The effects of plant cysteine proteinases on the nematode cuticle.

BACKGROUND: Plant-derived cysteine proteinases of the papain family (CPs) attack nematodes by digesting the cuticle, leading to rupture and death of the worm. The nematode cuticle is composed of collagens and cuticlins, but the specific molecular target(s) for the proteinases have yet to be identified. METHODS: This study followed the course of nematode cuticle disruption using immunohistochemistry, scanning electron microscopy and proteomics, using a free-living nematode, Caenorhabditis elegans and the murine GI nematode Heligmosomoides bakeri (H. polygyrus) as target organisms. RESULTS: Immunohistochemistry indicated that DPY-7 collagen is a target for CPs on the cuticle of C. elegans. The time course of loss of DPY-7 from the cuticle allowed us to use it to visualise the process of cuticle disruption. There was a marked difference in the time course of damage to the cuticles of the two species of nematode, with H. bakeri being more rapidly hydrolysed. In general, the CPs' mode of attack on the nematode cuticle was by degrading the structural proteins, leading to loss of integrity of the cuticle, and finally death of the nematode. Proteomic analysis failed conclusively to identify structural targets for CPs, but preliminary data suggested that COL-87 and CUT-19 may be important targets for the CPs, the digestion of which may contribute to cuticle disruption and death of the worm. Cuticle globin was also identified as a cuticular target. The presence of more than one target protein may slow the development of resistance against this new class of anthelmintic. CONCLUSIONS: Scanning electron microscopy and immunohistochemistry allowed the process of disruption of the cuticle to be followed with time. Cuticle collagens and cuticlins are molecular targets for plant cysteine proteinases. However, the presence of tyrosine cross-links in nematode cuticle proteins seriously impeded protein identification by proteomic analyses. Multiple cuticle targets exist, probably making resistance to this new anthelmintic slow to develop.

Conditional deletion of E11/Podoplanin in bone protects against ovariectomy-induced increases in osteoclast formation and activity.

E11/Podoplanin (Pdpn) is implicated in early osteocytogenesis and the formation of osteocyte dendrites. This dendritic network is critical for bone modelling/remodelling, through the production of receptor activator of nuclear factor κ B (RANK)-ligand (RANKL). Despite this, the role of Pdpn in the control of bone remodelling is yet to be established in vivo. Here we utilised bone-specific Pdpn conditional knockout mice (cKO) to examine the role of Pdpn in the bone loss associated with ovariectomy (OVX). MicroCT revealed that Pdpn deletion had no significant effect on OVX-induced changes in trabecular microarchitecture. Significant differences between genotypes were observed in the trabecular pattern factor (P<0.01) and structure model index (P<0.01). Phalloidin staining of F-actin revealed OVX to induce alterations in osteocyte morphology in both wild-type (WT) and cKO mice. Histological analysis revealed an expected significant increase in osteoclast number in WT mice (P<0.01, compared with sham). However, cKO mice were protected against such increases in osteoclast number. Consistent with this, serum levels of the bone resorption marker Ctx were significantly increased in WT mice following OVX (P<0.05), but were unmodified by OVX in cKO mice. Gene expression of the bone remodelling markers Rank, Rankl, Opg and Sost were unaffected by Pdpn deletion. Together, our data suggest that an intact osteocyte dendritic network is required for sustaining osteoclast formation and activity in the oestrogen-depleted state, through mechanisms potentially independent of RANKL expression. This work will enable a greater understanding of the role of osteocytes in bone loss induced by oestrogen deprivation.

A miRNA-145/TGF-ß1 negative feedback loop regulates the cancer-associated fibroblast phenotype.

The dissemination of cancer cells to local and distant sites depends on a complex and poorly understood interplay between malignant cells and the cellular and non-cellular components surrounding them, collectively termed the tumour microenvironment. One of the most abundant cell types of the tumour microenvironment is the fibroblast, which becomes corrupted by locally derived cues such as TGF-ß1 and acquires an altered, heterogeneous phenotype (cancer-associated fibroblasts, CAF) supportive of tumour cell invasion and metastasis. Efforts to develop new treatments targeting the tumour mesenchyme are hampered by a poor understanding of the mechanisms underlying the development of CAF. Here, we examine the contribution of microRNA to the development of experimentally-derived CAF and correlate this with changes observed in CAF derived from tumours. Exposure of primary normal human fibroblasts to TGF-ß1 resulted in the acquisition of a myofibroblastic CAF-like phenotype. This was associated with increased expression of miR-145, a miRNA predicted in silico to target multiple components of the TGF-ß signalling pathway. miR-145 was also overexpressed in CAF derived from oral cancers. Overexpression of miR-145 blocked TGF-ß1-induced myofibroblastic differentiation and reverted CAF towards a normal fibroblast phenotype. We conclude that miR-145 is a key regulator of the CAF phenotype, acting in a negative feedback loop to dampen acquisition of myofibroblastic traits, a key feature of CAF associated with poor disease outcome.

E11/Podoplanin Protein Stabilization Through Inhibition of the Proteasome Promotes Osteocyte Differentiation in Murine in Vitro Models.

The transmembrane glycoprotein E11 is considered critical in early osteoblast-osteocyte transitions (osteocytogenesis), however its function and regulatory mechanisms are still unknown. Using the late osteoblast MLO-A5 cell line we reveal increased E11 protein/mRNA expression (P < 0.001) concomitant with extensive osteocyte dendrite formation and matrix mineralization (P < 0.001). Transfection with E11 significantly increased mRNA levels (P < 0.001), but immunoblotting failed to detect any correlative increases in E11 protein levels, suggestive of post-translational degradation. We found that exogenous treatment of MLO-A5 and osteocytic IDG-SW3 cells with 10 µM ALLN (calpain and proteasome inhibitor) stabilized E11 protein levels and induced a profound increase in osteocytic dendrite formation (P < 0.001). Treatment with other calpain inhibitors failed to promote similar osteocytogenic changes, suggesting that these effects of ALLN rely upon its proteasome inhibitor actions. Accordingly we found that proteasome-selective inhibitors (MG132/lactacystin/ Bortezomib/Withaferin-A) produced similar dose-dependent increases in E11 protein levels in MLO-A5 and primary osteoblast cells. This proteasomal targeting was confirmed by immunoprecipitation of ubiquitinylated proteins, which included E11, and by increased levels of ubiquitinylated E11 protein upon addition of the proteasome inhibitors MG132/Bortezomib. Activation of RhoA, the small GTPase, was found to be increased concomitant with the peak in E11 levels and its downstream signaling was also observed to promote MLO-A5 cell dendrite formation. Our data indicate that a mechanism reliant upon blockade of proteasome-mediated E11 destabilization contributes to osteocytogenesis and that this may involve downstream targeting of RhoA. This work adds to our mechanistic understanding of the factors regulating bone homeostasis, which may lead to future therapeutic approaches.

Factors affecting the anthelmintic efficacy of papaya latex in vivo: host sex and intensity of infection.

The development of plant-derived cysteine proteinases, such as those in papaya latex, as novel anthelmintics requires that the variables affecting efficacy be fully evaluated. Here, we conducted two experiments, the first to test for any effect of host sex and the second to determine whether the intensity of the worm burden carried by mice would influence efficacy. In both experiments, we used the standard C3H mouse reference strain in which papaya latex supernatant (PLS) consistently shows >80 % reduction in Heligmosomoides bakeri worm burdens, but to broaden the perspective, we also included for comparison mice of other strains that are known to respond more poorly to treatment with papaya latex. Our results confirmed that there is a strong genetic influence affecting efficacy of PLS in removing adult worm burdens. However, there was no effect of host sex on efficacy (C3H and NIH) and no effect of infection intensity (C3H and BALB/c). These results offer optimism that plant-derived cysteine proteinases (CPs), such as these from papaya latex, can function as effective anthelmintics, with neither host sex nor infection intensity presenting further hurdles to impede their development for future medicinal and veterinary usage.

Host genetic influences on the anthelmintic efficacy of papaya-derived cysteine proteinases in mice.

Eight strains of mice, of contrasting genotypes, infected with Heligmosomoides bakeri were studied to determine whether the anthelmintic efficacy of papaya latex varied between inbred mouse strains and therefore whether there is an underlying genetic influence on the effectiveness of removing the intestinal nematode. Infected mice were treated with 330 nmol of crude papaya latex or with 240 nmol of papaya latex supernatant (PLS). Wide variation of response between different mouse strains was detected. Treatment was most effective in C3H (90·5-99·3% reduction in worm counts) and least effective in CD1 and BALB/c strains (36·0 and 40·5%, respectively). Cimetidine treatment did not improve anthelmintic efficacy of PLS in a poor drug responder mouse strain. Trypsin activity, pH and PLS activity did not differ significantly along the length of the gastro-intestinal (GI) tract between poor (BALB/c) and high (C3H) drug responder mouse strains. Our data indicate that there is a genetic component explaining between-mouse variation in the efficacy of a standard dose of PLS in removing worms, and therefore warrant some caution in developing this therapy for wider scale use in the livestock industry, and even in human medicine.

Cysteine proteinases from papaya (Carica papaya) in the treatment of experimental Trichuris suis infection in pigs: two randomized controlled trials.

BACKGROUND: Cysteine proteinases (CPs) from papaya (Carica papaya) possess anthelmintic properties against human soil-transmitted helminths (STH, Ascaris lumbricoides, Trichuris trichiura and hookworm), but there is a lack of supportive and up-to-date efficacy data. We therefore conducted two randomized controlled trials in pigs to assess the efficacy of papaya CPs against experimental infections with T. suis. METHODS: First, we assessed efficacy by means of egg (ERR) and adult worm reduction rate (WRR) of a single-oral dose of 450 µmol active CPs (CP450) against low (inoculum of 300 eggs) and high (inoculum of 3,000 eggs) intensity T. suis infections and compared the efficacy with those obtained after a single-oral dose of 400 mg albendazole (ALB). In the second trial, we determined and compared the efficacy of a series of CP doses (45 [CP45], 115 [CP115], 225 [CP225], and 450 [CP450] µmol) against high intensity infections. RESULTS: CP450 was highly efficacious against both levels of infection intensity, resulting in ERR and WRR of more than 97%. For both levels of infection intensity, CP450 was significantly more efficacious compared to ALB by means of WRR (low infection intensity: 99.0% vs. 39.0%; high infection intensity; 97.4% vs. 23.2%). When the efficacy was assessed by ERR, a significant difference was only observed for high intensity infections, CP450 being more efficacious than ALB (98.9% vs. 59.0%). For low infection intensities, there was no significant difference in ERR between CP450 (98.3%) and ALB (64.4%). The efficacy of CPs increased as a function of increasing dose. When determined by ERR, the efficacy ranged from 2.1% for CP45 to 99.2% for CP450. For WRR the results varied from -14.0% to 99.0%, respectively. Pairwise comparison revealed a significant difference in ERR and WRR only between CP45 and CP450, the latter being more efficacious. CONCLUSIONS: A single dose of 450 µmol CPs provided greater efficacy against T. suis infections in pigs than a single-oral dose of 400 mg ALB. Although these results highlight the possibility of papaya CPs for controlling human STH, further development is needed in order to obtain and validate an oral formulation for human application.

Anti-angiogenic properties of ADAMTS-4 in vitro.

Angiogenesis is an indispensable mechanism in development and in many pathologies, including cancer, synovitis and aberrant wound healing. Many angiogenic stimulators and inhibitors have been investigated, and some have progressed to the clinic. A disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) is a group of multifunctional proteinases. ADAMTS-1 and ADAMTS-8 have been reported to be anti-angiogenic. Here, we provide evidence that ADAMTS-4, like ADAMTS-1, is expressed by endothelial cells and binds to vascular endothelial groth factor (VEGF). Moreover, ADAMTS-4 inhibited human dermal microvascular endothelial cells (HuDMEC) VEGF-stimulated VEGF receptor (R) R2 phosphorylation, differentiation and migration, suggesting that ADAMTS-4 may be a novel anti-angiogenic molecule.

Glycosaminoglycan (GAG) binding surfaces for characterizing GAG-protein interactions.

Glycosaminoglycans play an important role in tissue organisation through interactions with a diverse range of proteins, growth factors and other chemokines. In this report, we demonstrate the GAG-binding 'fingerprint' of two important GAG-binding proteins - osteoprotogerin and TIMP-3. The technique uses a straightforward method for attaching GAGs to assay surfaces in a non-covalent manner using plasma polymerization that leaves the adsorbed GAG able to participate in subsequent ligand binding. We show that OPG and TIMP-3 bind preferentially to different GAGs in a simple ELISA and that this binding does not correlate directly with simple GAG properties such as degree of sulfation. The methods outlined in this report can be easily applied to tissue engineering scaffolds in order to exploit the potential of surface-bound GAGs in influencing the structure of engineered tissues.

Oral dosing with papaya latex is an effective anthelmintic treatment for sheep infected with Haemonchus contortus.

BACKGROUND: The cysteine proteinases in papaya latex have been shown to have potent anthelmintic properties in monogastric hosts such as rodents, pigs and humans, but this has not been demonstrated in ruminants. METHODS: In two experiments, sheep were infected concurrently with 5,000 infective larvae of Haemonchus contortus and 10,000 infective larvae of Trichostrongylus colubriformis and were then treated with the supernatant from a suspension of papaya latex from day 28 to day 32 post-infection. Faecal egg counts were monitored from a week before treatment until the end of the experiment and worm burdens were assessed on day 35 post-infection. RESULTS: We found that the soluble fraction of papaya latex had a potent in vivo effect on the abomasal nematode H. contortus, but not on the small intestinal nematode T. colubriformis. This effect was dose-dependent and at tolerated levels of gavage with papaya latex (117 µmol of active papaya latex supernatant for 4 days), the H. contortus worm burdens were reduced by 98%. Repeated treatment, daily for 4 days, was more effective than a single dose, but efficacy was not enhanced by concurrent treatment with the antacid cimetidine. CONCLUSIONS: Our results provide support for the idea that cysteine proteinases derived from papaya latex may be developed into novel anthelmintics for the treatment of lumenal stages of gastro-intestinal nematode infections in sheep, particularly those parasitizing the abomasum.

Androgen regulates ADAMTS15 gene expression in prostate cancer cells.

Prostate cancer is a major cause of mortality, largely as a consequence of metastases and transformation to androgen-independent growth. Metalloproteinases are implicated in cancer progression. A disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) are expressed in prostate cancer cells, with ADAMTS-1 and ADAMTS-15 being the most abundant. ADAMTS-15 but not ADAMTS-1 expression was downregulated by androgen in LNCaP prostate cancer cells, possibly through androgen response elements associated with the gene. ADAMTS-15 expression is predictive for survival in breast cancer, and the situation may be similar in prostate cancer, as androgen independence is usually due to aberrant signaling through its receptor.

Developing novel anthelmintics from plant cysteine proteinases.

Intestinal helminth infections of livestock and humans are predominantly controlled by treatment with three classes of synthetic drugs, but some livestock nematodes have now developed resistance to all three classes and there are signs that human hookworms are becoming less responsive to the two classes (benzimidazoles and the nicotinic acetylcholine agonists) that are licensed for treatment of humans. New anthelmintics are urgently needed, and whilst development of new synthetic drugs is ongoing, it is slow and there are no signs yet that novel compounds operating through different modes of action, will be available on the market in the current decade. The development of naturally-occurring compounds as medicines for human use and for treatment of animals is fraught with problems. In this paper we review the current status of cysteine proteinases from fruits and protective plant latices as novel anthelmintics, we consider some of the problems inherent in taking laboratory findings and those derived from folk-medicine to the market and we suggest that there is a wealth of new compounds still to be discovered that could be harvested to benefit humans and livestock.

Reactive oxygen species regulate neutrophil recruitment and survival in pneumococcal pneumonia.

RATIONALE: The role of NADPH oxidase activation in pneumonia is complex because reactive oxygen species contribute to both microbial killing and regulation of the acute pulmonary infiltrate. The relative importance of each role remains poorly defined in community-acquired pneumonia. OBJECTIVES: We evaluated the contribution of NADPH oxidase-derived reactive oxygen species to the pathogenesis of pneumococcal pneumonia, addressing both the contribution to microbial killing and regulation of the inflammatory response. METHODS: Mice deficient in the gp91(phox) component of the phagocyte NADPH oxidase were studied after pneumococcal challenge. MEASUREMENTS AND MAIN RESULTS: gp91(phox)(-/-) mice demonstrated no defect in microbial clearance as compared with wild-type C57BL/6 mice. A significant increase in bacterial clearance from the lungs of gp91(phox)(-/-) mice was associated with increased numbers of neutrophils in the lung, lower rates of neutrophil apoptosis, and enhanced activation. Marked alterations in pulmonary cytokine/chemokine expression were also noted in the lungs of gp91(phox)(-/-) mice, characterized by elevated levels of tumor necrosis factor-alpha, KC, macrophage inflammatory protein-2, monocyte chemotactic protein-1, and IL-6. The greater numbers of neutrophils in gp91(phox)(-/-) mice were not associated with increased lung injury. Levels of neutrophil elastase in bronchoalveolar lavage were not decreased in gp91(phox)(-/-) mice. CONCLUSIONS: During pneumococcal pneumonia, NADPH oxidase-derived reactive oxygen species are redundant for host defense but limit neutrophil recruitment and survival. Decreased NADPH oxidase-dependent reactive oxygen species production is well tolerated and improves disease outcome during pneumococcal pneumonia by removing neutrophils from the tight constraints of reactive oxygen species-mediated regulation.

In vitro anthelmintic effects of cysteine proteinases from plants against intestinal helminths of rodents.

Infections with gastrointestinal (GI) nematodes are amongst the most prevalent worldwide, especially in tropical climates. Control of these infections is primarily through treatment with anthelmintic drugs, but the rapid development of resistance to all the currently available classes of anthelmintic means that alternative treatments are urgently required. Cysteine proteinases from plants such as papaya, pineapple and fig are known to be substantially effective against three rodent GI nematodes, Heligmosomoides polygyrus, Trichuris muris and Protospirura muricola, both in vitro and in vivo. Here, based on in vitro motility assays and scanning electron microscopy, we extend these earlier reports, demonstrating the potency of this anthelmintic effect of plant cysteine proteinases against two GI helminths from different taxonomic groups - the canine hookworm, Ancylostoma ceylanicum, and the rodent cestode, Rodentolepis microstoma. In the case of hookworms, a mechanism of action targeting the surface layers of the cuticle indistinguishable from that reported earlier appears to be involved, and in the case of cestodes, the surface of the tegumental layers was also the principal location of damage. Hence, plant cysteine proteinases have a broad spectrum of activity against intestinal helminths (both nematodes and cestodes), a quality that reinforces their suitability for development as a much-needed novel treatment against GI helminths of humans and livestock.

The roles of proteolytic enzymes in the development of tumour-induced bone disease in breast and prostate cancer.

Tumour-induced bone disease is a common clinical feature of advanced breast and prostate cancer and is associated with considerable morbidity for the affected patients. Our understanding of the molecular mechanisms underlying the development of bone metastases is incomplete, but proteolytic enzymes are implicated in a number of processes involved in both bone metastasis and in normal bone turnover, including matrix degradation, cell migration, angiogenesis, tumour promotion and growth factor activation. Malignant as well as non-malignant cells in the primary and secondary sites express these enzymes, the activity of which may be regulated by soluble factors, cell- or matrix-associated components, as well as a number of cell signalling pathways. A number of secreted and cell surface-associated proteolytic enzymes are implicated in tumour-induced bone disease, including the matrix metalloproteinases, lysosomal cysteine proteinases and plasminogen activators. This review will introduce the role of proteolytic enzymes in normal bone turnover and give an overview of the studies in which their involvement and regulation in the development of bone metastases in breast and prostate cancer has been described. The results from trials involving protease inhibitors in clinical development will also be briefly discussed.

Human gastrointestinal nematode infections: are new control methods required?

Gastrointestinal (GI) nematode infections affect 50% of the human population worldwide, and cause great morbidity as well as hundreds of thousands of deaths. Despite modern medical practices, the proportion of the population infected with GI nematodes is not falling. This is due to a number of factors, the most important being the lack of good healthcare, sanitation and health education in many developing countries. A relatively new problem is the development of resistance to the small number of drugs available to treat GI nematode infections. Here we review the most important parasitic GI nematodes and the methods available to control them. In addition, we discuss the current status of new anthelmintic treatments, particularly the plant cysteine proteinases from various sources of latex-bearing plants and fruits.

Potentiation of TLR4 signalling by plasmin activity.

The potential for proteases to regulate mammalian TLR signalling is controversial. We found that inhibition of extracellular serine proteases did not reduce activation of TLR4, but observed that the protease plasmin, an important fibrinolytic plasma enzyme that also exerts proinflammatory functions in monocytes, potentiated TLR2 and TLR4 signalling in RAW264.7 macrophages. Plasmin enhanced endogenous production of TNFalpha and activation of an NF-kappaB reporter plasmid. These actions were prevented by inhibition of its proteolytic activity and were not recapitulated by agonists of protease-activated receptors. These studies link fibrinolysis and TLR signalling, identifying further mechanisms potentially involved in activation of innate immunity.

Agonists of toll-like receptor (TLR)2 and TLR4 are unable to modulate platelet activation by adenosine diphosphate and platelet activating factor.

Inappropriate platelet activation is a feature of acute and chronic diseases such as disseminated intravascular coagulation (DIC) and atherosclerosis. Since proinflammatory microbial-derived agonists can be involved in the pathogenesis of these diseases, we examined the potential role of TLR4 (mediating responses to LPS) and TLR2 (which responds to bacterial lipopeptides) in platelet activation. Our data suggested low-level expression of TLR2 and TLR4 on platelets, determined by flow cytometry, and we also observed expression of TLR4 on a megakaryocytic cell line by both flow cytometry and immunohistochemistry. Stimulation of the platelets with the TLR4 agonist LPS, and the synthetic TLR2 agonist Pam3CSK4, resulted in no platelet aggregation, no increase in CD62P surface expression and no increase in the cytosolic concentration of Ca2+. The TLR agonists were also unable to directly activate platelets primed with epinephrine, or pretreated with a low concentration of ADP or PAF. Pretreatment of platelets with LPS or Pam3CSK4 also failed to modulate the platelet response to submaximal concentrations of the classical platelet agonists ADP and PAF. We conclude that the TLR agonists LPS and Pam3CSK4 have no direct effect on platelet activation and that platelet TLRs may be a remnant from megakaryocytes. TLR2 and TLR4 agonists are thought to have a significant role in diseases such as atherosclerosis and DIC, but our research suggests that this is through a mechanism other than direct platelet activation or by modification of platelet responses to other agonists.

Inhibition of interleukin-1beta-stimulated collagenase and stromelysin expression in human tendon fibroblasts by epigallocatechin gallate ester.

The medicinal benefits of green tea (Camellia sinensis) consumption have been attributed to bioavailable polyphenols, notably epigallocatechin gallate (EGCG). We have assessed the effects of EGCG and its non-esterified counterpart EGC on the expression of the collagenases, matrix metalloproteinases (MMP)-1 and -13, and the stromelysin, MMP-3, in human tendon-derived fibroblasts. Interleukin (IL)-1beta increased MMP-1, -3 and -13 mRNA and output at least 30-fold. EGCG reduced this stimulation, by 20-30% at 2.5 microM and more than 80% at 25 microM, and had a smaller effect on MMP-2 mRNA expression, which was not stimulated by IL-1beta. In all experiments EGCG was at least 10-fold more potent than EGC. EGCG reduced the stimulation of p54 JNK/SAPK phosphorylation by IL-1beta but did not affect p38 MAPK phosphorylation, the degradation of IkappaB or the activating phosphorylation of NFkappaB. We conclude that EGCG reduces the IL-1-stimulated expression of both collagenase and stromelysin mRNA species, an effect which may be mediated by inhibition of the JNK/SAPK pathway. Taken together with previous reports of EGCG effects on the expression and/or activity of gelatinases and aggrecanases, our results underline the importance of extracellular matrix breakdown as a potential target for the actions of green tea polyphenols.