Hirudo Medicinalis Research Articles

Draft genome of the European medicinal leech Hirudo medicinalis (Annelida, Clitellata, Hirudiniformes) with emphasis on anticoagulants

The European medicinal leech has been used for medicinal purposes for millennia, and continues to be used today in modern hospital settings. Its utility is granted by the extremely potent anticoagulation factors that the leech secretes into the incision wound during feeding and, although a handful of studies have targeted certain anticoagulants, the full range of anticoagulation factors expressed by this species remains unknown. Here, we present the first draft genome of the European medicinal leech, Hirudo medicinalis, and estimate that we have sequenced between 79–94% of the full genome. Leveraging these data, we searched for anticoagulation factors across the genome of H. medicinalis. Following orthology determination through a series of BLAST searches, as well as phylogenetic analyses, we estimate that fully 15 different known anticoagulation factors are utilized by the species, and that 17 other proteins that have been linked to antihemostasis are also present in the genome. We underscore the utility of the draft genome for comparative studies of leeches and discuss our results in an evolutionary context.

Antinocyceptive effect of oxygen therapy and hirudotherapy in rehabilitation of patients with genital endometriosis

The results of treatment of patients (n = 100) suff ering from genital endometriosis are presented. The 1-st (control, n = 30) group of patients, against the background of standard drug therapy, received the Elevit® complex; The 2nd (main, n = 70) group of patients additionally received oxygen therapy procedures, in the form of oxygen inhalations from the Newlife apparatus and hirudotherapy using medical leeches. The course of treatment consisted of 10–12 procedures. The severity of pelvic pain in these patients was assessed on the YOUR scale in the period: before and after treatment, as well as after 1, 3, 6, 9 months. As a result, in patients in the 2-nd (main) group receiving complex treatment, the pain syndrome signifi cantly decreased, and it was also possible to predict its further decrease by two periods ahead, according to statistical regression analysis, compared with the treatment results in patients in 1-th (control) group.

Detailed ultrastructure of the Hirudo (Annelida: Hirudinea) salivary gland

Medical leeches have been widely used in medical applications and treatments for millennia. Studies on the salivary glands of blood-sucking leeches have focused on their bioactive secretions and mechanisms of action, with little attention to ultrastructure. In this study, we examined dissected embryonic and adult Hirudo verbana salivary glands by scanning electron microscopy (SEM). Gland cells of embryos were physically separated while adults displayed highly developed cell bunches in which each cell was connected to others by fine channels. Channels from each bunch combined to form a larger canal that opened to the jaw. Secreted material from these glands prevent blood from clotting and allow the adult to feed while sucking blood.

Hirudo verbana as an alternative model to dissect the relationship between innate immunity and regeneration

Given the key role of innate immunity in both defense against pathogens and tissue regeneration, innovative studies are becoming crucial to provide further information on how both processes are linked together and to clarify how immune cells perform the coordinated regulation of the aforementioned processes. The present review is mainly focused on two proteins that have been recently found to carry out critical functions in innate immune system regulation, i. e. the Allograft inflammatory factor-1 (AIF-1) and RNASET2, a protein belonging to the T2 ribonuclease family. Their crucial role in both the activation and modulation of the inflammatory response and in the remodeling of connective tissue during grafts and wound repair have been thoroughly investigated in the medicinal leech and will pave the way for novel therapeutic approaches to control immune and systemic responses to disease, injury, and bacterial infection, based on the functionalities of these biomolecules.

Effects of maternal age on reproductive performance of the southern medicinal leech, Hirudo verbana Carena, 1820

Medicinal leeches can be used to address important needs in medicine and veterinary medicine, as well as pharmaceutical and cosmetic industries. These leeches, however, are among the endangered species. The reproductive processes of the medicinal leeches are not well-known. The aim of the present study was to elucidate effects of maternal age on reproductive performance of medicinal leeches (Hirudo sp.). This study was conducted on Hirudo verbana which were 1, 2 and 3 years of age. The results from the study indicate the youngest maternal leeches have greater reproductive performance. Cocoon production was 4.30 ± 0.72, 3.00 ± 0.71 and 2.80 ± 0.65 cocoons/leech at 1-, 2- and 3-years of age, respectively. Offspring production was greater in younger leeches with there being 11.16 ± 0.63, 10.60 ± 0.96 and 7.14 ± 1.20 offspring produced per cocoon when the cocoon was produced by leeches that were 1, 2, and 3 years of age, respectively. With increasing age of leeches producing the cocoon, the number of cocoons from which there are no offspring produced and the rate of abnormalities of cocoons and morphological abnormalities of offspring increased. The mortality rate of offspring was 10% and 40% in offspring from cocoons produced by 1- and 3-year-old leeches, respectively, being greater when from cocoons produced by 3-year old leeches. Consistent with these findings, younger maternal leeches during their first reproductive phase after hatching from a cocoon have the greatest reproductive performance and should be utilized in leech aquaculture enterprises.

The hirudin-like factors HLF3 and HLF4\u2014hidden hirudins of European medicinal leeches

The hirudin-like factors 3 (HLF3) and 4 (HLF4) belong to a new class of leech-derived factors and are present in specimens of the three European medicinal leeches, Hirudo medicinalis, Hirudo verbana, and Hirudo orientalis, respectively. Here we describe the functional analysis of natural and synthetic variants of HLF3 and HLF4. Whereas the natural variants display only very low or no detectable anti-coagulatory activities, modifications within the N-termini in combination with an exchange of the central globular domain have the potency to greatly enhance the inhibitory effects of respective HLF3 and HLF4 variants on blood coagulation. Our results support previous observations on the crucial importance of all parts (both the N- and C-termini as well as the central globular domains) of hirudin and HLF molecules for thrombin inhibition.

Brief Electrical Stimulation Triggers an Effective Regeneration of Leech CNS.

The search for therapeutic strategies to promote neuronal regeneration following injuries toward functional recovery is of great importance. Brief low-frequency electrical stimulation (ES) has been reported as a useful method to improve neuronal regeneration in different animal models; however, the effect of ES on single neuron behavior has not been shown. Here, we study the effect of brief ES on neuronal regeneration of the CNS of adult medicinal leeches. Studying the regeneration of selected sets of identified neurons allow us to quantify the ES effect per cell type at the single-cell level. Chains of the CNS that were subjected to cut injury were observed for 3 d, and the spontaneous regeneration was compared with the electrically stimulated injured chains. We show that the ES improves the efficiency of regeneration of Retzius cells, as larger masses of the total branching tree traverse the injury site with better directed growth with no effect on the average branching tree length. No antero-posterior polarity was found along regeneration within the leech CNS. Moreover, the microglial cell distribution was examined revealing more microglial cells in proximity to the stimulation site compared with non-stimulated. Our results lay a foundation for future ES-based neuroregenerative therapies.

Draft genome sequences of Hirudo medicinalis and salivary transcriptome of three closely related medicinal leeches

BackgroundSalivary cell secretion (SCS) plays a critical role in blood feeding by medicinal leeches, making them of use for certain medical purposes even today.ResultsWe annotated the Hirudo medicinalis genome and performed RNA-seq on salivary cells isolated from three closely related leech species, H. medicinalis, Hirudo orientalis, and Hirudo verbana. Differential expression analysis verified by proteomics identified salivary cell-specific gene expression, many of which encode previously unknown salivary components. However, the genes encoding known anticoagulants have been found to be expressed not only in salivary cells. The function-related analysis of the unique salivary cell genes enabled an update of the concept of interactions between salivary proteins and components of haemostasis.ConclusionsHere we report a genome draft of Hirudo medicinalis and describe identification of novel salivary proteins and new homologs of genes encoding known anticoagulants in transcriptomes of three medicinal leech species. Our data provide new insights in genetics of blood-feeding lifestyle in leeches.

European Medicinal Leeches-New Roles in Modern Medicine.

Before the advent of modern medicine, natural resources were widely used by indigenous populations for the prevention and treatment of diseases. The associated knowledge, collectively described as folk medicine or traditional medicine, was largely based on trial-and-error testing of plant extracts (herbal remedies) and the use of invertebrates, particularly medicinal maggots of the blowfly Lucilia sericata and blood-sucking leeches. The widespread use of traditional medicine in the West declined as scientific advances allowed reproducible testing under controlled conditions and gave rise to the modern fields of biomedical research and pharmacology. However, many drugs are still derived from natural resources, and interest in traditional medicine has been renewed by the ability of researchers to investigate the medical potential of diverse species by high-throughput screening. Likewise, researchers are starting to look again at the benefits of maggot and leech therapy, based on the hypothesis that the use of such animals in traditional medicine is likely to reflect the presence of specific bioactive molecules that can be developed as drug leads. In this review, we consider the modern medical benefits of European medicinal leeches based on the systematic screening of their salivary proteins.

Rat peripheral blood leukocyte subset composition, hemogram, lymphoid organ and body morphometry after exposure to biologically active substances derived from Hirudo verbana saliva

Medical community has noted that the prevalence of allergic diseases by steadily tends to rise resulting from using synthetic drugs ecological catastrophe. In this regard, more and more attention is increasingly being paid to natural therapeutic approaches, one of which is based on applying hirudotherapy. There were used 40 pubescent outbred female rats and their 200 pups born during experiment. In particular, female rats underwent leeching in the subscapular region 2 weeks before and 2 weeks after mating by applying medical leeches weighing 400 ± 10 mg. Next, relevant progeny was examined at day 1, 15, 30, 45, and 60 after birth, whereas female rats– after weaning. Experiment was designed to assess diverse parameters in accordance with the age-related rat development: newborn pups – 1-5 days old, day 6-21 – suckling period, day 22-50 – onset of puberty, day 60 onward – puberty. After measuring body morphometric parameters, female rats were sacrificed by decapitation under ether narcosis on day 60, whereas rat pups – at various time points. followed by autopsy and collecting blood sample into a sterile centrifuge tube by using 2% Spofa crystalline heparin (8:1) to be further examined by standard assays: morphometry of rat body, spleen and thymus; measuring total leukocyte count; CBC with differential; RBC; hemoglobin level; color index. Statistical data processing was performed by calculating arithmetic mean, error of arithmetic mean, standard deviation by using SPSS version 21.0 and Microsoft Office Excel 2010 software. Significance of differences was estimated by using Student's t test, and set at p≤0.05. Our study demonstrated medical leech saliva components applied during hirudolotherapy exerted an immune-augmenting effect of on morphometric body, thymus and spleen parameters. Moreover, starting from day 1, total leukocyte count, RBC, and hemoglobin level were increased in the experimental group. Moreover, we convincingly demonstrated a stimulatory effect of medical leech saliva components on histogenetic reactions at various ontogenetic stages in both female rats and their progeny. In the former, such morphogenetic effect was maintained for more than 70 days after the last leeching: on week 2 after mating and 30 days after delivery. Changes in morphological and hematological evidence about enhanced morphogenetic function of immune system in response to saliva-derived biologically active substances released by medicinal leeches.

Cutaneous Pseudolymphoma As a Rare Adverse Effect of Medicinal Leech Therapy: A Case Report and Review of the Literature

Hirudotherapy (leech therapy) is one of the oldest practices in medical history, and nowadays it is used for several purposes in medicine. Salvage of flaps, wound healing, pain management, and treatment of varicose veins are among the common therapeutic applications of leeches. Complications associated with leech therapy include infections, bleeding, anemia, and allergic reaction. Cutaneous pseudolymphoma (benign proliferation of lymphoid cells in the skin) follows several underlying conditions. Although persistent arthropod bite reaction is one of the conditions associated with cutaneous pseudolymphoma, it has been rarely reported after medicinal leech therapy. Here we describe the case of a patient who presented with cutaneous pseudolymphoma after leech therapy as a rare cutaneous complication of hirudotherapy.

Characterization of a monoacylglycerol lipase in the medicinal leech, Hirudo verbana.

Endocannabinoids are a class of lipid neuromodulators found throughout the animal kingdom. Among the endocannabinoids, 2-arachydonoyl glycerol (2-AG) is the most prevalent endocannabinoid and monoacylglycerol lipase (MAGL) is a serine hydrolase primarily responsible for metabolizing 2-AG in mammals. In the medicinal leech, Hirudo verbana, 2-AG has been found to be an important and multi-functional modulator of synaptic transmission and behavior. However, very little is known about the molecular components of its synthesis and degradation. In this study we have identified cDNA in Hirudo that encodes a putative MAGL (HirMAGL). The encoded protein exhibits considerable sequence and structural conservation with mammalian forms of MAGL, especially in the catalytic triad that mediates 2-AG metabolism. Additionally, HirMAGL transcripts are detected in the Hirudo central nervous system. When expressed in HEK 293 cells HirMAGL segregates to the plasma membrane as expected. It also exhibits serine hydrolase activity that is blocked when a critical active site residue is mutated. HirMAGL also demonstrates the capacity to metabolize 2-AG and this capacity is also prevented when the active site is mutated. Finally, HirMAGL activity is inhibited by JZL184 and MJN110, specific inhibitors of mammalian MAGL. To our knowledge these findings represent the first characterization of an invertebrate form of MAGL and show that HirMAGL exhibits many of the same properties as mammalian MAGL's that are responsible for 2-AG metabolism.

Antimicrobial Role of RNASET2 Protein During Innate Immune Response in the Medicinal Leech Hirudo verbana.

The innate immune response represents a first-line defense against pathogen infection that has been widely conserved throughout evolution. Using the invertebrate Hirudo verbana (Annelida, Hirudinea) as an experimental model, we show here that the RNASET2 ribonuclease is directly involved in the immune response against Gram-positive bacteria. Injection of lipoteichoic acid (LTA), a key component of Gram-positive bacteria cell wall, into the leech body wall induced a massive migration of granulocytes and macrophages expressing TLR2 (the key receptor involved in the response to Gram-positive bacteria) toward the challenged/inoculated area. We hypothesized that the endogenous leech RNASET2 protein (HvRNASET2) might be involved in the antimicrobial response, as already described for other vertebrate ribonucleases, such as RNase3 and RNase7. In support of our hypothesis, HvRNASET2 was mainly localized in the granules of granulocytes, and its release in the extracellular matrix triggered the recruitment of macrophages toward the area stimulated with LTA. The activity of HvRNASET2 was also evaluated on Staphylococcus aureus living cells by means of light, transmission, and scanning electron microscopy analysis. HvRNASET2 injection triggered the formation of S. aureus clumps following a direct interaction with the bacterial cell wall, as demonstrated by immunogold assay. Taken together, our data support the notion that, during the early phase of leech immune response, granulocyte-released HvRNASET2 triggers bacterial clumps formation and, at the same time, actively recruits phagocytic macrophages in order to elicit a rapid and effective eradication of the infecting microorganisms from inoculated area.

Is the New England medicinal leech (Macrobdella sestertia) extinct?

Despite recent interest in invertebrate extinctions, most go undocumented due to chronic data deficiencies. Here, we investigate the absence of the New England medicinal leech (Macrobdella sestertia), a species not sighted in the last decade, but which has been rediscovered several times since it was described in 1886. We collate all known records (19 specimens from nine independent sightings) of this rare species, and use sighting-based extinction date estimators to evaluate the odds that populations of M. sestertia might persist. Using all available data and several different models, we find that while the species may be extinct, there is also not enough evidence to eliminate the hypothesis that the species remains extant. Given that the geographic range of M. sestertia is now known to extend much further south than previously thought, we recommend protections for M. sestertia should be expanded at state and potentially federal levels. We also recommend that systematists should continue to revisit Macrobdella records in natural history collections, which may turn up new evidence of its range, habitat preference, and possible persistence. We conclude by discussing implications of this work for the broader picture of leech conservation in Europe and North America.

Purinergic modulation of neuronal gap junction circuits in the CNS of the leech.

Gap junctions (GJs) are widely distributed in brains across the animal kingdom. To visualize the GJ- coupled networks of two major mechanosensory neurons in the ganglia of medicinal leeches, we injected these cells with the GJ-permeable tracer Neurobiotin. When diffusion time was limited to only 30min, tracer coupling was highly variable for both cells, suggesting a possible modulation of GJ permeability. In invertebrates the innexins (homologs of vertebrate pannexins) form the GJs. Because extracellular adenosine triphosphate (ATP) modulates pannexin and leech innexin hemichannel permeability and is released by leech glial cells following injury, we tested the effects of bath application of ATP after the injection of Neurobiotin and observed a significant increase in the number of neurons tracer coupled to the sensory neurons. This effect required the elevation of intracellular Ca2+ and could be produced by bath application of caffeine. Conversely, scavenging endogenous extracellular ATP with the ATPase apyrase decreased the number of coupled cells. ATP also increased electrical conductance and tracer permeability between the bilateral Retzius neurons. This modulatory effect of ATP on GJ coupling was blocked by siRNA knockdown of a P1-like adenosine receptor. Finally, exposure of leech ganglia to extracellular ATP induced a characteristic low frequency (<0.3Hz) rhythmic bursting activity that was roughly synchronous among multiple neurons, a behavior that was significantly attenuated by the GJ blocker octanol. These findings highlight the mediation by ATP of a robust physiological mechanism for modifying neuronal circuits by rapidly recruiting neurons into active networks and entraining synchronized bursting activity.

Effectiveness of medicinal leech therapy on infected postauricular ulcer: a case report

&lt;p class="abstract"&gt;Trauma induced auricular partial defect very common. Due to the aesthetic value, proper reconstruction has to be done preserving the symmetry of bilateral auricles. The reconstruction of auricle is difficult due to the intricate anatomy of the auricle, limited and inconsistent vascularization and the high ratio of cartilage to skin. Historically, Susruta was the first author to describe about auricular repair. In Ayurveda, medicinal leech therapy is praised for it’s usefulness for the healing of ulcers. In this case report, a male of 30 years with a trauma induced postauricular infected ulcer was successfully treated with the application of medicinal leeches for multiple sittings.&lt;/p&gt;&lt;p class="abstract"&gt; &lt;/p&gt;

Recombinant HvRNASET2 protein induces marked connective tissue remodelling in the invertebrate model Hirudo verbana.

The RNASET2 ribonuclease, belonging to the highly conserved RH/T2/s RNase gene family, has been recently shown to modulate inflammatory processes in both vertebrates and invertebrates. Indeed, the RNASET2 protein acts as a chemoattractor for macrophages in both in vitro and in vivo experimental settings and its expression significantly increases following bacterial infections. Moreover, we recently observed that injection of human recombinant RNASET2 protein in the body wall of the medicinal leech (a consolidated invertebrate model for both immune response and tissue regeneration) not only induced immune cell recruitment but also apparently triggered massive connective tissue remodelling as well. Based on these data, we evaluate here a possible role of leech recombinant RNASET2 protein (rHvRNASET2) in connective tissue remodelling by characterizing the cell types involved in this process through histochemical, morphological and immunofluorescent assays. Moreover, a time-course expression analysis of newly synthesized pro-collagen1α1 (COL1α1) and basic FGF receptor (bFGFR, a known fibroblast marker) following rHvRNASET2 injection in the leech body wall further supported the occurrence of rHvRNASET2-mediated matrix remodelling. Human MRC-5 fibroblast cells were also investigated in order to evaluate their pattern of collagen neosynthesis driven by rHvRNASET2 injection.Taken together, the data reported in this work provide compelling evidence in support of a pleiotropic role for RNASET2 in orchestrating an evolutionarily conserved crosstalk between inflammatory response and regenerative process, based on macrophage recruitment and fibroblast activation, coupled to a massive extracellular reorganization.

Neurotrophic (Neural Stimulating) and Neuromediator Effects of Hirudo medicinalis. Pathogenetic Mechanism of Treatment of Diseases of the Nervous System of the Human

Neurotrophic (Neural Stimulating) and Neuromediator Effects of Hirudo medicinalis. Pathogenetic Mechanism of Treatment of Diseases of the Nervous System of the Human

Insilico Studies on Antimicrobial Peptide (AMP) in Leeches

The earthworm immune system is robust and comprises of the coelom cytolytic factor (CCF), lysenin and antimicrobial peptides (AMPs, Ghosh in Environ Sci Pollut Res 25: 6196, 2018) have been reported from organisms belonging to Phylum Annelida including earthworms (Ghosh in Int J Pept Res Ther, http://doi.org/10.1007/s10989-019-09970-9, 2019) which reveal structural diversity. Leeches have been known of their medical importance and known to produce AMPs (Hung et al. in J Proteomics 103:216–226, 2014; Salzet Curr Med Chem 12(6):3055–3061, 2005), but information on their detailed structures were not observed in published literature. In this study we have conducted insilico studies to understand the (i) Physicochemical properties (ii) phylogenetic relation from peptide sequence (iii) molecular modelling studies by insilico approaches which reveals overall diversity in their structure despite their similar functional role. This report highlights the properties of all AMPs in leeches with known sequences and the importance of application of insilico tools, performing a mutation analysis affecting protein stability, to highlight the significance of K(8) amino acid of the AMP 536_2 from Hirudo medicinalis in maintaining protein stability and functional importance.

Effect of Unloading Condition on the Healing Process and Effectiveness of Platelet Rich Plasma as a Countermeasure: Study on In Vivo and In Vitro Wound Healing Models.

Wound healing is a very complex process that allows organisms to survive injuries. It is strictly regulated by a number of biochemical and physical factors, mechanical forces included. Studying wound healing in space is interesting for two main reasons: (i) defining tools, procedures, and protocols to manage serious wounds and burns eventually occurring in future long-lasting space exploration missions, without the possibility of timely medical evacuation to Earth; (ii) understanding the role of gravity and mechanical factors in the healing process and scarring, thus contributing to unravelling the mechanisms underlying the switching between perfect regeneration and imperfect repair with scarring. In the study presented here, a new in vivo sutured wound healing model in the leech (Hirudo medicinalis) has been used to evaluate the effect of unloading conditions on the healing process and the effectiveness of platelet rich plasma (PRP) as a countermeasure. The results reveal that microgravity caused a healing delay and structural alterations in the repair tissue, which were prevented by PRP treatment. Moreover, investigating the effects of microgravity and PRP on an in vitro wound healing model, it was found that PRP is able to counteract the microgravity-induced impairment in fibroblast migration to the wound site. This could be one of the mechanisms underlying the effectiveness of PRP in preventing healing impairment in unloading conditions.