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R M Maizels

Publications and source records attributed to R M Maizels.

At least 19 recordsLinked to original sources
PubMed1992

Identification of circulating parasite acetylcholinesterase in human and rodent filariasis.

In the present study, the enzyme acetylcholinesterase (AChE) from filarial parasites was identified in sera from humans infected with Onchocerca volvulus as well as in Mastomys natalensis infected with Brugia pahangi. The enzyme was present in immune complexes precipitated with cold 4% polyethylene glycol. The infected sera showed 3-4 times more AChE activity than did normal sera, and enzyme activity could be demonstrated in 5% polyacrylamide gels by specific staining. The enzyme from infected serum showed 3 times more activity when acetylthiocholine was used as the substrate as compared with butyrylthiocholine, whereas the enzyme activity present in normal serum was low and did not show this substrate specificity. Immunoprecipitation assays confirmed the presence of anti-AChE antibodies in the infected serum. The enzyme was further analysed by enzyme-linked immunosorbent assay and immunoblotting with rabbit antibodies to B. malayi AChE. Immunoblotting of the B. pahangi-infected serum revealed two closely located bands at about 200 kDa and one 95-kDa band, whereas in O. volvulus-infected serum, only one specific band was observed at about 200 kDa. The identification of parasite AChE may be particularly useful for diagnosis of the disease or for the study of the involvement of this enzyme in the host-parasite relationship.

Acetylcholinesterase↗
PubMed1992

Toxocara canis: monoclonal antibodies to carbohydrate epitopes of secreted (TES) antigens localize to different secretion-related structures in infective larvae.

The major secreted glycoproteins of Toxocara canis larvae appear to be derived from two specialized organs within the nematode organism. Using immunogold electron microscopy, we have analyzed the binding patterns of a panel of monoclonal antibodies (Tcn-1 to Tcn-8) reactive with Toxocara excretory-secretory (TES) antigens. We find, first, that the esophageal gland and lumen are strongly reactive with monoclonals Tcn-4, Tcn-5, and Tcn-8, and because the posterior portion of the gut is closed, we hypothesize that products of this gland are released through the oral aperture. Second, a distinct anti-TES antibody (Tcn-2) localizes solely to the midbody secretory column, which opens onto the cuticle at a secretory pore. Thus, the secretory apparatus is probably functional in this stage of parasite as an important source of TES products. Only one monoclonal, Tcn-7, can bind to both esophageal and secretory structures. In addition, another antibody, Tcn-3, binds both to the epicuticle and to a TES antigen, but our data do not directly determine whether antigens located in the cuticle are subsequently released. Thus there are at least two, and possibly three, independent sources of TES antigens within Toxocara larvae.

Animals↗
PubMed1992

Toxocara canis: a labile antigenic surface coat overlying the epicuticle of infective larvae.

An electron-dense coat covering the surface of Toxocara canis infective-stage larvae is described. This coat readily binds to cationized ferritin and ruthenium red, indicating a net negative charge and mucopolysaccharide content, and can be visualized by immuno-electron microscopy only if cryosectioning is employed. Monoclonal antibodies reactive to the surface of live larvae bind the surface coat but not the underlying cuticle in ultrathin cryosections. The surface coat is dissipated on exposure to ethanol, explaining the lack of surface reactivity of conventionally prepared immunoelectron microscopy sections of T. canis. Differential ethanol extraction of surface-iodinated larvae demonstrates that the major component associated with the coat is TES-120, a 120-kDa glycoprotein previously identified by surface iodination, which is also a dominant secreted product. The surface-labeled TES-70 glycoprotein is linked with a more hydrophobic stratum at the surface, while a prominent 32-kDa glycoprotein, TES-32, is more strongly represented within the cuticle itself. Antibody binding to the coat under physiological conditions results in the loss of the surface coat, but this process is arrested at 4 degrees C. This result gives a physical basis to earlier observations on the shedding of surface-bound antibodies by this parasite. An extracuticular surface coat has been demonstrated on Toxocara larvae prior to hatching from the egg and during all stages of in vitro culture, suggesting that it may play a role both in protecting the parasite on hatching in the gastrointestinal tract and on subsequent tissue invasion in evading host immune responses directed at surface antigens.

Animals↗
PubMed1992

Immunoepidemiology of Ascaris lumbricoides: relationships between antibody specificities, exposure and infection in a human community.

The serum antibody responses of 124 people naturally exposed to Ascaris lumbricoides infection were analysed by immunoprecipitation of radio-isotope labelled 3rd- and 4th-stage larval Ascaris suum excretory and secretory antigens (L3/4 ES). Profiles of antigens recognized were visualized by polyacrylamide gel electrophoresis (SDS-PAGE), and the band intensities of the 12 major precipitated antigens were individually scored. Most subjects were seropositive, but considerable variation was observed in the amount of total and individual ES antigens precipitated. The sex- and age-related profiles of antibody levels followed similar patterns to those of egg output. In addition, total antibody scores of individuals were closely correlated (r = 0.47-0.52) with their eggs per gram of faeces (e.p.g.) collected 4 months after blood samples were taken. These findings suggest that antibody levels against larval ES antigens reflect recent exposure and are consistent with the hypothesis that establishment of adult worms is proportional to the number of larvae that recently migrated through the lung.

Adolescent↗
PubMed1992

Lectin binding to secretory structures, the cuticle and the surface coat of Toxocara canis infective larvae.

Toxocara canis infective larvae are known to produce abundant glycosylated molecules which may be found associated with the surface or secreted into their environment. Using a range of fluorescein-conjugated and gold-conjugated lectins, the localization of particular carbohydrates was defined on the surface of live parasites, and internally at the ultrastructural level. Surface exposure of N-acetyl galactosamine and N-acetyl glucosamine was deduced by binding of FITC-conjugated Helix pomatia (HPA) and wheat-germ agglutinins (WGA). These sugars appear to be associated with a densely staining surface coat as conventional immuno-electron microscopy procedures dissipate this coat and reveal no surface binding site for these lectins. However, by using cryo-immuno-electron microscopical (C-IEM) techniques, the surface coat is retained and can be shown to bind WGA. The fluorescent lectins also revealed strong WGA binding to the secretory and amphidial pores, while the buccal opening and the cuticular alae bound HPA. Corresponding results were obtained at the ultra-structural level. Thus, HPA bound to the electron-dense area of the cuticle, areas of local cuticular thickening such as the alae and buccal labia, as well as to the oesophageal lumen. WGA also bound to the thickened cuticle of the alae and the buccal opening, but showed no reaction to either the electron-dense layer of the cuticle or the oesophageal lumen. Unlike HPA, WGA did bind specifically to the secretory column contents and the electron-dense regions of the lips associated with the chemosensory amphids. The compartmentalization of the sugars N-acetyl galactosamine and N-acetyl glucosamine, their sources and routes of surface expression and the possible association with the TES glycoprotein antigens are discussed.

Acetylgalactosamine↗
PubMed1992

Biosynthesis and glycosylation of serine/threonine-rich secreted proteins from Toxocara canis larvae.

Toxocara canis infective stage larvae continually produce excretory-secretory (TES) glycoproteins in long-term in vitro culture. The kinetics of synthesis and secretion were studied by metabolic labelling with radioactive [35S]methionine, [14C]serine and [14C]threonine. Maximal incorporation rates required overnight pre-incubation of parasites in medium depleted of the appropriate amino acid. Larvae rapidly incorporated isotope into their somatic tissues, but there was a minimum delay of 10 h before secretion of labelled antigens. Labelling with [14C]serine and [14C]threonine demonstrated a relative abundance of these amino acids in the major surface/secreted glycoproteins of this nematode (TES-32 and 120). Pulse-chase experiments suggested that TES-120 may be derived from a 58 kDa precursor, reflecting extensive posttranslational glycosylation. Inhibition of N-glycosylation with tunicamycin and digestion with N-glycanase provided evidence of N-glycosylation in the lower molecular weight ES components (TES-32, 55 and 70). These agents had no effect on the higher molecular weight components (TES-120 and 400) implying that for these molecules glycosylation is predominantly O-linked. The largest ES component (TES-400) was unusual, in incorporating serine and threonine but not methionine, and by exhibiting increased apparent molecular weight following pronase digestion; it is suggested that this molecule is a proteoglycan.

Animals↗
PubMed1992

Diethylcarbamazine (DEC): immunopharmacological interactions of an anti-filarial drug.

Anti-parasitic drugs may achieve their therapeutic effect either by direct activity against the pathogenic organism, or by altering host factors which lead to parasite killing. In this review, we discuss the evidence for an indirect mode of action for one major anti-filarial drug, diethylcarbamazine (DEC). The interpretation most consistent with existing data is that DEC alters arachidonic acid metabolism in microfilariae and in host endothelial cells. These changes may result in vasoconstriction and amplified endothelial adhesion leading to immobilization of microfilarial parasites, enhanced adherence and cytotoxic activity by host platelets and granulocytes. These events would represent activation of the innate, non-specific immune system, independent of the adaptive, antigen-specific, immune response. This model explains the paradox between rapid clearance in vivo and the lack of an in vitro effect, as well as the efficacy of DEC in non-immune animals. It may also account for the inconsistencies in the effects of DEC against different filariae in different host species. In addition, we discuss the significant side-effects often associated with treatment of heavily infected patients, and the longer-term changes in T-cell reactivity and the host-parasite relationship which follow successful treatment with DEC.

Animals↗
PubMed1991

Immune responses to filarial parasites.

Filarial nematode parasites are long-lived organisms which are responsible for one of the major tropical diseases. Their ability to survive may be related to the antigen-specific anergic state observed in carriers of the parasite. Severe disease symptoms are associated with the breakdown of anergy. Different stages of the parasite life cycle are anti-genetically distinct, and a state of concomitant immunity is therefore possible which prevents superinfection. The evidence for these conclusions is discussed, in the context of the need for an effective vaccine against filarial parasites which will avoid any risk of increasing disease severity.

Animals↗
PubMed1991

cDNA clones of Onchocerca volvulus low molecular weight antigens provide immunologically specific diagnostic probes.

We report here a panel of cDNA clones from Onchocerca volvulus which were isolated on the basis of being uniquely recognised by onchocerciasis sera and not by sera from patients infected with the major lymphatic filarial nematode parasite Wuchereria bancrofti. Over 90% of O. volvulus recombinants from a primary screen were found to cross-react with lymphatic filariasis sera and were discarded. The subset of specific clones, selected with pooled sera, was then screened with panels of individual patient sera. Individual onchocerciasis cases showed a highly heterogeneous pattern of recognition of recombinant peptides, but several clones were identified which could be combined in a cocktail of antigenic epitopes to successfully detect all infected cases in the study. All these clones encode low molecular weight proteins of the parasite, confirming earlier reports that antigens of this size class show greater species specificity. Several clones encode proteins of 20-23 kDa, the same molecular weight range as the major surface protein of adult worms. The two most commonly recognised clones, Ov22/31M and Ov20/36M were subcloned into the vector pNGS 8 which produces fusion proteins attached to a polyasparagine leader. The fusion peptides of both Ov22/31M and Ov20/36M were soluble and easily purified by gel filtration. Purified fusion protein was used in ELISA to assess reactivity of infected patients giving 90% sensitivity with 100% specificity.

Animals↗
PubMed1991

Characterization of proteolytic enzymes from larval and adult Nippostrongylus brasiliensis.

Proteases from infective larval (L3) and adult stages of Nippostrongylus brasiliensis were investigated with a combination of techniques involving gelatin degradation and cleavage of fluorogenic substrates. Analysis of L3 excretory-secretory (ES) products revealed enzymes of Mr 51, 58, 79, approximately 150 and approximately 250 kDa. Inhibition profiles indicate that the major 51 kDa protease is a metallo-enzyme. Significantly, little activity was present in larval somatic extracts, suggesting the synthesis of zymogens or precursor forms prior to secretion. Adult ES contained a distinct enzyme, of 50 kDa, and a number of other proteases were detected in somatic extracts of this stage, ranging from 51 to greater than 300 kDa. The largest of these adult somatic enzymes is also a putative metallo-protease. While nearly all enzymes from both L3 and adult are heat labile, incubation at 100 degrees C generated a previously unobserved activity at 20 kDa. Furthermore, a protease of similar size may be found in uninfected rat intestinal tissue, suggesting specific uptake of a host-associated enzyme by the parasite in the form of an inactive, heat-labile complex.

Animals↗
PubMed1991

Comparison of isolates and species of Toxocara and Toxascaris by biosynthetic labelling of somatic and ES proteins from infective larvae.

Infective-stage larvae of three different isolates of Toxocara canis were intrinsically ([35S]methionine) labelled in culture, to determine the presence of similarities or differences in the somatic and ES antigens expressed between larvae derived from different hosts and different geographical regions. Two other closely related ascaridids, Toxascaris leonina which infects cats and dogs, and Toxocara vitulorum (Neoascaris vitulorum) which infects cattle, were also compared to T. canis larvae by this method. Overall comparisons were made by 1- and 2-dimensional electrophoresis, while immunological cross-reactivities between the different species were analysed by radio-immunoprecipitation. Our results show that extensive physicochemical characteristics are shared between T. canis isolates, both from different hosts and different geographical locations. A substantial overlap was revealed when T. canis and T. vitulorum antigens were compared, whereas Toxascaris was found to produce a distinct antigen profile: this result was independent of whether methionine- or Iodogen-labelled products were being considered. Antigen recognition by polyclonal antibodies raised to all three species and to the cat ascaridid Toxocara cati, revealed considerable cross-reactivities. The cross-reactions were especially prominent between the Toxocara species, a fact further substantiated when reactivity of T. canis ES-specific monoclonal antibodies were tested against T. leonina and T. vitulorum antigens. The ES antigens of T. leonina were not recognized by the T. canis monoclonals, whereas the majority of these antibodies precipitated antigens of T. vitulorum. One which did not react with T. vitulorum was monoclonal antibody Tcn 2, indicating its species-specific reactivity and therefore its potential for the specific diagnosis of human toxocariasis.

Animals↗
PubMed1991

Characterization of nematode glycoproteins: the major O-glycans of Toxocara excretory-secretory antigens are O-methylated trisaccharides.

Toxocara excretory-secretory antigens (TES) were isolated from the culture media of T.canis and T.cati larvae and their O-glycan content was investigated using fast atom bombardment-mass spectrometry (FAB-MS), gas chromatography and electron impact mass spectrometry. The major oligosaccharides released by reductive elimination of T.canis TES glycoproteins were shown to be two, approximately equi-abundant, trisaccharides: 2-O-Me-Fucp(alpha 1----2)-4-O-Me-Galp(beta 1----3)GalNAcitol and 2-O-Me-Fucp(alpha 1----2)-Galp(beta 1----3)GalNAcitol. In contrast T.cati TES O-glycans are predominantly one component, shown by FAB-MS to be a di-O-methylated trisaccharide, which is probably identical to the di-O-methylated trisaccharide from T.canis. The O-methylated trisaccharides are strong candidates for the carbohydrate epitopes recognized by a panel of monoclonal antibodies which exhibit multiple reactivity against TES antigens. This study constitutes the first rigorous characterization of glycans from a parasitic nematode.

Animals↗
PubMed1991

Age-specific acquisition of immunity to infective larvae in a bancroftian filariasis endemic area of Papua New Guinea.

The development of antibodies to infective stages of the filarial parasite, Wuchereria bancrofti, with age of the host human population was studied by immunofluorescence, immunoprecipitation and immunoblotting assays. Among individuals under 20 years of age, few had detectable antibodies to the infective (L3) larval surface by IFA: only 2 out of 10 scored positive. However, all adults (over 20 years) were positive in this assay although the utilization of isotypes varied between different individuals. Whilst antibodies to the L3 surface are therefore acquired after prolonged exposure to infection (greater than 20 years), recognition patterns of L3 surface labelled antigens, measured by immunoprecipitation analysis iodinated proteins on SDS-PAGE, and of somatic L3 proteins on immunoblots, were equivalent in the two age groups. Thus, a critical surface antigen, recognised in an age-dependent manner, is present on the L6 cuticle but cannot be resolved as a conventional protein or glycoprotein constituent.

Adolescent↗
PubMed1990

Filarial-specific IgG4 response correlates with active Wuchereria bancrofti infection.

The filarial-specific humoral immune response of adult residents of two areas of Papua New Guinea, differing in transmission of Wuchereria bancrofti infection was compared. The majority of residents of the village of Bonahoi, in an area where transmission of filariasis had been interrupted by a 20-year insecticide spray program to control malaria, showed no parasitologic signs of active W. bancrofti infection and were negative for both circulating phosphorylcholine Ag and peripheral blood microfilariae. In contrast, adult residents of the village of Nanaha were in an area exposed to infection, and were phosphorylcholine-Ag- and microfilariae-positive. The antibody response of these two groups to both adult worm excretory/secretory (ES) Ag and somatic antigen extract was examined to determine which components of the filarial-specific immune response were dependent on active infection. Identification of these immune responses may point to immunologic methods to evaluate control programs for lymphatic filariasis. Adults from Bonahoi were found to have significant immune responses to [35S] methionine-labeled ES Ag by immunoprecipitation and to adult somatic antigen extracts by ELISA and by immunoblotting. This result is consistent with the fact that these individuals were previously exposed to and/or infected with W. bancrofti. Similarly, residents of the endemic village had detectable immune responses to these Ag irrespective of if they were microfilaremic. The most striking immunologic difference observed between the two groups was that residents of Bonahoi had a dramatically reduced filarial-specific IgG4 antibody response to both adult somatic Ag and adult ES Ag. These data suggest that longitudinal measurement of filarial-specific IgG4 levels may be a useful seroepidemiologic indicator of changes in W. bancrofti infection status.

Adolescent↗
PubMed1990

MHC and non-MHC-restricted recognition of filarial surface antigens in mice transplanted with adult Brugia malayi parasites.

We describe here the genetic control of humoral responses to filarial nematode Ag elicited by live adult Brugia malayi parasites in mice. Inbred and congenic mice of two different MHC haplotypes, H-2k and H-2d, were examined. Serologic analysis showed that the humoral responses to the major surface 29-kDa glycoprotein of adult parasites and a 40-kDa Ag from the surface of the microfilarial stage were restricted to mice with H-2k alleles (B10.BR, CBA/Ca, and CBA/N), whereas mice of the H-2d haplotype (B10.D2/n and BALB/c) were nonresponsive to these Ag. Conversely, internal adult Ag of molecular mass of 24 and 66 kDa were recognized only by animals with the H-2d haplotype. Apart from MHC-restricted recognition, the level of responses to phosphorylcholine and to a 15-kDa adult surface molecule were found to be influenced by non-MHC genes. A sharp restriction was also observed to an adult surface Ag complex of 17 to 200 kDa, which was recognized only by BALB/c mice. Thus, multiple examples of both H-2 and background genetic effects on the immune response to distinct filarial Ag can be found.

Animals↗
PubMed1990

Surface associated glycoproteins from Toxocara canis larval parasites.

The surface of infective larvae of Toxocara canis, the dog ascarid nematode, reveals relatively few exposed surface proteins which can be recovered in soluble form. The major components identified by surface labelling have molecular weights of 32, 55, 70 and 120 kilodaltons (kDa), and are all significantly glycosylated. All are recognised by the immune response in definitive (canine) and paratenic (murine or human) hosts. Expression of these antigens on the parasite surface begins after the larvae hatch from infective ova in vitro, and presumably in vivo. Each of these molecules may also be found in the set of secreted (ES) glycoconjugates released by larval parasites cultivated in vitro, and currently available biochemical and functional data on the surface/secreted ES glycoproteins are presented. Analysis with monoclonal antibodies (MAbs) confirms the identity of surface and ES molecules, and these MAbs show differing patterns of binding to the epicuticle, the cuticular matrix and to the oral orifice. Alternative mechanisms for antigen synthesis, insertion into the cuticle and export from the parasite are discussed.

Animals↗
PubMed1990

Cuticular localisation and turnover of the major surface glycoprotein (gp29) of adult Brugia malayi.

A polyclonal antiserum was raised to a gel purified preparation of the major water-soluble surface glycoprotein (gp29) of adult Brugia malayi, and used to define the stage specificity of expression, localisation (by immunoelectron microscopy) and the dynamics of biosynthesis and turnover via pulse-chase experiments. Gp29 was not detected in surface-labelled preparations of either pre- or post-parasitic third stage larvae (L3), but was present in fourth stage larvae (L4), where its mass was estimated to be 30 kDa by SDS-PAGE. In both L4 and adult worms, the protein resolved as 3 distinct species in 2-dimensional electrophoresis, with pIs from 6.5 to 7.5. Pulse-chase studies via metabolic labelling of adult worms with [35S]methionine in vitro indicated that gp29 was processed from a 32-kDa precursor to the mature molecule within 45 min and that it was secreted into culture medium within 5 h of synthesis. On extended culture, gp29 was converted to a 56-kDa product, presumably either by complex formation or covalent linkage with another secreted molecule. This higher molecular weight component had a more acidic pI of 4.5 and was insensitive to digestion with N-glycanase. Immunoelectron microscopy showed that gp29 was distributed throughout the cuticle and hypodermal cell layer of adult worms, suggesting that the protein was synthesised in the hypodermis, and that turnover into culture medium occurred through the cuticle. The protein appeared to concentrate at the distal cell membrane of the hypodermis, particularly at the stacked invaginations. Additional immunostaining was found on the basement membrane of the basal lamina of the intestine.

Animals↗