PubMed Health⌕ Search

Biomedical subjects

M E Selkirk

Publications and source records attributed to M E Selkirk.

70 records · Page 4Linked to original sources
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↗
PubMed1989

Heat shock cognate 70 is a prominent immunogen in Brugian filariasis.

A cDNA expression library constructed from RNA derived from adult stage Brugia pahangi (mixed sexes) was screened with pooled sera from chronic, amicrofilaremic cases of human lymphatic filariasis from the Indonesian island of Tanjungpinang, where Brugia malayi is endemic. Polyclonal antisera raised to purified beta-galactosidase fusion proteins from two of the most highly reactive clones identified a protein of Mr 70,000 in all stages examined (microfilariae, L3 and adults) of both B. malayi and Brugia pahangi. Derivation of the amino acid sequence from these two overlapping cDNAs identified the encoded protein as a member of the heat shock protein 70 family, and showed the closest similarity to the constitutively expressed "heat shock cognate 70" (hsc70) protein. Hybridization of hsc70 cDNAs to RNA and DNA from B. pahangi under stringent conditions identified a major transcript of 2.4 kb and revealed the existence of a family of related genes. In vitro culture of larval stages of B. pahangi at elevated temperatures (43 degrees C) resulted in increased expression of hsc70, and a classic heat shock response in which five proteins (mr 18,500, 22,000, 62,000, 70,000, and 85,000) were exclusively synthesized in microfilariae. Analysis of cross-reactivities by Western blotting implied that antibody generated by infection with B. malayi was directed at filarial-specific determinants of Brugia hsc70. However, ELISA with recombinant fusion proteins for both Plasmodium falciparum and Schistosoma mansoni hsc70 indicated that some individuals with Brugian or Bancroftian filariasis did produce antibodies which cross-reacted with plasmodial and schistosomal homologs. Thus filarial-specific antibody responses were not generated in all individuals, indicating that this molecule would not be suitable for diagnostic purposes. ELISA with a purified beta-galactosidase fusion protein from B. pahangi showed antibody responses to hsc70 across the clinical spectrum of filariasis. Alignment of the derived amino acid sequences from B. pahangi, P. falciparum, S. mansoni and rat hsc70 homologs, and comparison of the immunologic reactivity of the products of the two cDNA clones by Western blotting and ELISA suggested that these determinants were located primarily at the C terminus of the protein.

Amino Acid Sequence↗
PubMed1989

Filarial surface antigens: the major 29 kilodalton glycoprotein and a novel 17-200 kilodalton complex from adult Brugia malayi parasites.

Adult Brugia malayi nematode parasites possess a range of cuticular and epicuticular molecules which may be defined by various surface-labelling techniques. We present here evidence that at least two distinct antigens are associated with the surface, a glycoprotein of 29 kDa, and a complex of twelve components forming a regular series or 'ladder' between 17 and 200 kDa (17/200 kDa) which have not previously been described. Each of these products is antigenic in infected hosts, although responses in infected humans to the 17/200 kDa are relatively weak. Digestion of the 29 kDa antigen with proteases and endoglycosidases indicates that it is closely conserved between B. malayi and B. pahangi, and that it carries at least two N-linked oligosaccharide chains each of 1.5-2 kDa. By contrast, a smaller surface-labelled antigen of 15 kDa shows no glycosylation by either lectin adherence or endoglycosidase digestion assays. Trypsin treatment of intact, labelled parasites results in cleavage of 29 kDa molecules isolated 17/200 kDa 'ladder' to trypsin abolishes all bands except the 17 kDa base unit. Both the 29 kDa and 17/200 kDa antigens can be recovered as water-soluble molecules by homogenisation of the parasite in the absence of detergent, or by disruption of the cuticle with reducing agents such as 2-mercaptoethanol. In the presence of such agents, both the 17/200 kDa series and the 29 kDa glycoprotein are shed rapidly from intact parasites. Finally, two-dimensional electrophoretic analysis shows that while the 29 kDa glycoprotein is strongly basic and the 15 kDa acidic, the 17/200 kDa antigens form a related series of neutral pI.

Animals↗
PubMed1989

Identification, synthesis and immunogenicity of cuticular collagens from the filarial nematodes Brugia malayi and Brugia pahangi.

The major structural proteins of the cuticle of the filarial nematode parasites Brugia malayi and Brugia pahangi were identified by extrinsic iodination and sensitivity to clostridial collagenase. At least 16 acidic components were identified in adult worms by 2-dimensional electrophoresis, with molecular weights ranging from 35,000 to 160,000. These proteins appear to be cross-linked by disulphide bonds, and localised in the basal and inner cortical layers of the cuticle. The outer cortex, containing the epicuticle, is insoluble in 1% sodium dodecyl sulphate and 5% 2-mercaptoethanol, and can be isolated free of cellular material. Despite their inaccessibility to the immune system in intact worms, antibodies to the cuticular collagens are provoked in humans infected with a variety of filarial parasites. Immunological cross-reactivity was demonstrated between a 35 kDa component and human type IV (basement membrane) collagen. Autoantibodies to type IV collagen were detected in a number of individuals with lymphatic filariasis, although no correlation could be drawn with observed pathology. Synthesis of cuticular collagens is discontinuous, occurs at negligible levels in mature adult male worms, and does not appear to involve the production of small molecular weight precursors, in contrast to Caenorhabditis elegans. Hybridisation with a heterologous cDNA probe coding for the alpha 2 chain of chicken type 1 collagen suggests that they are encoded by a multigene family.

Animals↗
PubMed1989

Localization and immunogenicity of tubulin in the filarial nematodes Brugia malayi and B. pahangi.

Tubulin was identified in the filarial nematodes Brugia malayi and B. pahangi by several approaches. Initially, a monoclonal antibody (6D8) was selected for its unusual binding to B. malayi microfilariae in indirect immunofluorescence assays: 6D8 showed granular, heterogeneously dispersed fluorescence on fixed parasites but did not bind to unfixed microfilariae. The microfilarial sheath did not bind 6D8, although it did bind fluoresceinated wheatgerm agglutinin. By Western blotting against microfilarial sonicate, 6D8 reacted with a 50,000-55,000 mol. wt protein, and also bound to purified chicken brain beta-tubulin. Additionally, this monoclonal antibody reacted with a recombinant fusion protein expressed by a clone (Bpa-7) originally isolated from an adult B. pahangi cDNA expression library by its reaction with chronic human filariasis serum. This clone encodes a small 40 amino acid C-terminal segment corresponding to residues 409-449 of beta-tubulin, and shows complete amino acid sequence homology with vertebrate beta-tubulin from 409 to 430 but 55% divergence (six amino acid substitutions, four insertions and one deletion) from human and chicken beta-tubulin over positions 431-449 at the C terminus. Antibody to both parasite and vertebrate (chicken) tubulin was found in filarial infection sera, with higher levels of autoreactive antibody apparent in amicrofilaraemic individuals. Immunogold electron microscopy was then used to localize beta-tubulin in B. malayi microfilariae and adult worms. Tubulin was shown not to be exposed on the microfilarial sheath or in the cuticle of either stage, but was found to be abundant in the somatic tissues. In microfilariae, 6D8 bound myofibril structures under the hypodermal layer, and also bound within cell nuclei. In the adult stage, tubulin was associated with muscle blocks, as well as the intestinal brush border and the embryonic uterine microfilariae.

Amino Acid Sequence↗
PubMed1989

Secreted antigens of filarial nematodes: a survey and characterization of in vitro excreted/secreted products of adult Brugia malayi.

We report here a broad analysis of the excretory/secretory (E/S) products of adult Brugia malayi, collected by in-vitro cultivation of the parasite. Culture media and conditions were optimized, and non-essential amino acids were found to be crucial for efficient protein synthesis under cell- and serum-free culture conditions. A close correlation was found between total protein secretion, phosphorylcholine-bearing antigen release and lactate production on each day of culture, indicating that E/S molecules are actively secreted. Parasites cultured in vitro take 2-3 days to adjust to the new environment, and show peak levels of secretion at days 3 and 4. The active secretion of phosphorylcholine by the parasite therefore justifies the measurement of this molecule as an indication of active infection, possibly reflecting total worm burdens. By comparing metabolically labelled E/S from male and female worms, several molecules of low mol. wt, namely 10,000, 13,000, 14,000 and 22,000, together with high mol. wt components of above 12,000 were found to be female specific. Tracing the origin of the E/S products, several molecules were also found to be associated with the surface. Among these, there are at least two glycoproteins, 29,000 and 51,000 of which the 29,000 molecule is a major surface protein. The immunogenicity of the E/S was examined and antigenic cross-reactivity was found with sera from most filarial infections but not with non-filarial nematodiases such as hookworm or Trichinella. However, two molecules of low mol. wt, 15,000 and 19,000, were not recognized by anti-Onchocerca sera and appeared to be potential Brugia-specific diagnostic molecules. Possible functional roles of the adult E/S products were examined but we could find no evidence of protease activity in the E/S or glutathione S-transferase activity in either the E/S or in whole somatic extract.

Animals↗
PubMed1988

Circulating antibodies and antigens in Presbytis monkeys infected with the filarial parasite Wuchereria bancrofti.

Levels of circulating filarial antigen, and humoral antibody to other defined antigenic targets, were measured over the course of experimental infection of three Presbytis cristatus monkeys with Wuchereria bancrofti. Circulating antigen levels, measured with an anti-phosphorylcholine monoclonal antibody, varied widely although all animals were positive for some period of the infection. Circulating antigen levels tended to be inversely related to the titre of anti-phosphorylcholine antibody, and this trend was maintained even following acid dissociation and inactivation of immune complexed host antibody. Other antibody specificities were measured by Western blotting against somatic proteins and immunoprecipitation of surface antigens. Amongst somatic antigens, targets between 14,000 and 200,000 daltons were recognised by monkey antibodies, but no correlation with infection status could be discerned. Likewise when measuring the response to the 29,000 dalton major adult surface glycoprotein, one animal produced a rapid response but the others did not recognise it until late in infection. In general, the experimental findings are characterised by wide variability between individuals, as may perhaps be expected in a primate host for a human spectral disease.

Animals↗
PubMed1987

Antibody responses to human lymphatic filarial parasites.

The lymphatic filarial parasites, Brugia and Wuchereria, continue to present an immunological puzzle, particularly with respect to the development of natural resistance or damaging disease. We have approached this question by examining humoral responses to a few defined antigens of selected interest from these parasites, using sera from each category in the spectrum of filarial disease. Many antigens, such as the major adult surface protein of Mr 29,000 (29K), appear to be recognized at all stages of infection, but two components show interesting patterns of differential recognition. A triplet of proteins of Mr 65-75K associated with the microfilarial surface is preferentially bound by serum from patent microfilaraemic infections, whereas an unrelated 75K protein has been found to react only with antibody from amicrofilaraemic individuals. In general, however, the data obtained so far emphasize the importance of undertaking an antigenic analysis at the level of single epitopes. Such studies are now under way using recombinant proteins expressed in bacterial hosts.

Animals↗
PubMed1987

Secretory acetylcholinesterases from Brugia malayi adult and microfilarial parasites.

Brugia malayi, a lymphatic filarial parasite, secretes acetylcholinesterase during in vitro cultivation. A significant amount of enzyme activity was detected both in culture media and somatic extracts of adult and microfilarial stages of the parasite. The microfilarial stage produces three times more enzyme than adult parasites as a proportion of total protein. The enzyme has true acetylcholinesterase (AChE) activity as hydrolysis of acetylthiocholine is three times faster than butyrylthiocholine and is inhibited by eserine, a specific inhibitor of AChE. Secretory enzyme from adult female parasite excretory-secretory material (ES) was enriched 23 fold using copper chelating and concanavalin A (Con A) affinity chromatography. The Con A eluate showed a major protein band of 100 kDa and a minor 200 kDa component. The ES enzyme is antigenic and cross reacts with antibodies raised in mice against AChE from electric eel by enzyme-linked immunosorbent assay and immunoprecipitation. Immunoprecipitation of 125I-labelled microfilarial ES and adult ES with anti-electric eel AChE antibodies revealed three proteins of 30, 40 and 200 kDa in microfilariae and two proteins of 100 and 200 kDa in adult female ES. It appears that filarial secretory AChE exists in multiple molecular forms.

Acetylcholinesterase↗
PubMed1987

Cloned antigen genes of Brugia filarial parasites.

Human lymphatic filariasis is a chronic, potentially debilitating disease caused by Brugia and Wuchereria species of parasitic nematodes. The spectrum of clinical manifestations appears to be related to the immune response of individuals to invasive larvae, adult worms and circulating first-stage larvae (microfilariae). Potential immunopathological outcomes place constraints on vaccine development, emphasizing the need to understand the basis of immunity and pathology. Clones coding for a number of distinct antigenic proteins of Brugia pahangi and Brugia malayi have been isolated via immunological screening of a cDNA expression library. A small number of these expressed peptides show exclusive reactivity with antibody from amicrofilaraemic, potentially immune individuals. Surprisingly, a dominant immunogen isolated with human antibody is the filarial parasite homologue of heat shock protein (hsp) 70. This protein is constitutively expressed in both insect- and mammalian-dwelling parasitic stages, but does not appear to presented to the host immune system in intact worms.

Amino Acid Sequence↗
PubMed1983

In vitro induction of human helper T cell activity by Trypanosoma brucei.

Proliferation of peripheral blood T lymphocytes from unsensitized donors in response to fractions of Trypanosoma brucei was observed to be monocyte-dependent. The activated T cells displayed enhanced 'helper' activity, but no 'suppressor' activity during pokeweed mitogen (PWM)-induced immunoglobulin (IgM and IgG) synthesis, and were resistant to the inhibitory effects of theophylline on E rosette formation. Whilst in vivo studies have failed to reveal excessive T cell proliferation in patients with sleeping sickness, these results suggest a possible role for T cells in the induction of hypergammaglobulinaemia characteristic of this disease. Trypanosome fractions were not inhibitory to PWM-induced proliferation, and actually enhanced immunoglobulin synthesis. Thus it is unlikely that direct inhibition by the parasite per se is a major factor in the generation of immunosuppression by the T. brucei subgroup.

B-Lymphocytes↗
PubMed1981

Activation of human peripheral blood lymphocytes by a trypanosome-derived mitogen.

Activation of human peripheral blood lymphocytes from unsensitized donors by fractions of T.b rhodesiense has been demonstrated in vitro. Both T and B cells were stimulated to differentiate into blast cells. Purified T cells failed to proliferate, but did so when mitomycin C-treated non-T cells were added. Non-T cell proliferation (weak in comparison to the T cell response) was shown to be T-dependent. In unseparated cell cultures. cultures, IgM synthesis was enhanced, whilst IgG synthesis was unaltered. However, reconstitution of non-T cells with equal number of irradiated T cells resulted in enhanced IgG synthesis. Thus neither T nor B cells were activated directly, but required the co-operation of accessory cells to proliferate. The role of monocytes in the regulation of this response requires further assessment.

Cell Division↗
PubMed1980

Murine trypanosomiasis: cellular proliferation and functional depletion in the blood, peritoneum, and spleen related to changes in bone marrow stem cells.

Previous reports have described profound effects on the function of the lymphoid system, especially the spleen, in mice infected with Trypanosoma brucei. This study provides further evidence of major change in the cell populations of the blood, peritoneum, and bone marrow, but shows that at least some of the stem cells of the bone marrow survive the damage caused by trypanosomes and retain their ability to repopulate the animal. In these infected mice the initial parasitemia was terminated by day 11 and was followed by a subpatent period of approximately 7 days before a final, lethal parasitemia occurred. Lymphopenia preceded the initial and final waves of parasites in the blood, and there was a marked increase in circulating neutrophils and large mononuclear cells for 1 week after the termination of the first wave of bloodstream parasitemia and during the final lethal parasitemia. Dividing macrophages were detected in the peritoneum only briefly during week 1 of infection, but the total number of peritoneal cells was increased from day 8 until the mice died. The bone marrow is severely stressed by the parasite infection. Total cell numbers and spleen colony-forming cells in the bone marrow were profoundly depleted during the resolution of the first parasitemia, but both these parameters largely recovered during the subpatent period before the mice were killed by the disease. Immune function was restored gradually after treatment with Berenil late in infection. We conclude that the progenitors of lymphocytes as well as the mature cells are affected by trypanosomes, but that some of the early bone marrow stem cells escape and rapidly repopulate the peripheral organs upon removal of the parasites.

Animals↗
PubMed1980

Trypanotolerance in inbred mice: an immunological basis for variation in susceptibility to infection with Trypanosoma brucei.

Previous studies have suggested that the suppression of IgM responses induced by different strains of trypanosomes can be related to the virulence of the parasite in a defined mouse strain (Sacks et al. 1980). This study has examined immunosuppression induced by Trypanosoma brucei NIM7 in two mouse strains (C3H/He and [CBA/H x C57B1/6]F1) which differ considerably in their ability to survive infections. The results confirm our previous studies in that mice were able to survive infection whilst they maintained at last a residue of their ability to mount an IgM response to DNP-Ficoll, a T-independent antigen. Complete abrogation of the IgM response coincided with fulminating parasitaemia, resulting in the death of the host.

Animals↗