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G F Mitchell

Publications and source records attributed to G F Mitchell.

At least 127 records · Page 7Linked to original sources
PubMed1985

Schistosoma japonicum: the modulation of lung granuloma and inhibition of egg maturation in mice by human sera.

Human sera taken from patients with chronic schistosomiasis japonica have been demonstrated to have two effects on mice. Sera from those patients reduced the size of granuloma in mice sensitised for accelerated granuloma formation to eggs entrapped in the lungs of mice injected with the sera shortly before and at day 2 after intravenous egg challenge. The sera with this effect on the mouse lung granuloma models caused large segmented precipitates in the optimised circumoval precipitin test (COPT). Such sera also reduced the rate at which eggs matured in the liver and intestines of mice infected with S. japonicum. The results strongly support our postulate that a major cause of granuloma modulation in cases of chronic schistosomiasis japonica is antiembryonation immunity and that mice provide useful models for the analysis of our postulate. Identification of egg antigens responsible for the anti-embryonation effect should facilitate progress towards the development of a vaccine against granulomatous disease.

Animals↗
PubMed1984

The expression of Plasmodium falciparum bloodstage antigens in Escherichia coli.

A library of cDNA clones expressing proteins of the asexual blood stages of a Papua New Guinean isolate of Plasmodium falciparum (isolate FCQ27/PNG (FC27] was constructed in the bacteriophage vector lambda gt11-Amp3. In an in situ colony immunoassay, human serum was used to identify colonies producing natural immunogens. Sera from donors of defined clinical status, or reactive to a defined subset of natural immunogens were used to identify clones of particular interest (for example, clones reacting with convalescent but not with acute serum or clones expressing the isolate specific S-antigen of FC27). Antisera raised by immunizing mice and rabbits with cloned antigens were used to characterize the P. falciparum proteins corresponding to the antigen-positive clones. Nucleotide sequence analysis of an antigen found on the surface of cells infected with ring stage parasites revealed an unusual sequence coding for eight, four and three amino acid repeats rich in acidic amino acids. The discussion centres on the use of cloned antigens as tools for the analysis of the host-protective immune response and selection of candidate vaccine molecules.

Animals↗
PubMed1984

Identification of a cDNA clone encoding a mature blood stage antigen of Plasmodium falciparum by immunization of mice with bacterial lysates.

A cDNA library was constructed in pBR322 using mRNA from blood stages of a Papua New Guinean isolate of Plasmodium falciparum. Expression of parasite antigens was not directly detectable by conventional immunological assays. To circumvent this, mice were immunized with lysates of cDNA clones, and the antisera raised were assayed for anti-parasite reactivity. One cDNA clone was identified which reliably elicited antibodies to P. falciparum. The mouse antisera were used to characterize the native P. falciparum protein as a 120-kd protein, which is antigenic during natural infection. The protein occurs in late trophozoite and schizont stages and is found in isolates of the parasite from widely separated geographical areas. The genomic context of the antigen gene is conserved in the different isolates.

Animals↗
PubMed1984

Characterization of isolates and clones of Leishmania by analysis of kinetoplast DNA.

The genetic characterization of pathogenic isolates of Leishmania was attempted by analysis of the molecular properties of kinetoplast DNA (kDNA) minicircles. Unit minicircle size is not conserved during speciation of Leishmania since the minicircles of strains and clones of L t major are smaller (700 bp) than those found in certain strains of L mexicana ssp (820 bp), L donovani (850 bp) or L t tropica (900 bp). Schizodeme analysis of minicircles reveals a high degree of sequence divergence in kDNA of Leishmania with the degree of microheterogeneity varying between species. This sequence divergence allows the discrimination of species, strains, and clones of Leishmania into schizodemes . Southern blot hybridization experiments reveal that at high stringency overall minicircle sequence homology is conserved among clones and strains of one species (L t major) but not between different species. This property of minicircle DNA permits the use of kDNA probes as a species-specific diagnostic test for the identification of unknown Leishmania isolates. The properties of kDNA from an L t tropica strain LRC- L32 (a " recidiva " organism) are so diverged from those of L t major strains as to support the classification [22, 23] of L t tropica and L t major as separate species of Leishmania rather than subspecies of L tropica.

Animals↗
PubMed1984

Onchocerca gibsoni: increase of circulating egg antigen with chemotherapy in bovines.

Monoclonal antibodies directed to stage-specific surface antigens of Onchocerca gibsoni eggs were used in immunoradiometric assays to detect antigens in the sera of cattle infected with O. gibsoni. Two monoclonal antibodies detected antigens, presumably of egg origin, in sera. The target antigens appeared to be carbohydrate in nature and of variable molecular weights. Significant increases in levels of circulating egg antigens were found after treatment of infected cattle with benzimidazole compounds. These drugs cause disruption of embryogenesis and accelerated loss of worm uterine contents. In contrast, administration of either macrofilaricides or microfilaricides to infected cattle did not alter pretreatment levels of circulating egg antigens. Measurement of changes in levels of circulating antigens by immunoradiometric assays with stage-specific monoclonal antibodies provides a new means of assessing the efficacy of drugs and their site of action in onchocerciasis.

Animals↗
PubMed1984

Differences in the surface radioiodinated proteins of skin and uterine microfilariae of Onchocerca gibsoni.

Surface labeling studies using two populations of Onchocerca gibsoni microfilariae revealed important differences in major radioiodinated proteins. Small numbers of microfilariae harvested from the skin of cattle or the uteri of adult worms from skin nodules were purified, radioiodinated, solubilized and the proteins analysed by two dimensional gel electrophoresis and autoradiography. As reported previously, uterine microfilariae showed a complex profile of radioiodinated proteins, none of which appeared to be bovine albumin or immunoglobulin. In contrast, application of the same techniques to skin microfilariae demonstrated only one major labeled protein complex of approximate Mr 67 000. This protein complex was immunoprecipitated with an antiserum to bovine serum albumin. Surprisingly, fluorescence techniques failed to show bovine serum albumin on the surface of living microfilariae. Although the evidence is circumstantial at present, acquisition of host albumin (perhaps oriented in a particular way) may be a means whereby skin microfilariae evade immune effector mechanisms and, when living, generally fail to elicit inflammatory reactions in the skin of the host.

Animals↗
PubMed1984

Expression of Taenia taeniaeformis antigens in Escherichia coli.

Two important features of infection of mice with larvae of Taenia taeniaeformis are the ready demonstration of host protective antibodies and the ability to immunize susceptible strains of mice against first infection using crude parasite preparations. Candidate immunogens in established larvae and the invasive oncosphere have been identified by immunoprecipitation of radiolabeled parasite proteins with host-protective antibodies. To overcome the difficulties associated with purification of these antigens from parasite material, the alternative strategy of expressing parasite proteins in Escherichia coli has been adopted. Double stranded DNA complementary to mRNA from 28 day old liver larvae was inserted into the beta-galactosidase gene of the bacteriophage lambda Amp 3. Some recombinants express a fusion protein with additional parasite-encoded epitopes located at the C-terminal end of the beta-galactosidase protein. Four clones that reacted with antibodies in an E. coli colony immunoassay were selected for detailed characterization. Analysis of lysates of the selected clones by SDS-PAGE and Western blotting revealed that each clone produced an abundant fusion protein that reacted specifically with a hyperimmune anti-oncosphere serum. Sibling analysis revealed that the four antiserum-positive clones encoded three immunologically-distinct parasite antigens. The identity of the native protein of larvae encoded by one clone (designated TA10) was an abundant antigen of Mr 70,000. This approach allows the assessment of antigens expressed in E. coli as vaccines in susceptible strains of mice by direct immunization and challenge and thus the development of a model defined-antigen vaccine against a larval cestode parasite.

Animals↗
PubMed1984

Vaccination against cutaneous leishmaniasis in mice using nonpathogenic cloned promastigotes of Leishmania major and importance of route of injection.

In vaccination studies, mice have been injected by different routes with living promastigotes of nonpathogenic leishmania followed by cutaneous challenge with pathogenic promastigotes. Parasites used for vaccination have been promastigotes of the cloned parasite lines A12 and A52 derived from Leishmania major isolate L137, or long-term cultured promastigotes of the leishmaniasis recidiva isolate, L32 (L.t.tropica). None of these protozoa causes lesions after cutaneous injection to mice. Disease in previously injected mice has been monitored after cutaneous challenge with promastigotes of a virulent cloned line, V121, derived from isolate L137. Mice used were C57BL/6 (genetically resistant), BALB/c and BALB/c.H-2b (genetically susceptible) and BALB/c.H-2k (also genetically susceptible but sometimes less so than BALB/c). C57BL/6 mice were almost completely resistant to subsequent cutaneous disease when challenged after intraperitoneal injection of viable nonpathogenic cloned promastigotes. In contrast, BALB/c, BALB/c.H-2b and BALB/c.H-2k mice challenged after intravenous or intraperitoneal injection were only protected partially against cutaneous leishmaniasis. These vaccinated mice generally showed persistent low grade cutaneous disease for many months after challenge. High doses of viable L32 promastigotes injected intraperitoneally were also able to induce a degree of resistance to subsequent cutaneous leishmaniasis. Using any protocol, subcutaneous injections have been totally without protective effects as have been killed promastigotes injected by any route to mice. Subcutaneous injections appear to be ineffective rather than counterproductive in that mice injected by both the intravenous and subcutaneous routes with nonpathogenic living cloned promastigotes resemble mice injected by the intravenous route in their disease status following challenge.

Animals↗
PubMed1984

Immunization with Nematospiroides dubius adult worms plus pertussigen has different consequences in mice of various genotypes.

In the Nematospiroides dubius/mouse model of intestinal nematodiasis, young C57BL/6 male mice are highly susceptible in that they remain chronically infected after repeated oral administrations of infective third-stage larvae (L3). It was shown previously that a parenteral injection of small numbers of living adult worms, or their aqueous extracts, was highly effective at sensitizing these mice for accelerated rejection of adult worms but only when the mice were additionally injected with pertussigen from Bordetella pertussis. 'Host protective antigens' appear to be soluble proteins. This method of vaccination is largely ineffective in male mice of strains CBA/H and C3H/He and can actually be counterproductive in BALB/c mice. Thus, adult worms injected subcutaneously into male BALB/c mice will sensitize them for accelerated rejection and this effect is neutralized by simultaneous injection of such mice with pertussigen. The data highlight the known complexity of host-parasite relationships in intestinal parasitism and suggest that several different defects underlie the genetically based inability of some mice to reject N. dubius worms from the intestines following exposure to N. dubius antigens.

Animals↗
PubMed1984

Antibody responses to Toxocara canis using sera from parasite-infected mice and protection from toxocariasis by immunisation with ES antigens.

Evidence for serological cross-reactions between Toxocara canis and related nematode parasites has been sought using sera from infected mice and the immunodiagnostic excretory/secretory (ES) antigen of T. canis larvae. Sera from mice experimentally infected with either T. canis, T. cati, T. pteropodis, Toxascaris leonina or Ascaris suum were tested for the presence of antibodies to T. canis ES antigen by solid-phase radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA) using anti-mouse IgG. T. canis ES antigen was obtained from the medium used to culture T. canis larvae, this being the same source of antigen as has been used in a highly satisfactory immunodiagnostic test for human toxocariasis (visceral larva migrans). Sera from mice infected with either A. suum, T. cati or T. pteropodis showed definite cross-reactions with T. canis ES antigen in one or other of the two essays used. No reaction between anti-T. leonina sera and T. canis ES antigen was demonstrated, although infection levels with this parasite were low in mice. Concerning biological activity of ES antigen, it is of some interest that mice hyperimmunised with T. canis ES antigen in adjuvant were shown to be significantly resistant to infection by T. canis.

Animals↗
PubMed1984

Leishmania major: identification of stage-specific antigens and antigens shared by promastigotes and amastigotes.

A battery of antisera to Leishmania major was used to identify stage-specific antigens, or antigens expressed predominantly in amastigotes or promastigotes. At least 30 protein antigens common to amastigotes and promastigotes could be detected in 35S-methionine labelled preparations. They ranged in molecular weight from 25 000 to 165 000. Two amastigote specific antigens and five antigens expressed predominantly in the amastigote were detected in biosynthetically-labelled preparations. Five promastigote specific antigens were also identified. Antibodies from hyperimmunized mice that were resistant to reinfection with L. major recognized mainly antigens shared by the two life-cycle stages of the parasite. Analysis of parasite antigens on 'western blots' provided a different picture from that obtained by immunoprecipitation and gel electrophoresis of 35S-methionine labelled polypeptides. Only 19 antigens were detected and they were all shared by the two parasite forms. However, the abundance and immunogenicity of some of these antigens may be different in the two life-cycle stages of the parasite. Using the various sera, seven shared membrane antigens were identified in radio-iodinated preparations. Antibodies from hyperimmunized resistant mice that recognized shared antigens in 35S-methionine labelled preparations, detected four amastigote membrane antigens not detected by other sera. The function of the stage-specific antigens remains to be established. It is expected that individual antigens produced by recombinant DNA technology will allow these studies to proceed.

Animals↗
PubMed1984

Towards molecular vaccines against parasites.

A strategy is outlined for the identification of 'host protective antigens' of parasites using batteries of polyspecific sera from clinically and parasitologically defined individuals of the host population. Together with monospecific antibodies and gene cloning techniques, the approach aims to identify, characterize and produce defined antigens that, through active immunization, are necessary or sufficient for expression of host protective immunity on subsequent contact with the parasite population. The availability of a relevant mouse model system greatly increases the rate at which progress is made in the testing of vaccinating efficacy. Advantages and limitations of molecular, defined-antigen vaccines are discussed as are integral components of the strategy such as probes for the analysis and quantitation of parasite population heterogeneity and antigenic variability, identification of principal mechanisms of 'immune evasion' utilized by invasive and resident parasites, reliable diagnostic procedures, and predictable antigen delivery systems and adjuvants. Emphasis is placed on the value of combining epidemiological and clinical studies with immunochemical and molecular cloning approaches in achieving the molecular vaccine objective.

Animals↗
PubMed1984

Serological diagnosis of Echinococcus granulosus infection in sheep using cyst fluid antigen processed by antibody affinity chromatography.

Serum antibody responses in sheep naturally or experimentally infected with Echinococcus granulosus and/or other larval cestodes were examined using an enzyme-linked immunosorbent assay (ELISA) with various antigens prepared from sheep hydatid cyst fluid ( SHCF ). Serum donors included: sheep experimentally infected with E. granulosus and their age-matched non-infected controls; sheep experimentally infected with other helminth parasites; sheep naturally infected with E. granulosus both from Tasmania and the Australian mainland; sheep from Tasmania naturally infected with larval cestodes other than E. granulosus; and naturally reared sheep completely free from infection with larval cestodes. Attempts were made to eliminate serological reactions which were not specific for E. granulosus by using a series of antibody affinity chromatography steps to deplete crude SHCF antigen; these included adsorption with a monoclonal antibody, 3EgH 29-2, removal of host IgG using rabbit anti-sheep IgG antibody, and removal of antigens which bound non-specifically to normal sheep immunoglobulin. The final affinity-depleted antigen product was designated AD SHCF . Specific serological reactivity in infected sheep was very low. Affinity depletion of SHCF using 3EgH 29-2 did not appear to increase the specificity of serological diagnosis of E. granulosus infection when experimentally infected sheep were compared with their non-infected controls provided the latter were age-matched with experimental animals. The other affinity adsorption steps significantly reduced non-specific background binding to antigen by normal sheep serum. Despite this reduction in background in the ELISA, only low levels of antibody could be detected in naturally-infected sheep.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1984

Serological diagnosis and post-operative surveillance of human hydatid disease. II. The enzyme-linked immunosorbent assay (ELISA) using various antigens.

The ELISA using a urease conjugated antibody and 3 antigen preparations was examined for sensitivity and specificity in hydatid diagnosis. Four groups of sera tested were: 80 samples from patients with confirmed hydatid infection; 51 latex agglutination test (LA) +ve and immunoelectrophoresis test (IEP) -ve sera from patients with miscellaneous symptoms or disorders other than hydatid disease; 195 'normal' sera from healthy donors; 115 sera from persons infected with other parasites. Antigens tested included crude sheep hydatid cyst fluid (CSHCF), CSHCF partially purified by salt fractionation (PPSHCF) and CSHCF purified by sequential affinity chromatography depletion with rabbit anti-sheep IgG, 3EgH 29-2 anti-Echinococcus monoclonal antibody and 'normal' sheep Ig (ADSHCF). All 3 antigens showed high sensitivity in detecting antibody in serum from hydatid-infected patients, and gave excellent discrimination between these samples and the LA +ve IEP -ve sera and the 'normal' sera. Cross-reactions occurred with antibodies in the sera of patients with other parasitic infections, especially other larval cestodes and filarial parasites. Superior specificity was achieved with both CSHCF and ADSHCF, and ADSHCF reacted only with a single serum sample from an E. multilocularis patient. It was concluded that a combination of ELISA and IEP was useful for the diagnosis of hydatid disease, that ELISA at a single dilution could be useful as a screening test where other larval cestode infections were not prevalent and that ELISA was not of value for post-operative surveillance.

Antibodies↗
PubMed1984

Detection of circulating antigen in bancroftian filariasis by using a monoclonal antibody.

A monoclonal antibody designated Gib 13-5-2 (Gib 13) and directed against the cattle parasite Onchocerca gibsoni was used in a two-site immunoradiometric assay (IRMA) for detection of circulating antigen in the sera of Wuchereria bancrofti-infected individuals from Sri Lanka and Papua New Guinea. The microfilaremic patients were, in general, serum antigen positive by the Gib 13 IRMA. Among the amicrofilaremic patients, 47% of those with lymphedema, lymphangitis, hydrocele, etc., and 25% of those with elephantiasis had circulating antigen. Correlation of the presence of serum antigen with clinical status indicated that the Gib 13 target antigen in serum is probably an indicator of either active or early infection, or of both. The antigen was also detected in the urine of some patients. By sodium dodecyl sulphate polyacrylamide gel electrophoresis immunoblotting, Gib 13 target antigens of molecular weights 67,000 and 52,000 were identified.

Antibodies, Monoclonal↗
PubMed1984

Plasmodium falciparum complementary DNA clones expressed in Escherichia coli encode many distinct antigens.

A library of cDNA clones expressing antigens of the asexual blood-stages of Plasmodium falciparum (isolate FCQ27/PNG) was constructed in the bacteriophage vector gamma gt11-Amp3. Clones expressing P. falciparum antigens (as polypeptides fused to beta-galactosidase) were selected by their reactivity in an in situ colony immunoassay with affinity-purified malaria antibodies. A detailed analysis of 78 antigen-positive clones selected from approximately 10,000 recombinant clones has shown them to correspond to many different parasite antigens. cDNA hybridization studies on this array of 78 antigen-positive clones have so far identified 18 families of sibling clones with 22 clones as yet unassigned, the majority of which may represent additional unique sequences. Only about 20% of the clones synthesized abundant amounts of the malaria antigen/beta-galactosidase fused polypeptide but each multi-member family except one was represented by at least one clone producing a fused polypeptide in abundance. Antisera have been raised against cloned malaria antigens by immunizing mice and rabbits with bacterial lysates and purified fused polypeptides, respectively. These antisera have been used to characterize the antigens in P. falciparum that correspond to the various antigen-positive clones. The variety of distinct antigens recognized by these antisera confirms that the clone library contains coding sequences for many different antigens.

Animals↗