Hirudins and hirudin-like factors in Hirudinidae: implications for function and phylogenetic relationships.

Haematophagous leeches express a broad variety of bioactive factors that are released from the salivary gland cells into the wound of a host during feeding. Among these, hirudin is probably the best studied factor and, moreover, the only one that has successfully made the transition from nature to clinical use. Many components of the leech saliva still remain either poorly characterized or yet completely unknown. Only recently, a new class of leech-derived factors has been discovered in Hirudo medicinalis, the hirudin-like factors (HLFs). HLFs comprise typical structural features of hirudin but lack others. We were able to verify the expression of HLFs not only in two additional species of the genus Hirudo, but also in Hirudinaria manillensis. Various phylogenetic analyses based on gene and protein sequences support a sister group relationship between hirudins and HLFs. Although potential molecular targets of HLFs remain unknown, the presence of multiple isoforms in individual leeches of different genera points to key functions in the regulation of several processes associated with the blood-sucking life style of leeches.

More than just one: multiplicity of Hirudins and Hirudin-like Factors in the Medicinal Leech, Hirudo medicinalis.

Blood-sucking leeches like the medicinal leech, Hirudo medicinalis, have been used for medical purposes since ancient times. During feeding, medicinal leeches transfer a broad range of bioactive substances into the host's wound to prevent premature hemostasis and blood coagulation. Hirudin is probably the best known of these substances. Despite its long history of investigation, recombinant production and clinical use, there still exist conflicting data regarding the primary structure of hirudin. Entirely unclear is the potential biological significance of three different subtypes and many isoforms of hirudins that have been characterized so far. Furthermore, there is only incomplete information on their cDNA sequences and no information at all on gene structures and DNA sequences are available in the databases. Our efforts to fill these gaps revealed the presence of multiple hirudin-encoding genes in the genome of Hirudo medicinalis. We have strong evidence for the expression of all three subtypes of hirudin within individual leeches and for the expression of additional hirudins or hirudin-like factors that may have different biological functions and may be promising candidates for new drugs.

Be ready at any time: postprandial synthesis of salivary proteins in salivary gland cells of the haematophagous leech Hirudo verbana.

Sanguivorous leeches are ectoparasites having access to body fluids of potential hosts only infrequently. During feeding, salivary proteins are released from unicellular salivary glands into the wound. These substances, among them anti-coagulants, anti-inflammatory or anti-microbial agents, allow these animals proper feeding and long-term storage of host blood in their crops for several months. Using histological, protein biochemical and molecular techniques, we investigated whether synthesis of salivary proteins and refilling of salivary gland cells occur immediately after feeding or later when stored nutrients in the crop are getting scarce. The results of the histological analyses showed that gland cell area was significantly smaller right after feeding when compared with those in unfed animals. This parameter recovered quickly and reached the control level at 1 week after feeding. 2D gel electrophoresis and analysis of the abundance of individual proteins in extracts of leech tissues revealed that a subset of proteins that had been present in extracts of unfed animals virtually disappeared during feeding, but re-appeared within 1 week of feeding (most probably secretory proteins) while another subset did not change during the experimental period (most probably housekeeping proteins). Semi-quantitative PCR analysis of hirudin cDNA prepared from leech RNA samples revealed that the amount of hirudin transcripts increased immediately after feeding, peaked at 5 days after feeding and declined to control values thereafter. Our results indicate that bloodsucking leeches synthesize salivary proteins and refill their salivary gland cell reservoirs within a week of a blood meal to be prepared for another feeding opportunity.

Small bite, large impact-saliva and salivary molecules in the medicinal leech, Hirudo medicinalis.

Blood-sucking leeches have been used for medical purposes in humans for hundreds of years. Accordingly, one of the most prominent species has been named Hirudo medicinalis by Carl Linne in 1758. Feeding on vertebrate blood poses some serious problems to blood-sucking ectoparasites, as they have to penetrate the body surface of the host and to suppress the normal reactions of the host to such injuries (swelling, pain, inflammation) to remain undetected during the feeding period. Furthermore, the parasites have to take measures to inhibit the normal reactions in host tissues to blood vessel damage, namely hemostasis and blood coagulation (platelet aggregation and activation, activation of thrombin and formation of fibrin clots). During evolution, leeches have acquired the ability to control these processes in their hosts by transferring various bioactive substances to the host. These substances are supposedly produced in unicellular salivary gland cells and injected into the wound at the feeding site through tiny salivary ductule openings in the jaws that the leech uses to slice open the host body surface and to cut blood vessels in the depth of the wound. This review summarizes current knowledge about the salivary gland cells and the biological effects of individual saliva components as well as hints to the potential usefulness of some of these compounds for medical purposes.

Morphological Analysis Reveals a Compartmentalized Duct in the Venom Apparatus of the Wasp Spider (Argiope bruennichi).

Spiders are one of the most successful groups of venomous animals, but surprisingly few species have been examined in sufficient detail to determine the structure of their venom systems. To learn more about the venom system of the family Araneidae (orb-weavers), we selected the wasp spider (Argiope bruennichi) and examined the general structure and morphology of the venom apparatus by light microscopy. This revealed morphological features broadly similar to those reported in the small number of other spiders subject to similar investigations. However, detailed evaluation of the venom duct revealed the presence of four structurally distinct compartments. We propose that these subunits facilitate the expression and secretion of venom components, as previously reported for similar substructures in pit vipers and cone snails.

Cardiovascular adverse effects of lopinavir/ritonavir and hydroxychloroquine in COVID-19 patients: Cases from a single pharmacovigilance centre.

In this article we summarize the cardiovascular adverse events that were observed in three patients during their treatment for COVID-19 and discuss their association with lopinavir/ ritonavir (LPV/r) and hydroxychloroquine (HCQ). The cases were reported to our regional pharmacovigilance centre in April 2020. All three patients were above 75 years in age, male and multimorbid, and had been hospitalized for treatment of COVID-19. As part of their treatment, all of them received a very strictly monitored off-label therapy with LPV/r and HCQ, for which they had given their prior, written, informed consent. In one patient, erythromycin was also administered. All three patients developed a significant QTc time prolongation during or shortly after therapy with the above drugs. On account of this, the treatment had to be discontinued early in each case and QTc time recovered in all three patients.

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.

An Economic Dilemma Between Molecular Weapon Systems May Explain an Arachno-atypical Venom in Wasp Spiders (Argiope bruennichi).

Spiders use venom to subdue their prey, but little is known about the diversity of venoms in different spider families. Given the limited data available for orb-weaver spiders (Araneidae), we selected the wasp spider Argiope bruennichi for detailed analysis. Our strategy combined a transcriptomics pipeline based on multiple assemblies with a dual proteomics workflow involving parallel mass spectrometry techniques and electrophoretic profiling. We found that the remarkably simple venom of A. bruennichi has an atypical composition compared to other spider venoms, prominently featuring members of the cysteine-rich secretory protein, antigen 5 and pathogenesis-related protein 1 (CAP) superfamily and other, mostly high-molecular-weight proteins. We also detected a subset of potentially novel toxins similar to neuropeptides. We discuss the potential function of these proteins in the context of the unique hunting behavior of wasp spiders, which rely mostly on silk to trap their prey. We propose that the simplicity of the venom evolved to solve an economic dilemma between two competing yet metabolically expensive weapon systems. This study emphasizes the importance of cutting-edge methods to encompass the lineages of smaller venomous species that have yet to be characterized in detail, allowing us to understand the biology of their venom systems and to mine this prolific resource for translational research.

Phylogeny-Guided Selection of Priority Groups for Venom Bioprospecting: Harvesting Toxin Sequences in Tarantulas as a Case Study.

Animal venoms are promising sources of novel drug leads, but their translational potential is hampered by the low success rate of earlier biodiscovery programs, in part reflecting the narrow selection of targets for investigation. To increase the number of lead candidates, here we discuss a phylogeny-guided approach for the rational selection of venomous taxa, using tarantulas (family Theraphosidae) as a case study. We found that previous biodiscovery programs have prioritized the three subfamilies Ornithoctoninae, Selenocosmiinae, and Theraphosinae, which provide almost all of the toxin sequences currently available in public databases. The remaining subfamilies are poorly represented, if at all. These overlooked subfamilies include several that form entire clades of the theraphosid life tree, such as the subfamilies Eumenophorinae, Harpactirinae, and Stromatopelminae, indicating that biodiversity space has not been covered effectively for venom biodiscovery in Theraphosidae. Focusing on these underrepresented taxa will increase the likelihood that promising candidates with novel structures and mechanisms of action can be identified in future bioprospecting programs.

Hirudin and Decorsins of the North American Medicinal Leech Macrobdella decora: Gene Structure Reveals Homology to Hirudins and Hirudin-like Factors of Eurasian Medicinal Leeches.

Blood-sucking leeches, some of which are referred to as medicinal leeches, have caught attention not only because of their medical purposes, but also as study organisms to conduct research within fields as diverse as neurobiology, osmoregulation, ecology, and phylogeny. Of particular interest is the question whether hemophagy in leeches is of single origin or evolved independently several times. A key component in the saliva of hematophagous leeches is hirudin, a strong natural inhibitor of thrombin and hence the blood coagulation cascade. Multiple isoforms of hirudin have been described within and among several leech species and genera, often based on sequence data only. The identification of hirudin-like factors (HLFs) illustrated the necessity to underpin such predictions by functional tests. We overexpressed and purified the hirudin of the North American medicinal leech, Macrobdella decora, and proved its thrombin-inhibiting activity. In addition, analysis of the gene structure of both hirudin and some of the decorsins of M. decora clearly indicated conserved exon and intron positions when compared to genes of hirudins and HLFs of Eurasian medicinal leeches. Our data provide evidence for the incorporation of decorsins into the hirudin superfamily and support the concept of a single origin of blood feeding in jawed leeches.

May salivary gland secretory proteins from hematophagous leeches (Hirudo verbana) reach pharmacologically relevant concentrations in the vertebrate host?

Saliva of hematophagous leeches (Hirudo sp.) contains bioactive proteins which allow the leech proper feeding and storage of ingested blood, but may also exert effects in the host. Leech therapy is used to treat many different ailments in humans, although only a small fraction of salivary proteins are characterized yet. Moreover, we do not know whether complete transfer of salivary proteins stored in the unicellular salivary glands in a leech to the host during feeding may generate concentrations that are sufficiently high to affect physiological processes in the host. Our 3D reconstruction of a portion of internal leech tissue from histological sections revealed that one leech contains approx. 37,000 salivary gland cells. Using tissue slices from pig liver and mouse skeletal muscle for reference, we obtained data for protein densities in leech salivary gland cells. As individual salivary cells are voluminous (67,000 µm(3)) and the stored proteins are densely packed (approx. 500 µg/mm(3)), we extrapolated that a single leech may contain up to 1.2 mg of salivary proteins. Analyzing protein extracts of unfed or fed leeches by 2D electrophoresis, we calculated the relative molar amounts of individual salivary proteins in the mass range of 17-60 kDa which may be released from a single leech during feeding. Distribution of these salivary proteins in the host (assumed plasma volume of 5 l) may result in concentrations of individual compounds between 3 and 236 pmol/l. Such concentrations seem sufficiently high to exert biochemical interactions with target molecules in the host.