Search PubMed⌕ Search

Biomedical subjects

W Harnett

Publications and source records attributed to W Harnett.

At least 55 records · Page 3Linked to original sources
PubMed1997

Production of monoclonal antibodies against excretory-secretory products of adult male Onchocerca gibsoni.

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.

Animals↗
PubMed1996

Characterization of heterotrimeric G-proteins in adult Acanthocheilonema viteae.

Heterotrimeric G-proteins have been found in eukaryotic cells, from yeast to humans, but have received little attention, to date, with respect to parasitic organisms. We now present the first report of the characterization of heterotrimeric G-proteins expressed in a filarial nematode, Acanthocheilonema viteae. Using a combination of (i) affinity labelling with [alpha-32P]GTP; (ii) ADP-ribosylation with cholera toxin and pertussis toxin; (iii) Western blotting with a panel of anti-G-protein antibodies; and (iv) reverse transcriptase-PCR with degenerate G-protein oligonucleotide primers followed by hybridization analysis using oligonucleotides specific for individual G-protein subunits, we demonstrate that adult A. viteae expresses homologues of the beta 1- and/or beta 2-like subunits and alpha-subunits of the Gs, G1, Gq and G12 subfamilies found in mammals. The role which these G-proteins may play in the biology of the organism is discussed.

Adenosine Diphosphate Ribose↗
PubMed1996

Identification of Onchocerca volvulus collagen as an antigen mainly recognized by antibodies in chronic hyper-reactive onchodermatitis (sowda).

An Onchocerca volvulus expression library was differentially screened to identify a molecular marker distinguishing sowda (lichenified onchodermatitis) from other onchocerciasis forms. One clone, PG3, was recognized by pooled sera from patients with sowda, but not by pooled sera from patients with generalized onchocerciasis; it was not recognized by sera from patients with lymphatic filariasis or other helminth infections. The DNA of PG3 hybridized strongly with O. volvulus Eco RI-digested DNA, but not with DNA from Brugia spp., Trichinella spp., and humans. A weak reaction was observed with DNA from O. gibsoni and Acanthocheilonema viteae. The PG3 DNA sequence showed a high homology with both human and nematode collagens. Confirmation of the collagen-like nature of the sowda-specific PG3 product was obtained by amino terminal sequencing of the PG3 expression product, as well as by demonstrating its susceptibility to collagenase digestion. The characteristic recognition of the O. volvulus collagen specified by clone PG3 was confirmed by measuring antibody levels to the expressed product in individual sowda and generalized onchocerciasis sera, respectively. Identification of a nematode collagen antigen mainly recognized in sowda patients raises the possibility that this extreme form of dermatitis might arise through cross-reactivity between anti-O. volvulus collagen antibodies and human collagen. However, a relationship between the PG3 recognition by antibodies and the sowda pathogenesis could not be demonstrated.

Amino Acid Sequence↗
PubMed1996

Prevention of attachment of phosphorylcholine to a major excretory-secretory product of Acanthocheilonema viteae using tunicamycin.

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.

Animals↗
PubMed1996

Do excretory-secretory products of Onchocerca gibsoni contain phosphorylcholine attached to O-type glycans?

Excretory-secretory products (ES) of adult male Onchocerca gibsoni contain phosphorylcholine (PC). PC-ES are detected as a smear of M(r) approximately 60- approximately 200 kDa by western blotting employing a monoclonal antibody (mAb) against PC, suggesting that they are glycoproteins. Exposure of PC-ES to N-glycosidase F results in weak and inconsistent loss of binding of the mAb, indicating that unlike the situation with respect to ES of Acanthocheilonema viteae, PC is highly unlikely to be solely attached to N-type glycans. Conversely, treatment of O. gibsoni PC-ES with mild alkali, a strategy for removing O-type glycans, abolishes mAb binding. These results suggest that PC may be attached to O. gibsoni proteins mainly via O-type glycans, and raise the possibility that filarial parasites may vary with respect to their mode of attachment of PC. The implications of this with respect to the design of inhibitors of PC attachment for use as anti-filarial drugs, are discussed.

Amidohydrolases↗
PubMed1994

Biosynthetic radiolabelling of excretions-secretions of adult male Onchocerca gibsoni.

Maintenance of adult male worms of the bovine filarial parasite Onchocerca gibsoni in vitro, in the presence of radioactive precursors, resulted in the time-dependent excretion-secretion of radiolabelled parasite macromolecules (ES). Molecules labelled with amino acids ([35S] methionine, [3H] leucine) covered a wide range of molecular weights, whereas labelling with [3H] glucosamine produced predominantly molecules of high molecular weight. Many of the products were recognized by antibodies in two serum pools from humans infected with Onchocerca volvulus. O. gibsoni ES may therefore provide a substitute source of material for studies on the ES of the less readily available human parasite.

Animals↗
PubMed1994

Characterization of the surface polypeptides of Strongyloides ratti: a comparison of homogonic and heterogonic strains.

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.

Animals↗
PubMed1994

Some preliminary data on the nature/structure of the PC-glycan of the major excretory-secretory product of Acanthocheilonema viteae (ES-62).

The structure of the PC-glycan of the major excretory-secretory product (ES-62) of Acanthocheilonema viteae has been investigated using endoglycosidases and lectins. Results obtained raise the possibility that it may be of the high mannose type. This, and the insensitivity of the PC-glycan to treatments which remove PC or choline from bacterial PC-glycans, suggests that it may be more analogous to fungal, than to bacterial PC-containing glycans.

Animals↗
PubMed1993

Inhibition of murine B cell proliferation and down-regulation of protein kinase C levels by a phosphorylcholine-containing filarial excretory-secretory product.

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.

Animals↗
PubMed1993

Rapid changes in the surface of parasitic nematodes during transition from pre- to post-parasitic forms.

All mammalian-parasitic stages of a range of nematode species investigated (Brugia pahangi, Acanthocheilonema viteae, Strongyloides ratti, Nippostrongylus brasiliensis, Trichinella spiralis and Ostertagia ostertagi) labelled in a surface-restricted manner with the fluorescent lipid analogues 5-N-(octadecanoyl)aminofluorescein (AF18) or nitrobenzoxadiazole-cholesterol (NBD-chol), but failed to bind other similar probes. In contrast, the surfaces of the 'pre-parasitic' infective stages of these species had affinity for neither AF18 nor NBD-chol. This exclusion of lipid analogues changed rapidly upon exposure of the larvae to tissue culture conditions which mimic the mammalian tissue environment (e.g. RPMI 1640/37 degrees C) such that the above probes could then insert into the surface layer of the larvae. The dauer larva of Caenorhabditis elegans also excluded the probes, but became permissive to labelling upon stimulation to emerge from the dauer state. The time taken for the surface transformation to occur ranged from less than 10 min in the vector-borne parasites to approximately 5 h in those which enter by the oral route, with direct skin-penetrators occupying an intermediate position. In all cases, the alteration proceeded too rapidly for it to have been associated with a moult. Fluorescence Recovery After Photobleaching (FRAP) studies of A. viteae larvae showed that approximately 50% of the AF18 probe was free to diffuse within the plane of the surface immediately after transformation. This is only a transitory state because AF18 was found to be highly restricted in its lateral diffusion on the surface of adult parasites. In the larvae of S. ratti, the change in affinity for AF18 was accompanied by the rapid shedding of an otherwise stable surface coat of polyanionic material, here visualized by labelling with fluorescein-conjugated cationized ferritin. Incubation of larvae in lipid-rich host serum during the induction of transformation inhibited subsequent labelling with AF18. This possibly reflects competition for insertion sites and an in vivo propensity towards the acquisition of host lipid by invading parasites.

Animals↗
PubMed1991

Surface antigens of male worms and microfilariae of Onchocerca gibsoni.

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 dodecyl sulphate (SDS)-polyacrylamide 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 radiolabelled 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 of microfilarial 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.

Animals↗
PubMed1991

Studies on a 14-kilodalton surface protein of Onchocerca microfilariae.

A 14-kDa antigen present on the surface of uterine microfilariae of Onchocerca spp. has been identified using monoclonal antibodies. The antigen was also found in skin microfilariae, but in a masked or cryptic form. A complementary DNA clone encoding the epitope recognised by one of the monoclonal antibodies was identified in a lambda gt11 library. Nucleotide sequencing revealed that the 233-bp cDNA fragment codes for the carboxy-terminus of the antigen. The deduced amino acid sequence consists of three hydrophobic domains with high potential for beta-sheet formation. The amino-terminal hydrophobic domain is followed by 4 positively charged residues (positions 22-25) which contribute to the rather basic character of the protein. Another interesting feature of the polypeptide is its richness in phenylalanine (12.7%). From the sequence information, a synthetic peptide was synthesised which was recognised by one of the monoclonal antibodies directed against the 14-kDa antigen and a small number of sera from patients with onchocerciasis. The relevance of this to vaccination is discussed.

Amino Acid Sequence↗
PubMed1990

The surface lipid of parasitic nematodes: organization, and modifications during transition to the mammalian host environment.

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.

Animals↗
PubMed1990

Cloning of a species-specific DNA probe from Onchocerca gibsoni.

A genomic library of Onchocerca gibsoni has been prepared in the vector lambda-gt10 and has been screened for specific DNA sequences by hybridization with radiolabelled total genomic DNA from a number of Onchocerca species. A clone--fOGI--has been isolated which does not interact with DNA prepared from O. gutturosa, O. lienalis, O. ochengi, O. cervicalis or O. volvulus (both Liberian and Mexican isolates). In addition, no hybridization is observed with host (cattle) DNA. fOGI can detect as little as 100-200 pg of O. gibsoni DNA. It is thus concluded that fOGI has the sensitivity to detect microfilariae of O. gibsoni found in the skin of cattle and the specificity to differentiate them from closely related species living in the same environment.

Animals↗
PubMed1990

Association between circulating antigen and parasite load in a model filarial system, Acanthocheilonema viteae in jirds.

Jirds (Meriones libycus) were infected with various numbers of Acanthocheilonema viteae L3 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.

Animals↗
PubMed1990

Cloning of specific diagnostic antigens of Onchocerca volvulus.

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.

Animals↗
PubMed1989

An oligonucleotide probe specific for Onchocerca volvulus.

A genomic DNA library of a Liberian strain of Onchocerca volvulus was prepared in the vector bacteriophage lambda gt10. The library was differentially screened by hybridisation with radiolabelled total DNA from the homologous parasite, two heterologous Onchocerca 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.

Animals↗