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Intracellular proteolysis of ingested blood proteins is a crucial physiological process in ticks. In our model tick, Ixodes ricinus, cathepsin L (IrCL1) is part of a gut-associated multi-peptidase complex; its endopeptidase activity is important in the initial phase of haemoglobinolysis. We present the functional and biochemical characterisation of this enzyme. We show, by RNA interference (RNAi), that cathepsin L-like activity that peaks during the slow feeding period of females is associated with IrCL1. Recombinant IrCL1 was expressed in bacteria and yeast. Activity profiling with both peptidyl and physiological protein substrates (haemoglobin and albumin) revealed that IrCL1 is an acidic peptidase with a very low optimum pH (3-4) being unstable above pH 5. This suggests an endo/lysosomal localisation that was confirmed by indirect fluorescence microscopy that immunolocalised IrCL1 inside the vesicles of digestive gut cells. Cleavage specificity determined by a positional scanning synthetic combinatorial library and inhibition profile indicated that IrCL1 has the ligand-binding characteristics of the cathepsin L subfamily of cysteine peptidases. A non-redundant proteolytic function was demonstrated when IrCL1-silenced ticks had a decreased ability to feed compared with controls. The data suggest that IrCL1 may be a promising target against ticks and tick-borne pathogens., (Copyright © 2011 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.)

Cysteine proteases have been discovered in various bloodfeeding ectoparasites. Here, we assemble the available information about the function of these peptidases and reveal their role in hematophagy and parasite development. While most of the data shed light on key proteolytic events that play a role in arthropod physiology, we also report on the association of cysteine proteases with arthropod vectorial capacity. With emphasis on ticks, specifically Ixodes ricinus, we finally propose a model about the contribution of cysteine peptidases to blood digestion and how their concerted action with other tick midgut proteases leads to the absorbance of nutrients by the midgut epithelial cells.

Background: Ticks are vectors of a wide variety of pathogens causing severe diseases in humans and domestic animals. Intestinal digestion of the host blood is an essential process of tick physiology and also a limiting factor for pathogen transmission since the tick gut represents the primary site for pathogen infection and proliferation. Using the model tick Ixodes ricinus, the European Lyme disease vector, we have previously demonstrated by genetic and biochemical analyses that host blood is degraded in the tick gut by a network of acidic peptidases of the aspartic and cysteine classes., Results: This study reveals the digestive machinery of the I. ricinus during the course of blood-feeding on the host. The dynamic profiling of concentrations, activities and mRNA expressions of the major digestive enzymes demonstrates that the de novo synthesis of peptidases triggers the dramatic increase of the hemoglobinolytic activity along the feeding period. Overall hemoglobinolysis, as well as the activity of digestive peptidases are negligible at the early stage of feeding, but increase dramatically towards the end of the slow feeding period, reaching maxima in fully fed ticks. This finding contradicts the established opinion that blood digestion is reduced at the end of engorgement. Furthermore, we show that the digestive proteolysis is localized intracellularly throughout the whole duration of feeding., Conclusions: Results suggest that the egressing proteolytic system in the early stage of feeding and digestion is a potential target for efficient impairment, most likely by blocking its components via antibodies present in the host blood. Therefore, digestive enzymes are promising candidates for development of novel 'anti-tick' vaccines capable of tick control and even transmission of tick-borne pathogens.

Background: The possible emergence of resistance to the only available drug for schistosomiasis spurs drug discovery that has been recently incentivized by the availability of improved transcriptome and genome sequence information. Transient RNAi has emerged as a straightforward and important technique to interrogate that information through decreased or loss of gene function and identify potential drug targets. To date, RNAi studies in schistosome stages infecting humans have focused on single (or up to 3) genes of interest. Therefore, in the context of standardizing larger RNAi screens, data are limited on the extent of possible off-targeting effects, gene-to-gene variability in RNAi efficiency and the operational capabilities and limits of RNAi., Methodology/principal Findings: We investigated in vitro the sensitivity and selectivity of RNAi using double-stranded (ds)RNA (approximately 500 bp) designed to target 11 Schistosoma mansoni genes that are expressed in different tissues; the gut, tegument and otherwise. Among the genes investigated were 5 that had been previously predicted to be essential for parasite survival. We employed mechanically transformed schistosomula that are relevant to parasitism in humans, amenable to screen automation and easier to obtain in greater numbers than adult parasites. The operational parameters investigated included defined culture media for optimal parasite maintenance, transfection strategy, time- and dose-dependency of RNAi, and dosing limits. Of 7 defined culture media tested, Basch Medium 169 was optimal for parasite maintenance. RNAi was best achieved by co-incubating parasites and dsRNA (standardized to 30 µg/ml for 6 days); electroporation provided no added benefit. RNAi, including interference of more than one transcript, was selective to the gene target(s) within the pools of transcripts representative of each tissue. Concentrations of dsRNA above 90 µg/ml were directly toxic. RNAi efficiency was transcript-dependent (from 40 to >75% knockdown relative to controls) and this may have contributed to the lack of obvious phenotypes observed, even after prolonged incubations of 3 weeks. Within minutes of their mechanical preparation from cercariae, schistosomula accumulated fluorescent macromolecules in the gut indicating that the gut is an important route through which RNAi is expedited in the developing parasite., Conclusions: Transient RNAi operates gene-selectively in S. mansoni newly transformed schistosomula yet the sensitivity of individual gene targets varies. These findings and the operational parameters defined will facilitate larger RNAi screens.

Unlabelled: Crohn's disease activity index (CDAI) and serum C-reactive protein (CRP) levels are not prefect indicators of Crohn's disease severity. Magnetic resonance enteroclysis (MRE) is a method allowing simultaneous assessment of lesions involving an entire intestinal wall as well as intra- and extraintestinal spaces. This method, however, is not appropriate for monitoring the course of disease and therapeutic effects., The Aim of the Study: To evaluate which of the extensive panel of pro- and anti- inflammatory cytokines correlates with an actual severity of CD assessed by MRE. MATERIAL AND METHODS. 57 patients with endoscopically diagnosed ileocecal form of CD (28 women, age 29 + 11 yrs, range 18-62 yrs) hospitalized in 2007-2008. The mean CDAI was 293 + 119 points, range 18-503 points and serum CRP level was 17.5 + 31 mg/l, range 0.1-122 mg/l. MRE was performed in each patient not later than 3 days after entry to the study. The summarized score was calculated using standardized protocol, assessing the intestine wall thickness and length of its involvement (ileocecal region), pattern of mural contrast enhancement, presence of fistulas or other extraintestinal lesions and enlargement of mesenteric lymph nodes. At admission the blood was taken to measure following cytokines: IL-la, IL-1 receptor antagonist, IL-6, soluble IL-6 receptor, TNF-alpha, TNF-alpha type II receptor and IL-10., Results: In Spearman's correlation test the MRE score showed the strongest relationship with serum level of TNF-alpha type II receptor (r = 0.52, p < 0.001), correlating less significantly with IL-6 level (r = 0.37, p < 0.01) and CDAI (r = 0.40, p < 0.001)., Conclusion: TNFalpha receptor type II shows better correlation with the severity of ileocecal CD (assessed by MRE) than CDAI or serum levels of other cytokines and CRP.

Background: Ewing sarcoma is a malignant, small round cell bone tumor, presenting predominantly in children and adolescents. Ewing sarcoma may develop in every bone; diaphyses of long bones, ribs and flat bones are the main locations. Local and systemic clinical symptoms are nonspecific - pain, swelling, fever or ill-being. The aim of the study was to assess the role of radiography, computed tomography and magnetic resonance imaging in the analysis of bone lesions in children and young adults with Ewing sarcoma., Material/methods: Twenty-seven patients, aged between 1 year and 10 months, and 17 years and 2 months, with histologically verified Ewing sarcoma of the bone, referred to the Radiological Department of University Hospital No 6., John Paul II Upper Silesian Centre for Child Health Katowice, in the period from 1996 to 2007, were included in the study.Plain radiography was performed in every child, CT in 20 and MRI in 12 individuals. Tumour location, extension of the tumour, soft tissue mass, and periosteal reaction were taken into consideration in the evaluation of the lesion. In some cases, pathological features of the MRI and CT were compared. The prevalence of some radiological features was compared to the literature data., Results: THE MOST COMMON SITE OF TUMOR WAS: ribs (6 children), femoral bone (6 children), pelvis (4 children) and tibia (3 children). In 2 children, a primary tumor was diagnosed in the spine (multifocal in 1 child). X-rays revealed: periosteal reaction in 17 children (63%), soft tissue involvement in 19 children (70%), permeative component in 16 children (59%), and sclerotic component in 5 children (19%). In 10 children (37%), periosteal reaction was not detected. The examination revealed: soft tissue calcifications in 7 cases (26%), a well-delineated focus of destruction within bones in 3 children (11%), cortical thickening in 4 children (15%), cortical destruction in 4 children (15%), saucerisation in 3 children (11%), bone expansion in 3 children (11%), pathological fracture in 2 children (7%), cystic component in 1 child (4%), and vertebra plana in 1 child (4%).Reaction of tumors after i.v. contrast administration, shown on CT, was visible in 16 children - it was useful for a better description of the tumor and extension of the mass within the soft tissue. All MRI examinations (12 children) showed a heterogenous mass with ill-defined borders and a violated cortex. Low signal intensity of the tumor in a T1-weighted image and high signal intensity in a T2-weighted image was shown as well. Heterogenous enhancement of signal intensity on T1-weighted images could be observed after i.v. contrast administration. MRI EXAMINATIONS SHOWED: tumor in an adjacent soft tissue in 11 children, and involvement of the epiphyseal plate or of the joint cavity in 6 children., Conclusions: X-ray and MRI are essential in diagnostics. CT examination is more useful to estimate periosteal reactions and destruction of bone and marrow cavity, especially in flat bones. However, to recognise a malignancy, it is necessary to perform a histopathological examination. In doubtful cases, the examination has to be verified as well.

Aza-peptide Michael acceptors and epoxides with the general structure of YCO-Ala-Ala-AAsn-trans-CH horizontal lineCHCOR and YCO-Ala-Ala-AAsn-EP-COR, respectively, are shown to be potent inhibitors of asparaginyl endopeptidases (legumains) from the bloodfluke, Schistosoma mansoni (SmAE), and the hard tick, Ixodes ricinus (IrAE). Structure-activity relationships (SARs) were determined for a set of 41 aza-peptide Michael acceptors and eight aza-peptide epoxides. Both enzymes prefer disubstituted amides to monosubstituted amides in the P1' position, and potency increased as we increased the hydrophobicity of the inhibitor in this position. Extending the inhibitor to P5 resulted in increased potency, especially against IrAE, and both enzymes prefer small over large hydrophobic residues at P2. Aza-peptide Michael acceptor inhibitors are more potent than aza-peptide epoxide inhibitors, and for some of these compounds, second-order inhibiton rate constants are the fastest yet discovered. Given the central functions of these enzymes in both parasites, the data presented here may facilitate the eventual design of selective antiparasitic drugs.

Hemoglobin digestion is an essential process for blood-feeding parasites. Using chemical tools, we deconvoluted the intracellular hemoglobinolytic cascade in the tick Ixodes ricinus, a vector of Lyme disease and tick-borne encephalitis. In tick gut tissue, a network of peptidases was demonstrated through imaging with specific activity-based probes and activity profiling with peptidic substrates and inhibitors. This peptidase network is induced upon blood feeding and degrades hemoglobin at acidic pH. Selective inhibitors were applied to dissect the roles of the individual peptidases and to determine the peptidase-specific cleavage map of the hemoglobin molecule. The degradation pathway is initiated by endopeptidases of aspartic and cysteine class (cathepsin D supported by cathepsin L and legumain) and is continued by cysteine amino- and carboxy-dipeptidases (cathepsins C and B). The identified enzymes are potential targets to developing novel anti-tick vaccines.

The universal protease inhibitors of the alpha(2)-macroglobulin (alpha(2)M) family are evolutionarily conserved constituents of innate immunity, presumably because they guard organisms against undesired proteolytic attacks of a different origin. Here, we determined the primary structure of alpha(2)-macroglobulin from the hard tick Ixodes ricinus (IrAM) by sequencing of overlapping PCR products. Predicted disulfide and glycosylation patterns, post-translational cleavage and alternative splicing within its 'bait region' demonstrate that IrAM is closely related to the alpha(2)-macroglobulin from the soft tick Ornithodoros moubata. The IrAM message is expressed in all tick developmental stages and tissues, except for the gut, and the protein was detected to be mainly present in the hemolymph. Silencing of IrAM by dsRNA interference markedly reduced the phagocytosis of a potential pathogen, Chryseobacterium indologenes, by tick hemocytes both in vitro and in vivo. In contrast, phagocytosis of the Lyme disease spirochete Borrelia burgdorferi or a commensal bacteria Staphylococcus xylosus was not affected by the IrAM knock-down. Similar results were obtained upon deactivation of all thioester proteins in tick hemolymph by methylamine. We have further demonstrated that phagocytosis of C. indologenes is dependent on an active metalloprotease secreted by the bacteria. These data indicate that interaction of tick alpha(2)-macroglobulin with a protease of an invading pathogen is linked with cellular immune response.

Ticks are among the most important vectors of a wide range of human and animal diseases. During blood feeding, ticks are exposed to an enormous amount of free iron that must be appropriately used and detoxified. However, the mechanism of iron metabolism in ticks is poorly understood. Here, we show that ticks possess a complex system that efficiently utilizes, stores and transports non-heme iron within the tick body. We have characterized a new secreted ferritin (FER2) and an iron regulatory protein (IRP1) from the sheep tick, Ixodes ricinus, and have demonstrated their relationship to a previously described tick intracellular ferritin (FER1). By using RNA interference-mediated gene silencing in the tick, we show that synthesis of FER1, but not of FER2, is subject to IRP1-mediated translational control. Further, we find that depletion of FER2 from the tick plasma leads to a loss of FER1 expression in the salivary glands and ovaries that normally follows blood ingestion. We therefore suggest that secreted FER2 functions as the primary transporter of non-heme iron between the tick gut and the peripheral tissues. Silencing of the fer1, fer2, and irp1 genes by RNAi has an adverse impact on hatching rate and decreases postbloodmeal weight in tick females. Importantly, knockdown of fer2 dramatically impairs the ability of ticks to feed, thus making FER2 a promising candidate for development of an efficient anti-tick vaccine.

Aza-peptide Michael acceptors with the general structure of Cbz-Ala-Ala-AAsn- trans-CH=CHCOR are a new class of inhibitors specific for the asparaginyl endopeptidases (AE) (legumains). Structure-activity relationships (SARs) were characterized for a set of 31 aza-peptide Michael acceptors with AEs derived from three medically important parasites: the protist Trichomonas vaginalis, the hard tick Ixodes ricinus, and the flatworm Schistosoma mansoni. Despite arising from phylogenetically disparate organisms, all three AEs shared a remarkably similar SAR with lowest IC50 values extending into the picomolar range. The results suggest an evolutionary constraint on the topography of the prime side of the active site. SAR also revealed that esters in the P1' position are more potent than disubstituted amides and that monosubstituted amides and alkyl derivatives show little or no inhibition. The preferred P1' residues have aromatic substituents. Aza-asparaginyl Michael acceptors react with thiols, which provides insight into the mechanism of their inhibition of asparaginyl endopeptidases.

Background: Ticks are vectors for a variety of viral, bacterial and parasitic diseases in human and domestic animals. To survive and reproduce ticks feed on host blood, yet our understanding of the intestinal proteolytic machinery used to derive absorbable nutrients from the blood meal is poor. Intestinal digestive processes are limiting factors for pathogen transmission since the tick gut presents the primary site of infection. Moreover, digestive enzymes may find practical application as anti-tick vaccine targets., Results: Using the hard tick, Ixodes ricinus, we performed a functional activity scan of the peptidase complement in gut tissue extracts that demonstrated the presence of five types of peptidases of the cysteine and aspartic classes. We followed up with genetic screens of gut-derived cDNA to identify and clone genes encoding the cysteine peptidases cathepsins B, L and C, an asparaginyl endopeptidase (legumain), and the aspartic peptidase, cathepsin D. By RT-PCR, expression of asparaginyl endopeptidase and cathepsins B and D was restricted to gut tissue and to those developmental stages feeding on blood., Conclusion: Overall, our results demonstrate the presence of a network of cysteine and aspartic peptidases that conceivably operates to digest host blood proteins in a concerted manner. Significantly, the peptidase components of this digestive network are orthologous to those described in other parasites, including nematodes and flatworms. Accordingly, the present data and those available for other tick species support the notion of an evolutionary conservation of a cysteine/aspartic peptidase system for digestion that includes ticks, but differs from that of insects relying on serine peptidases.

Ticks are ectoparasitic blood-feeders and important vectors for pathogens including arboviruses, rickettsiae, spirochetes and protozoa. As obligate blood-feeders, one possible strategy to retard disease transmission is disruption of the parasite's ability to digest host proteins. However, the constituent peptidases in the parasite gut and their potential interplay in the digestion of the blood meal are poorly understood. We have characterised a novel asparaginyl endopeptidase (legumain) from the hard tick Ixodes ricinus (termed IrAE), which we believe is the first such characterisation of a clan CD family C13 cysteine peptidase (protease) in arthropods. By RT-PCR of different tissues, IrAE mRNA was only expressed in the tick gut. Indirect immunofluorescence and EM localised IrAE in the digestive vesicles of gut cells and within the peritrophic matrix. IrAE was functionally expressed in Pichia pastoris and reacted with a specific peptidyl fluorogenic substrate, and acyloxymethyl ketone and aza-asparagine Michael acceptor inhibitors. IrAE activity was unstable at pH > or = 6.0 and was shown to have a strict specificity for asparagine at P1 using a positional scanning synthetic combinatorial library. The enzyme hydrolyzed protein substrates with a pH optimum of 4.5, consistent with the pH of gut cell digestive vesicles. Thus, IrAE cleaved the major protein of the blood meal, hemoglobin, to a predominant peptide of 4kDa. Also, IrAE trans-processed and activated the zymogen form of Schistosoma mansoni cathepsin B1 -- an enzyme contributing to hemoglobin digestion in the gut of that bloodfluke. The possible functions of IrAE in the gut digestive processes of I. ricinus are compared with those suggested for other hematophagous parasites.

Two genes coding for cysteine peptidase inhibitors of the cystatin family (Om-cystatin 1 and 2) were isolated from a gut-specific cDNA library of the soft tick Ornithodoros moubata. Both cystatins were clearly down-regulated after a blood meal. Om-cystatin 1 is mainly expressed in the tick gut, while Om-cystatin 2 mRNA was also found in other tick tissues. Authentic Om-cystatin 2 was significantly more abundant than Om-cystatin 1 in the gut contents of fasting ticks and was associated with hemosome-derived residual bodies accumulated in the gut lumen. Om-cystatin 2 was also expressed by type 2 secretory cells in the salivary glands of unfed ticks. The inhibitory specificity of recombinant Om-cystatins 1 and 2 was tested with mammalian cysteine peptidases, as well as endogenous cysteine peptidases present in the tick gut. Both cystatins efficiently inhibited papain-like peptidases, including cathepsin B and H, but differed significantly in their affinity towards cathepsin C and failed to block asparaginyl endopeptidase. Our results suggest that the secreted cystatin isoinhibitors are involved in the regulation of multiple proteolytic targets in the tick digestive system and tick-host interaction.

The sequence of a alpha(2)-macroglobulin (alpha(2)M) from the soft tick Ornithodoros moubata (TAM) was determined by cloning and sequencing of overlapping polymerase chain reaction (PCR) and rapid amplification of cDNA ends PCR products. The TAM cDNA sequence is 4,944 bp long and contains one open reading frame coding for a protein precursor composed of 1,494 amino-acid residues, including a 24-residue signal sequence. The mature protein is cleaved into two subunits similarly to the C3 and C4 components of complement and fish alpha(2)Ms. Phylogeny analysis revealed that TAM is closely related to Limulus alpha(2)M and displays the highest similarity to the partial sequence of alpha(2)M from hard tick Ixodes scapularis. The comparison of conserved cysteine residues between TAM and human and Limulus alpha(2)Ms made it possible to predict the pattern of disulfide bridges and explain the atypical molecular arrangement of TAM. Four variants of the TAM bait region differing only in a short central segment were found; our data indicate that TAM exists as a single-copy gene in the tick genome and its bait region variants likely arise by alternative splicing. TAM is produced by tick hemocytes and it is also significantly expressed in salivary glands. TAM mRNA levels were shown to be up-regulated upon blood meal.

In this study, we show that administration of low-dose melphalan (l -PAM, l -phenylalanine mustard) to mice bearing a large MOPC-315 plasmacytoma led to a rapid up-regulation of B7-1 (CD80), but not B7-2 (CD86), expression on the surface of MOPC-315 tumor cells. This l -PAM-induced preferential up-regulation of B7-1 surface expression was due, at least in part, to a direct effect of l -PAM on the tumor cells, as in vitro exposure of MOPC-315 tumor cells to l -PAM led to the preferential up-regulation of B7-1 surface expression. Moreover, in vitro exposure of MOPC-315 tumor cells to two other anticancer modalities, gamma-irradiation and mitomycin C, resulted in the preferential up-regulation of B7-1 surface expression. This effect was not restricted to MOPC-315 tumor cells, as preferential up-regulation of B7-1 surface expression was observed also following in vitro exposure of the P815 mastocytoma (that is negative for both B7-1 and B7-2 surface expression) to any of the three anticancer modalities. The up-regulation of B7-1 surface expression following in vitro exposure of tumor cells to l -PAM, gamma-irradiation, or mitomycin C required de novo protein and RNA synthesis, and was associated with the accumulation of mRNA for B7-1 within 4-8 h, indicating that the regulation of B7-1 expression is at the RNA transcriptional level. These results have important implications for an additional immune-potentiating mechanism of these anticancer modalities in clinical setting.

We have previously shown that B7-2 (CD86) and, to a lesser extent, B7-1 (CD80) contribute to the curative effectiveness of low-dose melphalan (L-phenylalanine mustard) for mice bearing a large MOPC-315 tumor under conditions that lead to the acquisition of potent cytotoxic T lymphocyte (CTL) activity at the tumor site. Since B7-1 and B7-2 are expressed on both tumor cells and host antigen-presenting cells (APC), the current studies were undertaken to examine the relative importance of each costimulatory molecule on tumor cells and on host APC for the acquisition of anti-MOPC-315 CTL activity. Utilizing an in vitro system for the acquisition of CTL activity, we found that B7 expression on host APC is important for the development of CTL activity in stimulation cultures of spleen cells from low-dose-melphalan-treated MOPC-315 tumor bearers, although the expression of either B7-1 or B7-2 is sufficient. In addition, we found that B7-2, which is expressed at high levels on stimulator tumor cells, but not B7-1, which is expressed at much lower levels, is also important for the acquisition of CTL activity. However, the vast majority of the CTL activity acquired in vitro in response to stimulation with the B7-2-expressing MOPC-315 tumor cells was found to depend on B7-expressing host APC. Thus, it is likely that B7-2, which is expressed at high levels on MOPC-315 tumor cells, promotes the rapid lysis of MOPC-315 stimulator tumor cells, thereby making tumor-associated antigens more readily available for efficient presentation by B7-expressing host APC which, in turn, stimulate the acquisition of CTL activity by spleen cells from low-dose-melphalan-treated MOPC-315 tumor bearers.