Transcriptomic profiling and the first spatial expression analysis of candidate genes in the salivary gland of the East Asian medicinal leech, Hirudo nipponia.

Hirudo nipponia, a blood-sucking leech native to East Asia, possesses a rich repertoire of active ingredients in its saliva, showcasing significant medical potential due to its anticoagulant, anti-inflammatory, and antibacterial effects against human diseases. Despite previous studies on the transcriptomic and proteomic characteristics of leech saliva, which have identified medicinal compounds, our knowledge of tissue-specific transcriptomes and their spatial expression patterns remains incomplete. In this study, we conducted an extensive transcriptomic profiling of the salivary gland tissue in H. nipponia based on de novo assemblies of tissue-specific transcriptomes from the salivary gland, teeth, and general head region. Through gene ontology (GO) analysis and hierarchical clustering, we discovered a novel set of anti-coagulant factors-i.e., Hni-Antistasin, Hni-Ghilanten, Hni-Bdellin, Hni-Hirudin-as well as a previously unrecognized immune-related gene, Hni-GLIPR1 and uncharacterized salivary gland specific transcripts. By employing in situ hybridization, we provided the first visualization of gene expression sites within the salivary gland of H. nipponia. Our findings expand on our understanding of transcripts specifically expressed in the salivary gland of blood-sucking leeches, offering valuable resources for the exploration of previously unidentified substances with medicinal applications.

Revisiting the Asian Buffalo Leech (Hirudinaria manillensis) Genome: Focus on Antithrombotic Genes and Their Corresponding Proteins.

Leeches are well-known annelids due to their obligate blood-feeding habits. Some leech species secrete various biologically active substances which have important medical and pharmaceutical value in antithrombotic treatments. In this study, we provided a high-quality genome of the Asian buffalo leech (Hirudinaria manillensis), based on which we performed a systematic identification of potential antithrombotic genes and their corresponding proteins. Combining automatic and manual prediction, we identified 21 antithrombotic gene families including fourteen coagulation inhibitors, three platelet aggregation inhibitors, three fibrinolysis enhancers, and one tissue penetration enhancer. A total of 72 antithrombotic genes, including two pseudogenes, were identified, including most of their corresponding proteins forming three or more disulfide bonds. Three protein families (LDTI, antistasin, and granulin) had internal tandem repeats containing 6, 10, and 12 conserved cysteines, respectively. We also measured the anticoagulant activities of the five identified hirudins (hirudin_Hman1 ~ hirudin_Hman5). The results showed that three (hirudin_Hman1, hirudin_Hman2, and hirudin_Hman5), but not the remaining two, exhibited anticoagulant activities. Our study provides the most comprehensive collection of antithrombotic biomacromolecules from a leech to date. These results will greatly facilitate the research and application of leech derivatives for medical and pharmaceutical purposes in the treatment of thrombotic diseases.

Mast Cells and Basophils in the Defense against Ectoparasites: Efficient Degradation of Parasite Anticoagulants by the Connective Tissue Mast Cell Chymases.

Ticks, lice, flees, mosquitos, leeches and vampire bats need to prevent the host's blood coagulation during their feeding process. This is primarily achieved by injecting potent anticoagulant proteins. Basophils frequently accumulate at the site of tick feeding. However, this occurs only after the second encounter with the parasite involving an adaptive immune response and IgE. To study the potential role of basophils and mast cells in the defense against ticks and other ectoparasites, we produced anticoagulant proteins from three blood-feeding animals; tick, mosquito, and leech. We tested these anticoagulant proteins for their sensitivity to inactivation by a panel of hematopoietic serine proteases. The majority of the connective tissue mast cell proteases tested, originating from humans, dogs, rats, hamsters, and opossums, efficiently cleaved these anticoagulant proteins. Interestingly, the mucosal mast cell proteases that contain closely similar cleavage specificity, had little effect on these anticoagulant proteins. Ticks have been shown to produce serpins, serine protease inhibitors, upon a blood meal that efficiently inhibit the human mast cell chymase and cathepsin G, indicating that ticks have developed a strategy to inactivate these proteases. We show here that one of these tick serpins (IRS-2) shows broad activity against the majority of the mast cell chymotryptic enzymes and the neutrophil proteases from human to opossum. However, it had no effect on the mast cell tryptases or the basophil specific protease mMCP-8. The production of anticoagulants, proteases and anti-proteases by the parasite and the host presents a fascinating example of an arms race between the blood-feeding animals and the mammalian immune system with an apparent and potent role of the connective tissue mast cell chymases in the host defense.

NMR-Based Structural Characterization of a Two-Disulfide-Bonded Analogue of the FXIIIa Inhibitor Tridegin: New Insights into Structure-Activity Relationships.

The saliva of blood-sucking leeches contains a plethora of anticoagulant substances. One of these compounds derived from Haementeria ghilianii, the 66mer three-disulfide-bonded peptide tridegin, specifically inhibits the blood coagulation factor FXIIIa. Tridegin represents a potential tool for antithrombotic and thrombolytic therapy. We recently synthesized two-disulfide-bonded tridegin variants, which retained their inhibitory potential. For further lead optimization, however, structure information is required. We thus analyzed the structure of a two-disulfide-bonded tridegin isomer by solution 2D NMR spectroscopy in a combinatory approach with subsequent MD simulations. The isomer was studied using two fragments, i.e., the disulfide-bonded N-terminal (Lys1-Cys37) and the flexible C-terminal part (Arg38-Glu66), which allowed for a simplified, label-free NMR-structure elucidation of the 66mer peptide. The structural information was subsequently used in molecular modeling and docking studies to provide insights into the structure-activity relationships. The present study will prospectively support the development of anticoagulant-therapy-relevant compounds targeting FXIIIa.

Pharmacological activity and NMR solution structure of the leech peptide HSTX-I.

The role of voltage-gated sodium (NaV) channels in pain perception is indisputable. Of particular interest as targets for the development of pain therapeutics are the tetrodotoxin-resistant isoforms NaV1.8 and NaV1.9, based on animal as well as human genetic studies linking these ion channel subtypes to the pathogenesis of pain. However, only a limited number of inhibitors selectively targeting these channels have been reported. HSTX-I is a peptide toxin identified from saliva of the leech Haemadipsa sylvestris. The native 23-residue peptide, stabilised by two disulfide bonds, has been reported to inhibit rat NaV1.8 and mouse NaV1.9 with low micromolar activity, and may therefore represent a scaffold for development of novel modulators with activity at human tetrodotoxin-resistant NaV isoforms. We synthetically produced this hydrophobic peptide in high yield using a one-pot oxidation and single step purification and determined the three-dimensional solution structure of HSTX-I using NMR solution spectroscopy. However, in our hands, the synthetic HSTX-I displayed only very modest activity at human NaV1.8 and NaV1.9, and lacked analgesic efficacy in a murine model of inflammatory pain.

Changes in the Frequency of Rhythmic Excitation of Retzius Cells during Thermal Stimulation of Leech Skin.

Thermal stimulation of various parts of the skin in Hirudo medicinalis increases the frequency of spontaneous rhythmic excitation of Retzius neurons in leech ganglia. It was shown that the frequency of spontaneous rhythmic excitation of Retzius cells in the segmental ganglion increases only in response to thermal stimulation and returns to initial values upon cooling. This effect was also detected in neurons that are not directly connected by nerve fibers with the particular skin area. Changes in the frequency of spontaneous rhythmic excitation of Retzius cells in the segmental ganglion were observed during thermal stimulation of not only leech body, but also of the head and caudal suckers. These changes in spontaneous rhythmic excitation of Retzius cells in the segmental ganglion during thermal stimulation were observed in Hirudo medicinalis, but not in Macrobdella decora.

Temporal and spatial expression of the Fox gene family in the Leech Helobdella austinensis.

The Forkhead box (Fox) gene family is an evolutionarily ancient gene family named after the Drosophila melanogaster forkhead gene (fkh). Fox genes are highly conserved transcription factors critical for embryogenesis and carcinogenesis. In the current study, we report a whole-genome survey of Fox genes and their expression patterns in the leech Helobdella austienesis. Phylogenetic analysis suggests that some Fox genes of leeches are correlated with other Lophotrochozoa and vertebrate Fox genes. Here we have performed semiquantitative reverse transcription polymerase chain reaction and whole-mount in situ hybridization of Fox genes throughout the embryonic development of H. austinensis. We found that each one of the leech Fox genes (FoxA1, FoxA3, FoxC, FoxL2, FoxO1, and FoxO2) is expressed in a specific set of cells or tissue type. From Stages 9-11, Hau-FoxA1 was expressed in the foregut of the anterior region, and Hau-FoxL2 was expressed in mesodermal muscle fiber. Hau-FoxA3 was temporally expressed in the ventral neuroectoderm. At Stage 11, Hau-FoxC was expressed in the foregut. Hau-FoxO genes have a ubiquitous expression. Our results provide more insight on the evolutionary linkage and role of the Fox gene function in Bilateria.

Marine Leech Anticoagulant Diversity and Evolution.

Leeches (Annelida: Hirudinea) possess powerful salivary anticoagulants and, accordingly, are frequently employed in modern, authoritative medicine. Members of the almost exclusively marine family Piscicolidae account for 20% of leech species diversity, and they feed on host groups (e.g., sharks) not encountered by their freshwater and terrestrial counterparts. Moreover, some species of Ozobranchidae feed on endangered marine turtles and have been implicated as potential vectors for the tumor-associated turtle herpesvirus. In spite of their ecological importance and unique host associations, there is a distinct paucity of data regarding the salivary transcriptomes of either of these families. Using next-generation sequencing, we profiled transcribed, putative anticoagulants and other salivary bioactive compounds that have previously been linked to blood feeding from 7 piscicolid species (3 elasmobranch feeders; 4 non-cartilaginous fish feeders) and 1 ozobranchid species (2 samples). In total, 149 putative anticoagulants and bioactive loci were discovered in varying constellations throughout the different samples. The putative anticoagulants showed a broad spectrum of described antagonistic pathways, such as inhibition of factor Xa and platelet aggregation, which likely have similar bioactive roles in marine fish and turtles. A transcript with homology to ohanin, originally isolated from king cobras, was found in Cystobranchus vividus but is otherwise unknown from leeches. Estimation of selection pressures for the putative anticoagulants recovered evidence for both positive and purifying selection along several isolated branches in the gene trees, and positive selection was also estimated for a few select codons in a variety of marine species. Similarly, phylogenetic analyses of the amino acid sequences for several anticoagulants indicated divergent evolution.

Mechanism of ammonia excretion in the freshwater leech Nephelopsis obscura: characterization of a primitive Rh protein and effects of high environmental ammonia.

Remarkably little is known about nitrogenous excretion in freshwater invertebrates. In the current study, the nitrogen excretion mechanism in the carnivorous ribbon leech, Nephelopsis obscura, was investigated. Excretion experiments showed that the ribbon leech is ammonotelic, excreting 166.0 ± 8.6 nmol·grams fresh weight (gFW)(-1)·h(-1) ammonia and 14.7 ± 1.9 nmol·gFW(-1)·h(-1) urea. Exposure to high and low pH hampered and enhanced, respectively, ammonia excretion rates, indicating an acid-linked ammonia trapping mechanism across the skin epithelia. Accordingly, compared with body tissues, the skin exhibited elevated mRNA expression levels of a newly identified Rhesus protein and at least in tendency the Na(+)/K(+)-ATPase. Pharmacological experiments and enzyme assays suggested an ammonia excretion mechanism that involves the V-ATPase, Na(+)/K(+)-ATPase, and carbonic anhydrase, but not necessarily a functional microtubule system. Most importantly, functional expression studies of the identified Rh protein cloned from leech skin tissue revealed an ammonia transport capability of this protein when expressed in yeast. The leech Rh-ammonia transporter (NoRhp) is a member of the primitive Rh protein family, which is a sister group to the common ancestor of vertebrate ammonia-transporting Rh proteins. Exposure to high environmental ammonia (HEA) caused a new adjustment of body ammonia, accompanied with a decrease in NoRhp and Na(+)/K(+)-ATPase mRNA levels, but unaltered ammonia excretion rates. To our knowledge, this is only the second comprehensive study regarding the ammonia excretion mechanisms in a freshwater invertebrate, but our results show that basic processes of ammonia excretion appear to also be comparable to those found in freshwater fish, suggesting an early evolution of ionoregulatory mechanisms in freshwater organisms.

Arachidonic acid closes innexin/pannexin channels and thereby inhibits microglia cell movement to a nerve injury.

Pannexons are membrane channels formed by pannexins and are permeable to ATP. They have been implicated in various physiological and pathophysiological processes. Innexins, the invertebrate homologues of the pannexins, form innexons. Nerve injury induces calcium waves in glial cells, releasing ATP through glial pannexon/innexon channels. The ATP then activates microglia. More slowly, injury releases arachidonic acid (ArA). The present experiments show that ArA itself reduced the macroscopic membrane currents of innexin- and of pannexin-injected oocytes; ArA also blocked K(+) -induced release of ATP. In leeches, whose large glial cells have been favorable for studying control of microglia migration, ArA blocked glial dye-release and, evidently, ATP-release. A physiological consequence in the leech was block of microglial migration to nerve injuries. Exogenous ATP (100 µM) reversed the effect, for ATP causes activation and movement of microglia after nerve injury, but nitric oxide directs microglia to the lesion. It was not excluded that metabolites of ArA may also inhibit the channels. But for all these effects, ArA and its non-metabolizable analog eicosatetraynoic acid (ETYA) were indistinguishable. Therefore, ArA itself is an endogenous regulator of pannexons and innexons. ArA thus blocks release of ATP from glia after nerve injury and thereby, at least in leeches, stops microglia at lesions.

Structure of the leech protein saratin and characterization of its binding to collagen.

The leech protein Saratin from Hirudo medicinalis prevents thrombocyte aggregation by interfering with the first binding step of the thrombocytes to collagen by binding to collagen. We solved the three-dimensional structure of the leech protein Saratin in solution and identified its collagen binding site by NMR titration experiments. The NMR structure of Saratin consists of one alpha-helix and a five-stranded beta-sheet arranged in the topology betabetaalphabetabetabeta. The C-terminal region, of about 20 amino acids in length, adopts no regular structure. NMR titration experiments with collagen peptides show that the collagen interaction of Saratin takes place in a kind of notch that is formed by the end of the alpha-helix and the beta-sheet. NMR data-driven docking experiments to collagen model peptides were used to elucidate the putative binding mode of Saratin and collagen. Mainly, parts of the first and the end of the fifth beta-strand, the loop connecting the alpha-helix and the third beta-strand, and a short part of the loop connecting the fourth and fifth beta-strand participate in binding.

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.

Crystallization and preliminary X-ray crystallographic study of the leech protease inhibitor guamerin and its complex with bovine pancreatic chymotrypsin.

Guamerin, a small peptide inhibitor of the serine protease from Hirudo nipponia, was expressed in yeast and crystallized using the vapor diffusion method, with MPD as precipitant. The crystal was found to belong to the monoclinic P2(1) space group with unit cell parameters a=136.06, b=206.59, c=227.39 A, beta=105.03 degrees. The guamerin/bovine pancreatic chymotrypsin complex was also crystallized using PEG 8K as precipitant. The space group was identified as P2(1)2(1)2(1) with unit cell parameters of a=44.01, b=44.30, c=122.47 A. The diffraction data of the complex were collected up to a resolution of 2.4 A using a synchrotron-radiation source under cryogenic condition.

Molecular characterization of two novel antibacterial peptides inducible upon bacterial challenge in an annelid, the leech Theromyzon tessulatum.

Two novel antimicrobial peptides named theromacin and theromyzin were isolated and characterized from the coelomic liquid of the leech Theromyzon tessulatum. Theromacin is a 75-amino acid cationic peptide containing 10 cysteine residues arranged in a disulfide array showing no similarities with other known antimicrobial peptides. Theromyzin is an 86-amino acid linear peptide and constitutes the first anionic antimicrobial peptide observed in invertebrates. Both peptides exhibit activity directed against Gram-positive bacteria. Theromacin and theromyzin cDNAs code precursor molecules containing a putative signal sequence directly followed by the mature peptide. The enhancement of theromacin and theromyzin mRNA levels has been observed after blood meal ingestion and upon bacterial challenge. In situ hybridization revealed that both genes are expressed in large fat cells in contact with coelomic cavities. Gene products were immunodetected in large fat cells, in intestinal epithelia, and at the epidermis level. In addition, a rapid release of the peptides into the coelomic liquid was observed after bacterial challenge. The presence of antimicrobial peptide genes in leeches and their expression in a specific tissue functionally resembling the insect fat body provide evidence for the first time of an antibacterial response in a lophotrochozoan comparable to that of holometabola insects.

Chromacin-like peptide in leeches.

We demonstrate the presence in leech hemolymph of high levels of a peptide recognized by antiserum directed against bovine chromacin. The purification of the chromacin-like peptide was carried out by an acidic extraction, followed by solid phase and high pressure gel permeation chromatography and reversed-phase HPLC purification. Its sequence (GDFELPSIADPQATFESQRGPSAQQVDK) was established by a combination of techniques, including automated Edman degradation, MALDI-TOF measurement and DOT immunobinding assays with anti-chromogranin A. Mass spectrometry measurement revealed a m/z 3177Da, revealing the fact that the molecule is phosphorylated. ELISA titrations performed at each step of the purification revealed a major increase in the level of the peptide (ca. 125 nmol/microl of coelomic fluid) 15 min after LPS exposure. The increase in chromacin-like peptide levels is both time and concentration dependent. The level of this peptide decreased significantly 4 hours after LPS exposure. This report is the first discovery of a chromogranin derived like peptide in invertebrates.

Optimization of protease-inhibitor interactions by randomizing adventitious contacts.

Polypeptide protease inhibitors are often found to inhibit targets with which they did not coevolve, as in the case of high-affinity inhibition of bacterial subtilisin by the leech inhibitor eglin c. Two kinds of contacts exist in such complexes: (i) reactive site loop-active site contacts and (ii) interactions outside of these that form the broader enzyme-inhibitor interface. We hypothesized that the second class of "adventitious" contacts could be optimized to generate significant increases in affinity for a target enzyme or discrimination of an inhibitor for closely related target proteases. We began with a modified eglin c, Arg-42-Arg-45-eglin, in which the reactive site loop had been optimized for subtilisin-related processing proteases of the Kex2/furin family. We randomized 10 potential adventitious contact residues and screened for inhibition of soluble human furin. Substitutions at one of these sites, Y49, were also screened against yeast Kex2 and human PC7. These screens identified not only variants that exhibited increased affinity (up to 20-fold), but also species that exhibited enhanced selectivity, that is, increased discrimination between the target enzymes (up to 41-fold for furin versus PC7 and 20-fold for PC7 versus furin). One variant, Asp-49-Arg-42-Arg-45-eglin, exhibited a Ki of 310 pM for furin and blocked furin-dependent processing of von Willebrand factor in COS-1 cells when added to the culture medium of the cells. The exploitation of adventitious contact sites may provide a versatile technique for developing potent, selective inhibitors for newly discovered proteases and could in principle be applied to optimize numerous protein-protein interactions.

Effects of extracellular purines on ion transport across the integument of Hirudo medicinalis.

Little is known about the long-term regulation of epithelial ion transport in invertebrates and the specific mediators involved. For some years, we have been investigating the short-term regulation of transepithelial ion transport across the dorsal integument of the leech Hirudo medicinalis, and we have established a model of Na+ uptake. In the present study, we investigated the effect of long-term acclimation on transintegumental ion transport by adapting leeches to high-salinity conditions. We dissected segments of dorsal integument and measured ion currents in Ussing chamber experiments. Electrophysiological variables, such as transepithelial potential (V(T)) and short-circuit-current (I(sc)), were profoundly affected by adaptation to high-salinity conditions. The total transepithelial Na+ current (I(Na)) decreased from 7.66+/-0.82 to 4.6+/-0.54 microA cm(-2) in preparations adapted to high salinity. The involvement of epithelial Na+ channels was determined as current inhibition (I(ami)) by apical application of amiloride; Na+ channels were equally active in control epithelia and epithelia from leeches adapted to high salinity. Removal of Ca2+ from the apical solutions, which is believed to reduce intracellular Ca2+ concentrations, equalized transepithelial variables between high-salt-adapted integuments and control integuments. Extracellular purines regulate transepithelial Cl- secretion and Na+ absorption. In a variety of tissues we tested ATP and adenosine for their effects on epithelial transport. Examination of integuments from pondwater- and high-salinity-adapted leeches revealed different sensitivities for these purines. Apical and basolateral application of ATP both stimulated transepithelial Na+ uptake and I(ami). Adenosine upregulated non-Na+ currents and acted from the basolateral side only. Apical Ca2+-free conditions attenuated these effects of purines on transepithelial currents. Extracellular UTP had no effect on ion transport.

The ouabain-induced [Ca2+]i increase in leech Retzius neurones is mediated by voltage-dependent Ca2+ channels.

In leech Retzius neurones the inhibition of the Na+/K+ pump by ouabain causes an increase in the cytosolic free calcium concentration ([Ca2+]i). To elucidate the mechanism of this increase we investigated the changes in [Ca2+]i (measured by Fura-2) and in membrane potential that were induced by inhibiting the Na+/K+ pump in bathing solutions of different ionic composition. The results show that Na+/K+ pump inhibition induced a [Ca2+]i increase only if the cells depolarized sufficiently in the presence of extracellular Ca2+. Specifically, the relationship between [Ca2+]i and the membrane potential upon Na+/K+ pump inhibition closely matched the corresponding relationship upon activation of the voltage-dependent Ca2+ channels by raising the extracellular K+ concentration. It is concluded that the [Ca2+]i increase caused by inhibiting the Na+/K+ pump in leech Retzius neurones is exclusively due to Ca2+ influx through voltage-dependent Ca2+ channels.

The complex dynamic network of microtubule and microfilament cytasters of the leech zygote.

The organization of the cytoskeleton in the early first interphase zygote and its involvement in organelle redistribution were studied in the glossiphoniid leech Theromyzon trizonare by confocal and electron microscopy, immunofluorescence, and time-lapse video imaging after microinjection of labeled tubulin and/or actin and loading with a mitotracker. The cytoskeleton consists of an inner or endoplasmic and an outer or ectoplasmic domain. The inner domain consists of a monaster whose fibers retract from the zygote periphery by the end of the early first interphase. The outer domain is built upon a network of microtubules and microfilaments cytasters. Short pulses of microinjected labeled actin or tubulin and Taxol treatment demonstrate that cytasters are centers of microtubule and microfilament nucleation. Immunostaining with anti-centrophilin, anti-BX-63, and anti-AH-6 indicates that the network of cytasters includes centrosomal antigens. Cytasters move in an orderly fashion at speeds of 0.5-2 micrometer/min, in an energy-dependent process retarded and finally blocked by the ATP analogue AMP-PNP and high concentrations of Taxol. Colliding cytasters fuse and form larger cytoskeletal nucleation centers. The leech zygote is a highly compartmentalized cell whose cytasters function as articulated components of a very dynamic cytoskeletal system engaged in bulk transportation of organelles during ooplasmic segregation.

Lipopolysaccharide-dependent induction of leech leukocytes that cross-react with vertebrate cellular differentiation markers.

We have designed experiments to characterise leech leukocytes that mediate inflammatory responses. Shortly after inflicting injury to the body wall in the presence of lipopolysaccharides, many cells resembling macrophages, NK cells and granulocytes of vertebrates and many invertebrates migrated to the lesioned area. Nuclei of migrating cells incorporated bromodeoxyuridine. Using human monoclonal antibodies, macrophage-like cells were positive for CD25, CD14, CD61, CD68, CD11b and CD11c. NK-like cells were positive for CD25, CD56, CD57 and CD16, and granulocytes were positive for CD11b and CD11c. In blots of leech extracts, the CD25 monoclonal antibody recognised a band of about 55 kD; the CD56 monoclonal antibody, two bands of about 140 and 210 kD; the CD57 monoclonal antibody, two bands of about 106 and 70 kD; the CD14 monoclonal antibody, a band of about 50 kD; the CD16 monoclonal antibody, a band of about 60 kD. CD61 and CD68 both recognised a band of about 110 kD; CD11b recognised a band of 200 kD, and CD11c, a band of 180 kD.