Leeches Baicalobdella torquata feed on hemolymph but have a low effect on the cellular immune response of amphipod Eulimnogammarus verrucosus from Lake Baikal.

Lake Baikal is one of the largest and oldest freshwater reservoirs on the planet with a huge endemic diversity of amphipods (Amphipoda, Crustacea). These crustaceans have various symbiotic relationships, including the rarely described phenomenon of leech parasitism on amphipods. It is known that leeches feeding on hemolymph of crustacean hosts can influence their physiology, especially under stressful conditions. Here we show that leeches Baicalobdella torquata (Grube, 1871) found on gills of Eulimnogammarus verrucosus (Gerstfeldt, 1858), one of the most abundant amphipods in the Baikal littoral zone, indeed feed on the hemolymph of their host. However, the leech infection had no effect on immune parameters such as hemocyte concentration or phenoloxidase activity and also did not affect glycogen content. The intensity of hemocyte reaction to foreign bodies in a primary culture was identical between leech-free and leech-infected animals. Artificial infection with leeches also had only a subtle effect on the course of a model microbial infection in terms of hemocyte concentration and composition. Despite we cannot fully exclude deleterious effects of the parasites, our study indicates a low influence of a few leeches on E. verrucosus and shows that leech-infected amphipods can be used at least for some types of ecophysiological experiments.

[Effect of Astragali Radix on growth, immunity and related gene expression of Whitmania pigra].

To explore the effect and mechanism of Astragali Radix on growth and immunity of Whitmania pigra, 0, 0.01%, 0.03%, 0.05%, 0.07%, 0.09% of Astragali Radix were added to the daily feeding of Wh. pigra. After 60 days of feeding, the growth performance, activities of digestive enzyme and anti-reverse enzyme, inner quality, the expression levels of GH, IGF-1 and digestive enzyme-related genes were measured. Meanwhile, the effects of heat stress on the living conditions of Wh. pigra were observed and counted, and the expression levels of HSP70 and immune related genes were measured. The results showed that the final weight, weight gain rate, specific growth rate, activities of digestive enzyme and anti-reverse enzyme, the expression levels of GH, IGF-1 and digestive enzyme-related genes in the Astragali Radix group were higher than those in the control group, and with the increase of Astragali Radix concentration, the above-mentioned indexes increased initially and then decreased, and significantly higher in the 0.05% of Astragali Radix group than in the other groups (P<0.05). There was no significant difference in the inner quality of Wh. pigra between the Astragali Radix and control groups. The survival rate of Wh. pigra was negatively correlated with heat stress treatment duration. With the prolongation of heat stress treatment duration, the expression levels of HSP70 and immune related genes were increased first and then decreased, and peaked at 24 h. The survival rate and the expression levels of HSP70 and immune related genes in the Astragali Radix group were higher than those in the control group, and was significantly higher in the 0.05% of Astragali Radix group than in the other groups (P<0.05). In conclusion, Astragali Radix can increase the activities of digestive enzyme and anti-reverse enzyme, the expression levels of related genes, growth performance, and immunity to heat stress of Wh. pigra. It is suggested to add 0.05% of Astragali Radix in the actual production of Wh. pigra to improve the production profit.

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.

Reciprocal immune benefit based on complementary production of antibiotics by the leech Hirudo verbana and its gut symbiont Aeromonas veronii.

The medicinal leech has established a long-term mutualistic association with Aeromonas veronii, a versatile bacterium which can also display free-living waterborne and fish- or human-pathogenic lifestyles. Here, we investigated the role of antibiotics in the dynamics of interaction between the leech and its gut symbiont Aeromonas. By combining biochemical and molecular approaches, we isolated and identified for the first time the antimicrobial peptides (AMPs) produced by the leech digestive tract and by its symbiont Aeromonas. Immunohistochemistry data and PCR analyses evidenced that leech AMP genes are induced in the gut epithelial cells when Aeromonas load is low (starved animals), while repressed when Aeromonas abundance is the highest (post blood feeding). The asynchronous production of AMPs by both partners suggests that these antibiotic substances (i) provide them with reciprocal protection against invasive bacteria and (ii) contribute to the unusual simplicity of the gut microflora of the leech. This immune benefit substantially reinforces the evidence of an evolutionarily stable association between H. verbana and A. veronii. Altogether these data may provide insights into the processes making the association with an Aeromonas species in the digestive tract either deleterious or beneficial.

Effect of medicinal leeches' antigens on the proliferative response of human blood mononuclear cells and cytokine production in vitro.

UNLABELLED: The purpose of research is to study the influence of plant mitogens and antigens of the water-salt extract from the bodies of Hirudo verbana, H. medicinalis, H. orientalis on the reaction of lymphocyte blast-transformation and the synthesis of pro-inflammatory cytokines (IL-1ß, IL-8, TNF-α) in patients' cell culture supernatants before and after hirudotherapy. RESEARCH METHODS: the reaction of lymphocyte blast-transformation; the determination of proinflammatory cytokines by ELISA. After hirudotherapy increased values of the reaction of lymphocyte blasttransformation have got bigger in all stimulated types of cell culture, but reactivity on medicinal leeches antigens proceeded to unproductive immunogenesis (apoptosis and necrosis). In supernatants of mitogen- and antigen-stimulated lymphocyte cultures induced high levels of pro-inflammatory cytokines after hirudotherapy have decreased adequately to apoptotic induction of lymphocytes by medicinal leeches' antigens.

Morphological and functional characterization of leech circulating blood cells: role in immunity and neural repair.

Unlike most invertebrates, annelids possess a closed vascular system distinct from the coelomic liquid. The morphology and the function of leech blood cells are reported here. We have demonstrated the presence of a unique cell type which participates in various immune processes. In contrast to the mammalian spinal cord, the leech CNS is able to regenerate and restore function after injury. The close contact of the blood with the nerve cord also led us to explore the participation of blood in neural repair. Our data evidenced that, in addition to exerting peripheral immune functions, leech blood optimizes CNS neural repair through the release of neurotrophic substances. Circulating blood cells also appeared able to infiltrate the injured CNS where, in conjunction with microglia, they limit the formation of a scar. In mammals, CNS injury leads to the generation of a glial scar that blocks the mechanism of regeneration by preventing axonal regrowth. The results presented here constitute the first description of neuroimmune functions of invertebrate blood cells. Understanding the basic function of the peripheral circulating cells and their interactions with lesioned CNS in the leech would allow us to acquire insights into the complexity of the neuroimmune response of the injured mammalian brain.

Characterization and immune function of two intracellular sensors, HmTLR1 and HmNLR, in the injured CNS of an invertebrate.

Unlike mammals, the CNS of the medicinal leech can regenerate damaged neurites, thus restoring neural functions. Our group recently demonstrated that the injured leech nerve cord is able to mount an immune response, which promotes the regenerative processes. This defense mechanism is microorganism-specific, suggesting that the leech CNS is able to discriminate among microbial components. We report here the characterization of two receptors potentially implicated in this detection: HmTLR1 and HmNLR. Interestingly, HmTLR1 presents an endosomal distribution in neurons and appears as a chimera combining the mammalian intraendosomal domain of TLR3 and the cytoplasmic section of TLR13, while HmNLR is cytosolic and has the highest homology to NLRC3 receptors. Both receptors show patterns of induction upon stimulation that suggest their involvement in the leech neuroimmune response. This work constitutes the first demonstration in an invertebrate of (i) an intracellular TLR and (ii) a cytosolic PRR related to the NLR family.

Leech immunity: from brain to peripheral responses.

In the present chapter, we will emphasize the immune response in two compartments (Central nervous system and peripheral system) in two blood sucking leeches i.e., the medicinal leech and the bird leech Theromyzon tessulatum. In the medicinal leech, the neuroimmune response has been described in the context of septic trauma at the cellular and humoral levels through microglia, Toll-like, cannabinoids and chemoattractant factors activation and modulation. In the bird leech, the antimicrobial responses have been dissected at the cellular and molecular levels. Altogether, this chapter presents a complete integrate immune response from the brain and the systemic compartments with high similarity to the vertebrates one. These points that the neuroimmune and immune responses evolved sooner than can be expected.

Deciphering the immune function and regulation by a TLR of the cytokine EMAPII in the lesioned central nervous system using a leech model.

A highly conserved ortholog of the human complex p43/endothelial monocyte-activating polypeptide II (EMAPII) was characterized in the CNS of the leech Hirudo medicinalis. As observed in mammals, the leech complex is processed to release the cytokine HmEMAPII. Taking advantages of these similarities, we have attempted to elucidate the role of EMAPII in the CNS using the leech model. Although EMAPII is considered a modulator of inflammatory reactions within the peripheral innate immune response in humans, its function in CNS immunity has yet to be described. Chemotaxis assays were conducted, revealing the ability of EMAPII to exert a chemoattractant effect on both leech and human microglial cells, indicating a novel function of this cytokine in the human brain. Quantitative RT-PCR analysis together with in situ hybridization and immunohistochemistry approaches showed that bacterial challenge induced the expression of HmEMAPII at the lesion site where microglial cells accumulated. Moreover, gene silencing experiments have demonstrated that the gene expression of HmEMAPII is under the control of a signaling pathway associated with the TLR HmTLR1, newly characterized in the CNS of our model. To the best of our knowledge, this is the first report showing evidence for (1) the chemoattractant properties of EMAPII on leech and human microglial cells, (2) the regulation by a TLR of the expression of a gene encoding a cytokine in the CNS of an invertebrate, and (3) an immune function of a TLR in a lophotrochozoan model.

Cathepsin L and cystatin B gene expression discriminates immune coelomic cells in the leech Theromyzon tessulatum.

Previous studies evidenced that cystatin B-like gene is specifically expressed and induced in large circulating coelomic cells following bacterial challenge in the leech Theromyzon tessulatum. In order to understand the role of that cysteine proteinase inhibitor during immune response, we investigated the existence of members of cathepsin family. We cloned a cathepsin L-like gene and studied its tissue distribution. Immunohistochemical studies using anti-cathepsin L and anti-cystatin B antibodies and ultrastructural results demonstrated the presence of three distinct coelomic cell populations: (1) the chloragocytes, which were initially defined as large coelomocytes, (2) the granular amoebocytes and (3) small coelomic cells. Among those cells, while chloragocytes contain cystatin B and cathepsin L, granular amoebocytes contain only cathepsin L and the third cell population contains neither cathepsin nor inhibitor. Finally, results evidenced that cathepsin L immunopositive granular amoebocytes are chemoattracted to the site of injury and phagocyte bacteria.

Neuropeptide-derived antimicrobial peptides from invertebrates for biomedical applications.

Since the beginning of the 20th century, important medicinal progress has led medical doctors to think that the end of devastating epidemics has arrived. In 1930, the discovery of sulfamides and penicillin opened a wide area of applications able to fight against bacterial infections. However, almost all antibiotics were baffled by the great ability to adaptation of bacteria (1) and the emergence of new bacterial agents, discovered with up-dated technologies. The living world is perpetually in co-evolution and since more than 3 billion years, bacteria have developed resistance mechanisms to overcome external aggressions. Thus, in the middle of the 80th century, multi-resistant bacteria appeared and disseminated out from hospitals. In this context, researches have been developed in order to find new antimicrobial substances to destroy such new types of bacteria. Thus, several groups have turned their focus on invertebrates, which co-evoluad with human and have appeared on the planet since a long time. Evidence of new families of antimicrobial substances isolated from invertebrates different to the classical cationic peptide family i.e. dipeptides and anionic peptides been given. Moreover, these molecules are also present in human and may serve in the innate immune response as an important survival strategy.

Transcriptomic analysis in the leech Theromyzon tessulatum: involvement of cystatin B in innate immunity.

At the present time, there is little information on mechanisms of innate immunity in invertebrate groups other than insects, especially annelids. In the present study, we have performed a transcriptomic study of the immune response in the leech Theromyzon tessulatum after bacterial challenge, by a combination of differential display RT (reverse transcriptase)-PCR and cDNA microarrays. The results show relevant modulations concerning several known and unknown genes. Indeed, threonine deaminase, malate dehydrogenase, cystatin B, polyadenylate-binding protein and alpha-tubulin-like genes are up-regulated after immunostimulation. We focused on cystatin B (stefin B), which is an inhibitor of cysteine proteinases involved in the vertebrate immune response. We have cloned the full-length cDNA and named the T. tessulatum gene as Tt-cysb. Main structural features of cystatins were identified in the derived amino acid sequence of Tt-cysb cDNA; namely, a glycine residue in the N-terminus and a consensus sequence of Gln-Xaa-Val-Xaa-Gly (QXVXG) corresponding to the catalytic site. Moreover, Tt-cysb is the first cystatin B gene characterized in invertebrates. We have determined by in situ hybridization and immunocytochemistry that Tt-cysb is only expressed in large coelomic cells. In addition, this analysis confirmed that Tt-cysb is up-regulated after bacterial challenge, and that increased expression occurs only in coelomic cells. These data demonstrate that the innate immune response in the leech involves a cysteine proteinase inhibitor that is not found in ecdysozoan models, such as Drosophila melanogaster or Caenorhabditis elegans, and so underlines the great need for information about innate immunity mechanisms in different invertebrate groups.

[Effect of the antibodies to gangliosides on the Retzius neuron electrical activity and the functional activity of sodium channels for incoming current in leech]. / Analiz vliianiia antitel k gangliozidam na élektricheskuiu aktivnost' neirona Rettsiusa piiavki i na funktsional'nuiu aktivnost' natrievykh kanalov vkhodiashchego toka.

In the present study, the effect of antibodies to gangliosides on the Retzius neurons of the leech was investigated to study the spike activity and the functional activity of the Na-channels which generate the spike. A forty-minute incubation of the Retzius neurons in a 20% solution of antiganglioside serum in a Ringer solution provoked appearance of a double spike (a spike with two parts) connected with a decrease of the speed of the activation of the tetrodotoxin (TTX)-sensitive Nachannels. The high frequency synaptic activation of the neuron (10 Hz during 10 minutes) under the plasticity exchange of the gate system of the TTX-sensitive Na-channels. As a result of this, there was a disturbance of the habituation of the Retzius neuron to the high-frequency stimulation.

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.

[Contact dermatitis with Hirudo medicinalis]. / Eczéma de contact à Hirudo medicinalis.

BACKGROUND: Hirudine is the first natural anti-coagulant ever described. It is used for its anti-coagulant properties in plastic surgery or for the treatment of post-phlebitic diseases. Natural hirudine is extracted from the saliva of the Hirudo medicinalis leech, but it can also be found in crushed leech and included in a cream (Hirucrème). Side effects to hirudine are considered to be rare. CASE REPORT: We report a contact eczema caused by an extract from the medicinal Hirudo medicinalis leech. This was confirmed by the patch tests. However, we noticed a negativity of these tests with two analogs of the recombinant hirudine. DISCUSSION: Several cases of contact dermatitis with Hirucreme have been described. The analogs of recombinant hirudine, which share similar biological activity, have a very close molecular structure. They are indicated via the systemic route for thrombopenia related to heparin for the prevention of severe thromboses. The negative patch tests does not allow definite conclusion, but they prove that these molecules do not always lead to cross-allergies.

Elements of angiotensin system are involved in leeches and mollusks immune response modulation.

We present immunocytochemical, biochemical and cellular evidences for the presence of a renin-angiotensin system (RAS) in coelomocytes of invertebrates (leech, Theromyzon tessulatum and mollusk Mytilus edulis). Leech coelomocytes are immunoreactive to polyclonal antisera raised against the T. tessulatum angiotensin-converting enzyme (ACE) and leech brain angiotensin II (AII) and a commercial anti-AT1 receptor. Biochemically, renin, ACE- and AT1-like receptor were identified in the leech immune cells. We further demonstrate that leech AII (10(-6) M) alone does not initiate nitric oxide (NO) release in invertebrate immunocytes but does only after pre-exposing the cells to IL-1 (15.9+/-2.6 nM; P<0.005 vs. 1.1 nM when AII is added alone). Similar results were obtained with human leukocytes (14.5+/-2.7 nM; P<0.005 IL-1+AII vs. 0.9 nM when AII is added alone). Then, an immunocytochemical study performed at the structural and ultrastructural levels confirmed the presence in same immune cells all the molecules of the renin-angiotensin system (RAS) in leeches as epitopes to IL-1-like protein and IL-1-like receptor. This is the first report in invertebrates and of a co-action between cytokines like substances and neuropeptides in an immune process and the involvement of the RAS in modulation of the immune response.

Cellular localization of a renin-like enzyme in leeches.

OBJECTIVES: The purpose of this study was to localize in leeches the renin-like enzyme previously characterized as well as the leech angiotensin-converting like enzyme (ACE). METHODS: Immunocytochemical as well as whole mount experiments were performed with an antibody raised against a fragment of the leech renin-like enzyme (VLWAAEKTQLDTGSS) and with anti-leech ACE. RESULTS: Anti-leech renin stains the vascular pole of the glomerulus and the afferent arteriole of the rat kidney. Immunostaining of leech sections revealed labeling in neurons and glial cells of the central nervous system (CNS), immunocytes and the nephridial canal, canaliculi and the periphery of the ciliated funnel, as well as the epithelium lining nephridia. Co-localization between antibodies raised against this fragment and a fragment of leech angiotensin-converting enzyme was demonstrated in neurons and glial cells of the leech CNS, as in vertebrates MAIN FINDING: Leech renin is localized in leeches like in vertebrate in the excretory system and in the nervous system. CONCLUSIONS: Our findings suggest the presence of a renin-angiotensin system involved in osmoregulation in leeches.

Molecular crosstalk in host-parasite relationships: schistosome- and leech-host interactions.

The host-parasite relationship is based on subtle interplay between parasite survival strategies and host defense mechanisms. In this context, parasites often use the same or similar immune signaling molecules and/or molecular mimicry to escape host immunosurveillance. Both processes represent an adaptive strategy to ensure host immunocompatibility. This bidirectional communication between parasites and their hosts includes the renin-angiotensin, opioid and opiate systems. Here, Michel Salzet, André Capron and George Stefano review recent work on the interaction of common signaling mechanisms in schistosomes, leeches and their host.

Evidence of opiates and opioid neuropeptides and their immune effects in parasitic invertebrates representing three different phyla: Schistosoma mansoni, Theromyzon tessulatum, Trichinella spiralis.

Studies done in our laboratories have demonstrated that the parasitic trematode, Schistosoma mansoni is capable of producing several proopiomelanocortin (POMC) peptides including beta-endorphin, adrenocorticotropin (ACTH), melanocyte stimulating hormone (alphaMSH) and enkephalin as well as morphine. Some of these opioids have been demonstrated to be immunosuppressive and may play an important part in immune evasion by these parasites. The parasitic nematode Trichinella spiralis also produces immune suppressive substances in vitro as well as causes immune suppression in its encysted stage in vivo. We recently have demonstrated the presence of morphine in both infected mice and in the nematode by HPLC and RIA. In a recent study of the leech Theromyzon tessulatum, we demonstrated the presence of proopiomelanocortin (POMC) and its derived peptides, ACTH and alphaMSH, in the immune tissues. The peptide was cloned and extensively purified by HPGPC and reversed-phase HPLC, and then sequenced. The 25.4 kDa protein was purified by gel permeation chromatography, anti-ACTH-affinity column separation followed by reversed-phase HPLC. Its amino acid determination was performed by Edman degradation, enzymatic treatments and electrospray mass spectrometry. The structure of the leech POMC-like precursor and its derived peptides demonstrates considerable amino acid sequence similarity with mammalian POMC. Taken together, these studies demonstrate that opiates and opioid neuropetides are present in invertebrates and their immunoregulatory actions have been conserved during evolution. The role of opiates and opioid peptides in immune and behavior modification of hosts is also discussed.