals. These peptides are flanked by potential proteolytic signal sites for the various known enzymes confirming that annelids neuropeptide precursors are processed in a similar manner to that described in mammals i.e. implicating prohormone convertase enzymes.

Neurovascular immunology: a 25-year odyssey.

Small vasoactive neurotransmitter molecules with a long evolutionary history of involvement in biological defense and inflammation play important roles in the up-regulation and down-regulation of the immune response and with similar effects on neuronal and lymphocyte transmembrane signaling molecules and mechanisms. The longest acting of these stimulatory molecules on lymphocyte traffic, substance P and bradykinin, also are transmitters of impulses relating to heat and pain. Heat and pain have been primordial stimuli to learning and memory--immunological as well as CNS.

In vitro antigen-binding properties of coelomocytes of Eisenia foetida (Annelida).

Annelids are capable of cellular and humoral defence reactions against foreign antigens. The main aim of this study was to characterise the antigen-binding properties of coelomocytes of Eisenia foetida by means of quantitative autoradiography and direct measurement of radioactivity. It was found that the antigen-binding capacity was significantly increased after antigen stimulation. Furthermore, the preincubation of coelomocytes with non-labelled proteins reduced the binding of radiolabelled antigen. The highest level of inhibition was found when the same protein was used for preincubation. These results indicate that antigen-binding properties are to some extent specific.

The proteins in Lumbricus terrestris and Eisenia foetida coelomic fluids and on coelomocytes reacting with sheep and goat IgG molecules.

Sheep and goat fast migrating IgG molecules and their F(ab')2 fragments react with L. terrestris and E. foetida coelomic fluid proteins. The reaction was not mediated by antibody binding site but by some other part of the IgG molecule, since affinity purified antibodies of different antigen specificities as well as IgG preparations from normal sheep sera reacted with the same coelomic fluid proteins. The presence of at least two reacting proteins was detected by immunoelectrophoresis and immunoblotting analysis. Molecular weights of these proteins were 47 and 50 kD in L. terrestris and 42 kD in E. foetida. Sheep antibodies and IgG molecules also bind to the Sheep antibodies and IgG molecules also bind to the surface of about 2% of coelomic leukocytes and the immunoblotting analysis showed that the IgG binding proteins in coelomic fluids and in cell lysates were similar.

Specific immune recognition by lymphocytes: an evolutionary perspective.

In this review, we analyze data pertinent to the origins of specific immune recognition by lymphocytes. The phenomena of immunity in invertebrates and the cells that might be involved in these processes are considered. We conclude that the existence of vertebrate-type immunocompetent lymphocytes in invertebrates is not yet proven. All vertebrates apparently possess immunologically competent lymphocytes, and the following conclusions may be drawn: (1) Specific antigen recognition by lymphocytes of all vertebrates appears to be mediated by membrane-bound immunoglobulins. These immunoglobulins are, in all probability, distinct from secreted, serum immunoglobulins (antibodies), although serum and surface immunoglobulins share combining sites for antigen which are formed of variable regions. (2) There is evidence for similar functional divisions among the immune systems of all vertebrates, as reflected in the results of anatomical, functional, and physicochemical investigations of lymphocytes from animals as diverse as fish and mammals.