Plant-nematode interactions: environmental signals detected by the nematode's chemosensory organs control changes in the surface cuticle and behaviour.

Plant parasitic nematodes have developed the capacity to sense and respond to chemical signals of host origin and the ability to orientate towards plant roots enhances the nematode's chance of survival. Root exudates contain a range of compounds which mediate belowground interactions with pathogenic and beneficial soil organisms. Chemical components of root exudates may deter one organism while attracting another and these compounds alter nematode behaviour and can either attract nematodes to the roots or result in repellence, motility inhibition or even death. In vitro, plant signals present in root exudates, trigger a rapid alteration of the surface cuticle of Meloidogyne incognita and the same changes were also induced by indole-acetic acid (IAA). IAA binds to the chemosensory organs of M. incognito and it is possible that IAA acts as a signal that orientates the nematode on the root surface in the rhizosphere and/or inside the root tissue and thereby promotes nematode infection.

Nematicidal effects of cysteine proteinases against sedentary plant parasitic nematodes.

Cysteine proteinases from the fruit and latex of plants, such as papaya, pineapple and fig, have previously been shown to have substantial anthelmintic efficacy, in vitro and in vivo, against a range of animal parasitic nematodes. In this paper, we describe the in vitro effects of these plant extracts against 2 sedentary plant parasitic nematodes of the genera Meloidogyne and Globodera. All the plant extracts examined caused digestion of the cuticle and decreased the activity of the tested nematodes. The specific inhibitor of cysteine proteinases, E-64, blocked this activity completely, indicating that it was essentially mediated by cysteine proteinases. In vitro, plant cysteine proteinases are active against second-stage juveniles of M. incognita and M. javanica, and some cysteine proteinases also affect the second-stage juveniles of Globodera rostochiensis. It is not known yet whether these plant extracts will interfere with, or prevent invasion of, host plants.

Characterization of Meloidogyne javanica surface coat with antibodies and their effect on nematode behaviour.

The surface coat of the 2nd-stage juveniles (J2) of plant-parasitic nematodes is considered to be involved in interactions with microorganisms in the soil and rhizosphere, as well as with the host plant. Characterization of surface antigens might be important in the development of new nematode control strategies. In this study, polyclonal and monoclonal antibodies raised against Meloidogyne javanica, M. incognita and other plant-parasitic nematodes were tested for their binding to the surface coat and secreted-excreted products of M. javanica. Some of the monoclonal and polyclonal antibodies raised against M. incognita showed cross-reactivity with the surface coat of M. javanica. Western blot analysis of M. javanica surface coat extracts revealed labelling of several polypeptides with a 48 kDa main band for the polyclonal antibody IACR-PC Mi 373, and a 55 kDa main band for PC Mj E2. Further characterization of the antigens recognized by the polyclonal antibody PC Mj E2, in planta, showed that they were present in the parasitic stages J2 and J3 and that the surface coat was shed during root penetration. The hypodermis of the infective juveniles was labelled by PC Mj E2 and the monoclonal antibody IACR-Misec 3F.4, suggesting that these surface antigens are produced in the hypodermis. Nematode behaviour was affected by all the antibodies that bound to the surface coat of the pre-parasitic J2, and we demonstrated that the movement pattern of the M. javanica J2 was affected by these antibodies. Continuous binding of the antibodies to the M. javanica surface inhibited the infection of Arabidopsis thaliana roots on agar plates.

Characterisation of a collagen gene subfamily from the potato cyst nematode Globodera pallida.

We have isolated two full-length genomic DNA sequences, which encode the cuticle collagen proteins GP-COL-1 and GP-COL-2, from the potato cyst nematode Globodera pallida. A third, partial collagen gene ORF termed gp-col-t(t=truncated) has also been isolated and appears to represent an unexpressed pseudogene. The gp-col-1 and gp-col-2 genes both contain three short (<97 bp) introns which disrupt coding regions predicted to specify proteins with molecular weights of 33 and 32.7 kDa respectively. All three sequences show high similarity to each other and to the previously isolated G. pallida cDNA clone gp-col-8. The conserved pattern of cysteine residues and non-(Gly-X-Y)(n) region sequence similarity observed in all four G. pallida genes suggests that these molecules form part of the same subfamily of collagens. Southern analysis indicates that this subfamily is likely to contain further members. The G. pallida collagen sequences show striking similarity to twelve genes from Caenorhabditis elegans which collectively represent the recently classified Group 1a collagen subfamily. No data exists on the function of this subfamily in C. elegans. gp-col-1 and gp-col-2 are developmentally regulated with transcripts of both genes detected in adult virgin and gravid females but not in pre-parasitic second stage juveniles. A similar expression pattern is observed for the Group 1a collagen lemmi 5 from Meloidogyne incognita perhaps indicating a generic link between subfamily and function during the various changes in cuticular structure which accompany nematode growth and reproduction. Immunochemical studies indicate that the GP-COL-1 protein is specifically located in the hypodermis of G. pallida adult females.

Identification and characterization of excreted-secreted products and surface coat antigens of animal and plant-parasitic nematodes.

Nematode surface coat (SC) proteins and excreted-secreted products (E-S) are likely to play important roles in the host-parasite interaction and considerable similarities can be found in SC proteins and E-S products from certain plant and animal parasitic nematodes. Monoclonal antibodies raised to E-S products of plant-parasitic nematodes were shown to cross-react with E-S products and the surface coats of the animal parasites Trichinella spiralis and Haemonchus contortus. Most of the antibodies recognized carbohydrate epitopes but the activity of 2 MAbs (IACR-CCNj.2a.15 and IACR-Misec.8D.3) which recognized proteic epitopes in these nematodes were further characterized. Antibody 2a.15 recognized the SC and oral exudate of Meloidogyne incognita, T. spiralis and H. contortus. This antigen was also immunolocalized in the lining of the oesophagus and gut and in the exudate present during ecdysis of H. contortus L3. Antibody 8D.3 reacted with the SC of these nematodes on cryosections but on live nematodes the immunofluorescence was very patchy and was shed from the nematode SC.

Health of the Nation Outcome Scales (HoNOS). Research and development.

BACKGROUND: An instrument was required to quantify and thus potentially measure progress towards a Health of the Nation target, set by the Department of Health, "to improve significantly the health and social functioning of mentally ill people". METHOD: A first draft was created in consultation with experts and on the basis of literature review. This version was improved during four stages of testing: two preliminary stages, a large field trial involving 2706 patients (rated by 492 clinicians) and tests of the final Health of the Nation Outcome Scales (HoNOS), which included an independent study (n = 197) of reliability and relationship to other instruments. RESULTS: The resulting 12-item instrument is simple to use, covers clinical problems and social functioning with reasonable adequacy, has been generally acceptable to clinicians who have used it, is sensitive to change or the lack of it, showed good reliability in independent trials and compared reasonably well with equivalent items in the Brief Psychiatric Rating Scales and Role Functioning Scales. CONCLUSIONS: The key test for HoNOS is that clinicians should want to use it for their own purposes. In general, it has passed that test. A further possibility, that HoNOS data collected routinely as part of a minimum data set, for example for the Care Programme Approach, could also be useful in anonymized and aggregated form for public health purposes, is therefore testable but has not yet been tested.

Protocol for assessing services for people with severe mental illness.

BACKGROUND: The Clinical Standards Advisory Group was asked by UK health ministers to advise on the standards of clinical care being achieved for people with schizophrenia. A subcommittee commissioned a review of standards, followed by research into how far these were reflected in contracts and met by providers. METHOD: No comprehensive but practical set of standards was found. A protocol of 143 items of good service practice was constructed, and applied by teams visiting services in II UK districts. The team appraisals were summarised in 20 key points, each scored 0 (absent) to 4 (excellent performance). Seven points were used to assess standards of commissioning and 13 for standards of service provision. RESULTS: When placed into rank order, the mean key point scores for commissioners and providers in the same district tended to be very similar. Total district scores were then used to assign districts to one of three groups. Four performed reasonably well, five were moderate and two were poor. CONCLUSIONS: One of the key elements associated with these differences was the local level of morale. After wide consultation, a revised protocol of 26 key points for direct rating was drawn up and has since been further tested.

Schistosoma mansoni: anomalous immunogenic properties of a 27 kDa larval serine protease associated with protective immunity.

A cationic Schistosoma mansoni cercarial antigen was shown to be a serine protease as it was capable of hydrolysing N-acetyl-DL-phenylalanine beta-naphthyl ester (NAPBNE) after precipitation by immunoelectrophoresis, and this reaction was modulated by the serine protease inhibitors phenylmethanesulfonyl fluoride (PMSF) and diisopropylfluorophosphate (DEP). The antigen in the immunoprecipitin arcs could also be radio-isotope labelled with tritiated DFP. The peptidolytic enzyme identified in immunoelectrophoresis with polyspecific sera and radio-isotope labelled with tritiated DFP had a relative molecular size of approximately 27 kDa in sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), and evidence obtained after partial purification, SDS-PAGE and immunoblotting supported this size estimate for the enzyme. A rabbit antiserum raised against the peptidolytic antigen reacted against a doublet of antigens at 27/28 kDa in immunoelectrophoresis arcs and against an antigen of 60 kDa in Western immunoblots of crude cercarial homogenate. However, the latter serum precipitated the cationic antigen in immunoelectrophoresed cercarial homogenates only after pre-incubation of the homogenates with PMSF. Fractions containing the partially purified protease also degraded radio-isotope labelled human IgG. The reactivity of a range of polyspecific and monospecific rabbit antisera in Western blots with larval extracts indicated that antibody responses against the 27/28 kDa doublet may be modulated. When immunized with material which contained the 27 kDa enzyme as a major constituent, and which was secreted by S. mansoni cercariae during transformation, only 5 of 16 mice produced antibody to this antigen that was detectable in Western blots. The 5 antibody 'responder' mice were significantly (P < 0.001) protected against challenge with a percutaneous infection of S. mansoni cercariae compared with a group of a mice also immunized with CTF, but which had not produced antibodies against the 27/28 kDa doublet. The results indicate that the 27 kDa serine protease of S. mansoni larvae is a target that is sensitive to immunological attack.

Isolation and characterization of a putative collagen gene from the potato cyst nematode Globodera pallida.

A cDNA clone encoding a full length putative collagen has been isolated in a screen of a mixed stage Globodera pallida expression library. Comparison of the deduced amino acid sequence of this molecule with other collagens suggests it is a cuticular collagen and a member of the col-8 subfamily of collagen genes. Northern blots show the gene is expressed specifically in gravid, adult females of the parasite as compared to second (invasive) stage juveniles and virgin females. Preliminary immunocytochemical studies indicate this collagen is present in areas other than the cuticle; these findings and the potential functional role of this collagen are discussed.

Identification and in situ and in vitro characterization of secreted proteins produced by plant-parasitic nematodes.

Secretions of plant-parasitic nematodes which are released into plant tissue may play critical roles in plant-nematode interactions. The identification and characterization of these molecules are of fundamental importance and may help to facilitate the development of novel strategies to interfere with nematode infection of plants and thereby decrease nematode-induced damage to crops. An antibody-based approach was used to isolate molecules present on the nematode surface and in nematode secretions. Monoclonal antibodies (MAbs) were produced to secretions and to whole Heterodera avenae 2nd-stage juveniles; several of these MAbs recognized molecules present in nematode secretions produced in vitro. Three of these molecules have been partly characterized in H. avenae, Globodera rostochiensis, G. pallida and Meloidogyne incognita. A MAb reacting with the surfaces of these nematodes recognized antigens of different molecular weight in each of the species tested. This difference in antigenicity might be related to specific functions in these nematodes. Preliminary results show that this antibody also localized the antigen in root cells surrounding the feeding site induced by M. incognita in Arabidopsis thaliana.

Sm480: a high molecular weight Schistosoma mansoni antigen associated with protective immunity.

Rabbit antisera were raised against an antigen present in Schistosoma mansoni adult worms and eggs, and were shown to yield a single immunoprecipitin arc in immunoelectrophoresis and immunodiffusion against S. mansoni egg and worm antigen extracts. The antisera conferred partial but significant protection (22-30%) against a S. mansoni challenge when transferred to mice five and six days after the mice had been infected percutaneously with 200 cercariae. The egg and the worm forms of the antigen were immunologically cross-reactive, but the egg antigen possessed peptidolytic activity that could be inhibited with serine protease inhibitors. In indirect immunofluorescence the rabbit antisera reacted with surfaces of cercariae, five-day old lung-stage schistosomula, miracidia and praziquantel-treated adult worms. Gel-filtration chromatography demonstrated a relative molecular size of approximately 480 kDa for both the egg and worm forms of the antigen, and lectin-affinity chromatography indicated both were glycosylated. The serine protease activity and large relative molecular size of egg Sm480 were confirmed by a combination of radiolabelling with tritiated di-isopropyl fluorophosphate, immunoelectrophoresis and polyacrylamide gel electrophoresis.

Proteases in the schistosome life cycle: a paradigm for tumour metastasis.

Cancers and parasites have a number of properties in common, particularly those that relate to their respective capacities to evade host defence mechanisms. This review highlights the similarities between metastatic tumours and schistosomes in particular, and describes the role that proteases may have in the migration, growth, survival and transmission of the different stages of the schistosome life-cycle in the vertebrate host. An elastase-like serine protease of schistosome larvae has been particularly well characterized, and its substrate profile and other properties are indicative of a role in facilitating migration of the parasite through skin tissue early after infection. The primary structures of a cathepsin B-like enzyme, and a putative 'haemoglobinase' found in adult worms have also recently been derived, these enzymes being responsible for degradation of haemoglobin in erythrocytes upon which the adults feed. Adult schistosome worms reside and produce eggs intravascularly, and the processes that mediate the extravasation and subsequent migration of the egg through host tissue are dependent on both blood platelets and the immune response. Fibrino(geno)lytic enzymatic activity that is present in the egg could modulate the thrombogenic potential that eggs might have as a result of their capacity to cause platelet aggregation in vitro and in vivo. The roles of other proteases and peptidases that have been found in schistosome larvae, worms and eggs are less clear. Some of these enzymes may modulate immunological and haemostatic defence mechanisms and thus prolong survival of the parasite, and the consequences of the interactions between schistosomes and host protease inhibitors could also be immune modulatory.

Immunological identification of Schistosoma mansoni peptidases.

Precipitin arcs formed after immunoelectrophoresis of Schistosoma mansoni egg, worm and cercarial antigens with polyspecific rabbit antisera have been 'stained' at neutral pH with chromogenic enzyme substrates specific for proteases and peptidases. A total of 7 antigenically distinct enzymes with peptidolytic properties were identified. An enzyme which hydrolyzed phenylalanine naphthyl ester bonds was found in adult worm extracts, but appeared to be host-derived in so far as it was also immunoprecipitable from the same extracts by rabbit anti-normal mouse plasma. Three more phenylalanine naphthyl esterases were found, all stage-specific; one was in cercarial homogenates and two were in egg homogenates. A leucine naphthylamidase was also found in egg extracts, and it was antigenically distinct from, and did not react on dipeptide substrates that were hydrolyzed by two other leucine aminopeptidases present in extracts of all three stages of S. mansoni. The method provides a simple means of distinguishing constituents of complex mixtures of antigens by a combination of their immunological and biochemical properties.