Search

E-S 62, a major excretory-secretory product of the rodent filarial parasite, Acanthocheilonema viteae, inhibits the polyclonal activation (DNA synthesis) of mouse B cells by mitogenic anti-Ig antibodies. This effect appears to be due at least in part to the phosphorylcholine (PC) moiety of the molecule, because it can be mimicked by PC-BSA or PC-chloride. Activation of the B cells by LPS is not influenced by the presence of E-S 62/PC, suggesting that they may target some aspect of signaling via the Ag receptor. E-S 62/PC failed to inhibit surface Ig-mediated generation of the second messenger, inositol triphosphate, indicating that their target may not be the early biochemical events associated with activation. Exposure to E-S 62/PC was found to lead to a reduction in the level of protein kinase C, an important downstream regulatory enzyme, in anti-Ig-treated cells. This protein-kinase C down-regulation may be the biochemical mechanism underlying E-S 62/PC-mediated inhibition of surface Ig-activated B cells.

Living adult males and microfilariae of the cattle filarial parasite Onchocerca gibsoni were externally labelled with radioactive iodine using the iodogen and Bolton-Hunter procedures. Characterization of labelled surface proteins by sodium dodecyi sulphate (SDS)-polyaerylamide gel electrophoresis revealed clear cut differences in the two life cycle stages. In addition, the two radiolabelling procedures yielded some differences in the profiles of radiolabelied surface proteins for both adults and microfilariae. Immunoprecipitation analysis revealed a number of labelled antigens recognized by antibodies in human onchocerciasis serum pools, thereby demonstrating the usefulness of O. gibsoni as a model in Onchocerca volvulus vaccine studies. The reactivity ofmicrofilarial antigens extended to antibodies from other human nematode infections, whereas male surface antigens, particularly those of low molecular weight, were Onchocerca specific. This indicates that O. gibsoni can provide a convenient source of specific diagnostic antigen.

SUMMARYJirds (Meriones libycus) were infected with various numbers ofAcanthocheilonema viteaeL3 stage parasites. During the course of the ensuing 16 weeks, blood samples were collected at 2 weekly intervals and the amount of the major parasite excretory–secretory product (E–S 62) and antibodies directed against it measured. After 16 weeks, animals were sacrificed and the size of the mature worm burden established. In spite of interaction between E–S 62 and host antibody, a statistically significant relationship was found to exist between the amount of E–S 62 present in the bloodstream and the size of the parasite load. It is suggested that the detectable antigen level is more influenced by the size of the worm burden than the presence of antibody and that antibody is only likely to affect adversely antigen measurement in situations where the amount released is relatively low. Examples of this are early in infection and in low-level infections. These ideas are discussed in relation to the development and assessment of serological assays which attempt to predict parasite burden in human filarial infections.

Specific, serological diagnosis is one of the main goals in onchocerciasis research. To date this objective has been hampered by (a) scarcity of parasite material, and (b) antigenic cross-reaction between Onchocerca volvulus and other nematode species. In order to obtain specific antigens, and in amounts suitable for study, molecular biological techniques have been adopted. A lambda gt11 cDNA expression library prepared from O. volvulus adult female worms was screened using infected human sera from onchocerciasis patients and rabbit hyperimmune sera raised against Onchocerca and genus-specific Onchocerca antigen extracts. Five clones were selected and their inserts expressed as beta-galactosidase fusion proteins. The fusion proteins were examined using individual sera from patients with O. volvulus or Wuchereria bancrofti infections. Three of the fusion proteins were recognised by more than 80% of O. volvulus sera and exhibited weak reactivity with a few W. bancrofti sera. One of these three clones was recognised to a significantly greater degree by sera from sowda than from generalised onchocerciasis patients.

The biophysical properties of the surface lipid of a range of nematode species and their developmental stages were examined, using fluorescent lipid probes and fluorescence recovery after photobleaching (FRAP). These methods can be applied to living, intact parasites, and the analysis confined to lipid on the outermost surface. In all cases, surface lipid was unusual in its selectivity for the insertion of the lipid probes. In addition, a polar lipid probe was generally not free to diffuse in the plane of the surface, in contrast to a non-polar lipid probe which was free to diffuse. This is evidence that the surface lipid layer is heterogeneous, and possibly comprises lipid domains. The infective larvae of Acanthocheilonema viteae, Nippostrongylus brasiliensis, Trichinella spiralis and Ostertagia ostertagi were found to exhibit a rapid change in lipophilicity upon exposure to conditions simulating entry into a mammalian host environment. Parasitic nematodes, therefore, present their hosts not only with a highly unusual biological surface, but also one which can be rapidly re-organised upon a change of environment.

The longevity of filarial nematodes is dependent on secreted immunomodulatory products. Previous investigation of one such product, ES-62, has suggested a critical role for post-translationally attached phosphorylcholine (PC) moieties. In order to further investigate this, ES-62 lacking PC was produced, using the Pichia pastoris recombinant gene expression system. Unlike parasite-derived ES-62, which is tetrameric the recombinant material was found to consist of a mixture of apparently stable tetramers, dimers and monomers. Nevertheless, the recombinant protein was considered to be an adequate PC-free ES-62 as it was recognized by existing antisera against the parasite-derived protein. However, subsequent to this, recognition of parasite-derived ES-62 by antibodies produced against the recombinant protein was found to be absent. In an attempt to explain this, recombinant ES-62 was subjected to structural analysis and was found to (i) contain 3 changes in amino acid composition; (ii) demonstrate significant alterations in glycosylation; (iii) show major differences in protein secondary structure. The effects of these alterations in relation to the observed change in immunogenicity were investigated and are discussed. The data presented clearly show that recognition by existing antibodies is insufficient proof that recombinant proteins can be used to mimic parasite-derived material in studies on nematode immunology and vaccination.

Meeting abstract on a novel therapeutic approach to cytokine modulation in articular inflammation. Discovering safe, novel immunomodulators that are effective in RA is currently a major therapeutic objective. Long-term immune system deviation is most striking in the host-parasite relationship, in which microbes may coexist with a human host. ES-62 exhibited powerful immunomodulation of CIA, preventing initiation of inflammatory arthritis. Crucially, ES-62 suppressed even established disease. These effects were due to inhibition of cytokine release, specifically TNF-α, and reversal of collagen specific Th1 responses associated with reduced expression of IFN-γ. The physiologic relevance of these observations was confirmed, as ES-62 down-regulated the release of proinflammatory cytokines (TNF-α and IL-6) from patient-derived samples.

Nine monoclonal antibodies (mabs) have been produced against excretory-secretory products (ES) of adult male Onchocerca gibsoni. These mabs fail to interact with the highly cross-reactive phosphorylcholine (PC) group and with ES of the related rodent filarial parasites Acanthocheilonema viteae and Litomosoides carinii. Eight of the mabs are of the IgG isotype: 1 is an IgM. Three of the mabs, OGMES 4, 9, and 10, were each found by immunoprecipitation/SDS-PAGE analysis of [3H] leucine-labeled ES, to recognize a triplet of polypeptides of molecular weight 120, approximately 210, and approximately 260 kDa. No recognition was observed by any mab when [3H] glucosamine was employed as the radiolabel for ES. Western blotting employing [125I] as indicator system demonstrated that OGMES 7 recognized a molecule of 27 kDa, and OGMES 1, a molecule of approximately 210 kDa, albeit faintly. These mabs may be of value to researchers working on the isolation, characterization, and detection in the bloodstream of Onchocerca volvulus ES.

ES-62, a major excretory-secretory (ES) product of Acanthocheilonema viteae, consists of a protein backbone with N-linked carbohydrate and the immunomodulatory group phosphorylcholine (PC); it can, therefore, be biosynthetically labeled with radioactive leucine, glucosamine, or choline. Incubation of worms with tunicamycin results in an ES product whose secretion is partially blocked, which demonstrates reduced molecular weight when employing leucine as radiolabel, and which lacks radioactivity when employing glucosamine or choline as label. Furthermore, the retained ES product can be detected in somatic extracts of parasites exposed to tunicamycin, by its reactivity for antibodies against the whole parasite product but not by antibodies against PC alone. These results support the idea that PC is attached to ES-62 via an N-linked glycan and hence are consistent with the recent observation that PC can be removed from ES-62 by the sugar-cleaving enzyme, N-glycosidase F. The implications of this structural information with respect to designing inhibitors of PC attachment for use as chemotherapeutic agents, and also the advantage of such material in raising antibodies to filarial ES, are discussed.

The endemicity of human onchocerciasis in NorthI Cameroon can be related to the presence of cattle. In the Adamawa highland, an area (if intensive cattle breeding, human onchocerciasis is hypoendemic, although the trans­ mission of O. volvulus infective third-stage larvae is much higher than in the hyperendemic savanna area, where cattle breeding is almost non-existing (Wahl, 1991). Onchocerciasis-associated eye lesions and blindness very rarely occur in the highland, whilst in the savanna region, severe onchocerciasis in the human population is frequent (Renz et al., 1987). Simulium squamosum, the vector of human onchocerciasis in this area also transmits O. ochengi, a cattle filaria, which out-numbers the infective larvae of O. volvulus in man-biting flies by a factor of over ten in the highland area. It is believed, that the ongoing and intensive transmission of this bovine species to man induces cross-protective immunity against forthcoming lar­ vae of O. volvulus (cf. abstracts Renz et al., Wahl et al). In this study we first compared the protein composition of O. ochengi and O. volvulus and then examined the antigen recognition by sera from patients living in the highland (cattle breeding) or savanna area. It was of particular inte­ rest to identify O. ochengi antigens that refer to contact of highland patients with this cattle filaria. Using one- and two dimensional electrophoresis the two filariae showed a high homology. However some speciesspecific proteins could be identified in O. ochengi and O. volvulus extracts, with molecular weights of 32, 6.3, 5.3 kDa and 16, 94, 9.1, 5.6 and 5.1 kDa, respectively. Sera from cattle recognized a major O. ochengi-specific antigen duplett of 28 kl)a, whilst other specific antigens migrated between 10-20 kDa. Using Tricine-SDS-PAGE and immunoblotting human sera identified a possible O. ochengispecific antigen of 7 kDa. Comparison of the two patient groups from the savanna (with high microfilarial density in the skin) and highland (few microfilariae or skin-negative) revealed distinct diffe­ rences in respect to their antigen recognition ; low molecu­ lar weight antigens of both filaria species were recognized

The antigenic cross-reactivity of the excretions-secretions (E-S) of Litomosoides carinii was investigated. Immunoprecipitation using pooled sera from a number of human filarial infections in combination with sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis revealed variations in the cross-reactivity of individual molecules. Some were specific to L. carinii, the major examples in this category being two E-S components of 140 and 160 kDa released by day 40- to 42-day-old female worms. Another, a high-molecular-weight product of 26- to 28-day E-S, was broadly cross-reactive. A third group appeared to exhibit reactivity to antibody to some but not all human filarial parasites. The most striking examples of this were two distinct 14-kDa products that bound solely to antibodies in an onchocerciasis serum pool. These results are discussed in relation to the use of cross-reacting molecules in investigating the immunisation potential, defining the function, and evaluating the diagnostic utility of human filarial E-S.

Antigenic, and hence possible evolutionary, relationships amongst various TPP-dependent non-oxidative α-keto acid decarboxylases were determined by the Ouchterlony double diffusion method and by measuring the degree of antibody-induced enzyme inhibition. The results show that: (a) phenylglyoxylate decarboxylases of various wild-type strains of Acinetobacter calcoaceticus are antigenically indistinguishable; (b) there seems to be no antigenic cross-reactivity between the phenylglyoxylate decarboxylase of A. calcoaceticus and of Pseudomonas aeruginosa or Pseudomonas putida; and (c) no antigenic homology can be detected amongst phenylglyoxylate decarboxylase and phenylpyruvate decarboxylase of A. calcoaceticus and pyruvate decarboxylase of brewers' yeast.

The effects of. a variety of lipophilic compounds on the young stage (schistosomulum) and adult Schistosoma mansoni have been studied by measuring the release of51Cr and125I-labelled wheat-germ agglutinin from labelled parasites. The compounds could be classified into three groups, one of which described reagents which affected only the schistosomulum. It is concluded that during development, changes occur in the organization of the lipid phase of the parasite membrane

Graphical abstract Highlights ► A worm-derived product, ES-62, protects against allergic airway inflammation induced by ovalbumin in mice. ► Protection is associated with resetting of the Th1/Th2 balance and correlates with suppression of Th17 responses. ► The study provides important information on the mechanism of action of a parasitic helminth-derived immunomodulator. ► The immunomodulator offers novel and safe therapeutic potential in the treatment of allergic diseases., We previously demonstrated inhibition of ovalbumin-induced allergic airway hyper-responsiveness in the mouse using ES-62, a phosphorylcholine-containing glycoprotein secreted by the filarial nematode, Acanthocheilonema viteae. This inhibition correlated with ES-62-induced mast cell desensitisation, although the degree to which this reflected direct targeting of mast cells remained unclear as suppression of the Th2 phenotype of the inflammatory response, as measured by eosinophilia and IL-4 levels in the lungs, was also observed. We now show that inhibition of the lung Th2 phenotype is reflected in ex vivo analyses of draining lymph node recall cultures and accompanied by a decrease in the serum levels of total and ovalbumin-specific IgE. Moreover, ES-62 also suppresses the lung infiltration by neutrophils that is associated with severe asthma and is generally refractory to conventional anti-inflammatory therapies, including steroids. Protection against Th2-associated airway inflammation does not reflect induction of regulatory T cell responses (there is no increased IL-10 or Foxp3 expression) but rather a switch in polarisation towards increased Tbet expression and IFNγ production. This ES-62-driven switch in the Th1/Th2 balance is accompanied by decreased IL-17 responses, a finding in line with reports that IFNγ and IL-17 are counter-regulatory. Consistent with ES-62 mediating its effects via IFNγ-mediated suppression of pathogenic Th2/Th17 responses, we found that neutralising anti-IFNγ antibodies blocked protection against airway inflammation in terms of pro-inflammatory cell infiltration, particularly by neutrophils, and lung pathology. Collectively, these studies indicate that ES-62, or more likely small molecule analogues, could have therapeutic potential in asthma, in particular for those subtypes of patients (e.g. smokers, steroid-resistant) who are refractory to current treatments.

Although fairly expensive (around US$4.00 per single dose) praziquantel is now the most favoured drug against all forms of schistosomiasis, and against many other helminth infections. It is now marketed by four companies: E. Merck and Bayer (F.R.G.), Ames-Myers (USA), and Shin-Poon Pharmaceuticals (S. Korea). Administration of praziquantel typically causes paralysis of susceptible worms, or damage to their tegument, making them more vulnerable to host enzymes or antibody-dependent immune effector mechanisms. Other effects may also be involved. Here, Bill Harnett reviews the range of anthelmintic effects displayed by this remarkable drug.

SUMMARYThe class-specific antibody responses of 3 strains of mice (C57/Bl10, BALB/C and CBA/N) known to vary in their ability to control the microfilaraemia which follows the subcutaneous transplantation of adult femaleDipetalonema viteaehas been investigated. The 3 mouse strains showed significant variation (a) in total levels of immunoglobulins and (b) in ability to recognize individual radio-isotope-labelled antigens as measured by coprecipitation. Within each mouse strain it was noted that antigens could vary with respect to the nature of the isotype of the antibody response which they elicited. Furthermore, by comparing results obtained from class-specific coprecipitation with surface ELISA it was found that a similar variation between responses to individual epitopes was also likely. No differences were observed in the humoral response of the 3 mouse strains which could explain the known resistance of the C57/B110 strain; reasons for this are discussed.

A genomic DNA library of a Liberian strain of Onchocerca volvulus was prepared in the vector bacteriophage λgt10. The library was differentially screened by hubridisation with radiolabelled total DNA from the homologous parasite, two heterologous Onchocerco parasites ( Onchocerca gibsoni and Onchocerca gutturosa ) and human liver cells. A clone (C1A1) was isolated whose binding to O. volvulus DNA was at least 50 times stronger than to the other parasite DNA samples. No binding was observed with human DNA. The insert of C1A1 was subcloned into the filamentous phage vector M13 mp18 and sequenced. Two oligonucleotides, each corresponding to a unique region of 60 nucleotides (out of a total of 154) were synthesised and examined for hybridisation with three different geographical isolates of O. volvulus (including forest and savannah strains) and six other Onchocerca spp. One of the oligonucleotides (C1A1-2) was found to hybridise to the three O. volvulus isolates with an intensity in the region of 300 times greater than to any other Onchocerca spp. Since the other species include the two which may be most closely related to O. volvulus , i.e., O. gibsoni and Onchocerca ochengi , it is concluded that C1A1-2 is likely to represent a truly species-specific probe.

It was found that retinol at concentrations of 0.2–1.0 mg · ml −1 caused significant 51 Cr release from schistosomula, while adult worms appeared unaffected. Retinol was shown, by spectrofluorimetry and fluorescence microscopy, to be absorbed into the membrane systems of both schistosomulum and adult worm, particularly when the parasites were incubated in retinol dissolved in non-ionic detergents (Tweens 20, 40 and 80). The retinol within the adult membrane could be induced to cause detectable 51 Cr and 125 I wheat germ agglutinin release if the adult was treated with retinol in combination with Tween 20. The effect of the combination of Tween 20 and retinol, was synergistic for the release of both isotopes. This synergism was also observed when haemolysis of human erythrocytes was measured. Thus it is possible to greatly enhance the effect on the schistosome and the erythrocyte membrane of one membrane-active compound by presenting it in combination with another. This may have implications in chemotherapy when membrane active drugs are employed.

Incubation of adult male Schistosoma mansoni for 24 hr in medium containing newborn calf serum or normal human plasma resulted in an increase in the amount of parasite antigen exposed at the worm surface. No effect was observed on the amount of host antigen which was present. The increase in the exposure of parasite antigens takes place progressively over 24 hr and is partially dependent on the presence of lipoproteins in the culture medium. The possibility is discussed that the increase is due to environmentally induced changes in surface membrane lipid composition.

Summary An indirect radiolabelled antibody method has been developed to measure the effects of aldehydes on the amount of antigen detectable at the surface of adult male Schistosoma mansoni. Incubation of schistosomes in formaldehyde (0·01–10% wt/vol.) or glutaraldehyde (0·01–0·1% wt/vol.) was found to result in increased exposure of parasite antigens with a concomitant decrease in the amount of surface located host RBC antigens. Those concentrations of formaldehyde or glutaraldehyde which were most effective in removing or displacing host antigens were also most able to expose parasite antigens. These findings are consistent with the hypothesis that host antigens mask parasite antigens at the surface of the adult schistosome.

Surface iodination, extraction and SDS-PAGE analysis techniques were employed to characterize and compare the surface polypeptides of two strains of Strongyloides ratti. Third stage infective larvae and parasitic adults of homogonic and heterogonic strains were studied using a variety of surface labelling procedures and detergents for the extraction of labelled molecules. Profiles obtained from SDS-PAGE analysis demonstrated that homogonic and heterogonic strains of S. ratti have identical surface antigens.

Phosphorylcholine (PC) is found attached to N-type glycans of proteins secreted by filarial nematodes, where it appears to act as an immunomodulator. Based on information on the structure and biosynthesis of the PC-glycan of a major secreted protein, ES-62, strategies were designed with potential for preparing PC-free material to better understand the importance of PC in filarial nematode immunomodulation. The strategies involve either enzymatic removal of PC or inhibition of its attachment during ES-62 synthesis. No method tested was found to be 100% effective although approximately 70% removal was obtained by culturing worms in Et18OCH3. Reasons for failure to obtain complete absence of PC moieties are discussed in relation to the structure and synthesis of PC-glycans and in addition PC-glycan biosynthesis is briefly commented on as a target for chemotherapy.

A thorough study of parasite antigens is a prerequisite for control programmes based on protection by vaccination, accurate serodiagnosis and perhaps immune modulation to diminish pathological sequelae. Stage specific surface secreted and somatic antigens may be of particular value in proceeding towards these goals. The design of vaccines is most appropriately focused on surface antigens. With respect to pathology, certain antigens must stimulate humoral and, or cellular immune responses which are responsible for the undesirable immunopathologic consequences of the disease. The ultimate objective, therefore, is identification of those particular antigens followed by appropriate down regulation of the immune system in order to delete such potentially harmful immunological reactions. The relevant illustration presented in this context is an interesting correlation between one particular clinical condition of onchocerciasis (“sowda”) and the serological response, defined both in terms of the parasite antigen and an immunoglobulin class restricted antibody response. Current parasitological methods of diagnosis consistently underestimate parasite prevalence. Failure to detect low level patent infections incurs the risk of having a reservoir capable of perpetuating infections. There is, then, an urgent requirement for accurate serodiagnosis, to be used in association with, and for the evaluation of, drug treatment and vector elimination in parasite control programmes. Given the high sensitivity of current immunoassay technology, the only bar to establishing the necessary immunological tests is the choice of suitably specific antibody-antigen systems. Once these are identified, a combination of recombinant nucleic acid biochemistry and hybridoma technology should provide the necessary reagents for inexpensive, robust and specific diagnostic tests. In addition, it may not be many years beore the ubiquitous RIA and ELISA technology gives way to the newly developing biosensor systems. Finally, given the sensitivity and specificity of today's nucleic acid hybridization techniques, we may soon expect to see specific identification of infective larvae in their vectors of this, a cloned DNA probe specific for Onchocerca volvulus , and with potential for the detection of infective larvae in blackflies is described.

Summary available: p.xxii.

ES-62 is a phosphorylcholine (PC)-containing glycoprotein secreted by the rodent filarial nematode Acanthocheilonema viteae which is able to inhibit antigen receptor-stimulated proliferation of B and T lymphocytes in vitro and in vivo. The active component of ES-62 appears to be PC, as the results obtained with ES-62 are broadly mimicked by PC conjugated to bovine serum albumin or PC alone. Such desensitization of lymphocyte responsiveness appears to reflect an uncoupling of the antigen receptors from key intracellular proliferative signaling events, such as the phosphoinositide-3-kinase, protein kinase C and Ras mitogen-activating protein kinase pathways. ES-62 mediates such immunomodulatory effects at concentrations equivalent to those found for PC-containing molecules in the bloodstream of parasitized humans and, thus, ES-62 provides a model system for dissecting the mechanisms of immune evasion induced by related PC-containing glycoproteins expressed by human filarial nematodes.

The filarial nematodes Wuchereria bancrofti, Brugia malayi and Onchocerca volvulus represent major public health problems in the Tropics. Effective diagnosis of infection with these parasites is required both for administration of drugs to infected individuals and for monitoring of control programs. However parasitological diagnosis is associated with a number of problems including frequently inadequate sensitivity, long pre-patency of infection and inconvenience for patients. For these reasons there has been considerable effort expended in developing other forms of diagnosis, in particular immunoassays for measuring antibody and circulating parasite antigen as well as molecular-biology-based assays for detecting parasite DNA. This article reviews the progress and achievements obtained to date. The latter include the development of ELISAs employing recombinant antigen for detection of antibody to O. volvulus which have both high sensitivity and specificity, the commercial availability of immunoassays to measure circulating antigen in W. bancrofti infection and the generation of specific DNA-based detection systems for all three parasites.