Explore the vast range of titles available.

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. Graphical Abstract

Background Subchondral bone (SCB) thickening is one of the earliest detectable changes in osteoarthritic joints and is considered a potential trigger for subsequent articular cartilage degeneration. In this manuscript, we examine whether disruption to the SCB osteocyte network contributes to the initiation and pathogenesis of osteoarthritis. Methods We examined expression patterns of the glycoprotein E11/podoplanin by immunohistochemical labelling in murine, human and canine osteoarthritis models. We also examined the effects of twice-weekly administration of Bortezomib, a proteasome inhibitor which stabilises osteocyte E11 levels, to C57/BL6 wild-type male mice (1 mg/kg/day) for 8 weeks after surgical destabilisation of the medial meniscus. By inducing osteoarthritis-like changes in the right knee joint of 12-week-old male E11 hypomorphic mice (and corresponding controls) using a post-traumatic joint loading model, we also investigated whether a bone-specific E11 deletion in mice increases joint vulnerability to osteoarthritis. Articular cartilage degradation and osteophyte formation were assessed by histology and in line with the OARSI grading system. Results Our studies reveal increased E11 expression in osteocytes of human and canine osteoarthritic SCB. We found that Bortezomib administration had no effect on surgically-induced osteoarthritis, potentially due to a lack of the expected stabilisation of E11 in the SCB. We also found, in concordance with our previous work, wild-type mice exhibited significant load-induced articular cartilage lesions on the lateral femoral condyle ( p  < 0.01) and osteophyte formation. In contrast, E11 hypomorphic mice did not develop osteophytes or any corresponding articular lesions. Conclusions Overall, these data suggest that an intact osteocyte network in the SCB contributes to the development of mechanically-driven osteoarthritis. Further, the data presented here indicate that the molecular pathways that preserve the osteocyte network, such as those driven by E11, may be targeted to limit osteoarthritis pathogenesis.

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.

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.

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.

Airway remodelling is a feature of chronic asthma comprising smooth muscle hypertrophy and deposition of extracellular matrix (ECM) proteins. Matrix metalloproteinases (MMPs) breakdown ECM, are involved in tissue remodelling and have been implicated in airway remodelling. Although mesenchymal cells are an important source of MMPs, little data are available on airway smooth muscle (ASM) derived MMPs. We therefore investigated MMP and tissue inhibitor of metalloproteinase (TIMP) production and activity in human ASM cells. MMPs and TIMPs were examined using quantitative real‐time RT–PCR, Western blotting, zymography and a quench fluorescence (QF) assay of total MMP activity. The most abundant MMPs were pro‐MMP‐2, pro‐ MMP‐3, active MMP‐3 and MT1‐MMP. TIMP‐1 and TIMP‐2 expression was low in cell lysates but high in conditioned medium. High TIMP secretion was confirmed by the ability of ASM‐conditioned medium to inhibit recombinant MMP‐2 in a QF assay. Thrombin increased MMP activity by activation of pro‐MMP‐2 independent of the conventional smooth muscle thrombin receptors PAR 1 and 4. In conclusion, ASM cells express pro‐MMP‐2, pro and active MMP‐3, MMP‐9 and MT1‐MMP. Unstimulated cells secrete excess TIMP 1 and 2, preventing proteolytic activity. MMP‐2 can be activated by thrombin which may contribute to airway remodelling. British Journal of Pharmacology (2004) 142, 1318–1324. doi:10.1038/sj.bjp.0705883

Abstract 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 gp91phox component of the phagocyte NADPH oxidase were studied after pneumococcal challenge. Measurements and Main Results gp91phox−/− 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 gp91phox−/− 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 gp91phox−/− mice, characterized by elevated levels of tumor necrosis factor-α, KC, macrophage inflammatory protein-2, monocyte chemotactic protein-1, and IL-6. The greater numbers of neutrophils in gp91phox−/− mice were not associated with increased lung injury. Levels of neutrophil elastase in bronchoalveolar lavage were not decreased in gp91phox−/− 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.

Living skin-equivalent grafts consisting of fibroblasts cast in collagen lattices and seeded with epidermal cells were successfully grafted onto the donors of the cells. The grafts were vascularized, did not evoke a homograft reaction, inhibited wound contraction, filled the wound space, and persisted.

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.

Magnetic methods offer rapid non‐destructive in‐situ measurement of engineering residual stress, applicable in industrial environments, though limited to ferromagnetic materials. The technology can be incorporated into permanent monitors, scanners and hand held devices for use on components and structures. These benefits are tempered by the burden of calibration for the target material(s), the magneto‐elastic response yielding a non‐linear tensor relationship between stress and measured properties. This chapter appraises the Magnetic and Acoustic Barkhausen Noise and the MAPS methods. The measured spatial resolution and depth are reviewed and significant attention is paid to all aspects influencing ultimate accuracy such as material, magnetic and stress history, probe stand‐off and temperature before giving examples. Finally some application examples are given.