Derivation of highly mefloquine-resistant lines from Plasmodium falciparum in vitro.

Serial passage of a multidrug-resistant clone of Plasmodium falciparum in concentrations of mefloquine hydrochloride ranging from 30 to 2,400 ng/ml resulted in the derivation of increasingly resistant parasite lines in vitro. Parasite lines isolated in mefloquine concentrations greater than 300 ng/ml demonstrated increased vacuolization, enhanced pigment production, and increased growth rates as compared with the progenitor clone, W2-mef. Although microdilution incorporation assays demonstrated that the 50% inhibitory concentration (IC50) of mefloquine were similar for all lines, the IC90, IC95, and IC99 levels were significantly increased. Growth rate assays performed in 5% hematocrit suspensions demonstrated different levels of mefloquine resistance among these lines. Under these conditions the most resistant line, Mef 2.4, grew efficiently in approximately 10-fold higher concentrations of mefloquine than the progenitor clone W2-mef. Analysis of drug susceptibility profiles to mefloquine hydrochloride, chloroquine diphosphate, quinine sulfate, and halofantrine hydrochloride indicated that selection for high levels of mefloquine resistance had resulted in significant increases in resistance to halofantrine and increased sensitivity to chloroquine. The phenotypic changes demonstrated in the most resistant line, Mef 2.4, reflect a multidrug resistant-like phenotype, and appear to mimic changes recently reported in drug susceptibility profiles of recrudescent isolates following mefloquine treatment failures in Thailand.

Reverse transcription of trypanosome variable antigen mRNAs initiated by a specific oligonucleotide primer.

African trypanosomes change their antigenicity by successively expressing different members of a group of related but highly diverse proteins, the variant surface glycoproteins (VSGs). We describe a synthetic oligonucleotide that can prime specifically reverse transcription of VSG mRNA out of total trypanosome poly(A)+ RNA. The specificity of this priming was verified by cDNA sequence analysis of the transcription products and by the demonstration of variant-specific hybridization of the individual cDNAs to cellular RNA. The oligonucleotide primer also was used as a probe for the conserved sequence found on these VSG mRNAs in trypanosome genomic DNA libraries. A large number of primer-positive clones were detected in a Trypanosoma gambiense genomic library, but very few positive signals were found in a library of Trypanosoma congolense genomic DNA.

Purification and cell-free translation of mRNA coding for a variant specific antigen from Trypanosoma brucei gambiense.

Messenger RNA (mRNA) coding for a variant specific surface antigen (VSA) from Trypanosoma brucei gambiense was isolated from total trypanosomal polyribosomes by indirect immunoprecipitation. An IgG fraction of antisera to purified VSA was obtained by ion exchange chromatography and Protein A-agarose affinity chromatography. These antibodies were then subjected to affinity chromatography on a VSA-agarose column to remove non-specific IgG. Polyribosomes from the same antigenic variant of T. b. gambiense were isolated from a cell lysate and those polysomes bearing nascent VSA were bound to the IgG by gentle mixing and the complexes formed were retrieved by precipitation with fixed Staphylococcus aureus cells. The VSA-specific mRNA was separated from these complexes by dissociation of the polysomes, deproteinization, and affinity chromatography on oligo(dT)-cellulose. The mRNA isolated in this way was shown to be undegraded, active in protein synthesis and homogeneous electrophoretically. The products of the cell-free translation of this mRNA were precipitable by specific IgG but not by antiserum to a heterologous VSA. The translation product was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel exclusion chromatography. The molecular weight of the mRNA was measured by electron microscopy, agarose and polyacrylamide gel electrophoresis. Enough highly purified mRNA can be isolated in this manner to be used for hybridization analysis of the VSA gene(s).

Tegument-associated antigen modulation by adult male Schistosoma mansoni.

Adult Schistosoma mansoni from mice were incubated in rabbit anti-mouse immunoglobulin class or subclass-specific sera. The rabbit antibody binding was then visualized by incubation of the labeled worms in fluoresceinated Staphylococcus aureus, which associated with the Fc portion of the rabbit antibody molecule by means of bacterial cell wall Protein A. The assay system was found to be simple, inexpensive, specific, repeatable, and rapidly accomplished. By using this technique, previous work documenting the presence of mouse IgG1, IgG2a, IgG2b, IgG3, IgA, and IgM on the tegumental surface of adult worms of mouse origin was confirmed. It was further confirmed that the complexing of rabbit antibody to the mouse antigen led to the shedding of that antigen from the parasite's surface membrane within 20 min at 37 degrees C. This shedding phenomenon was specific for only those antigens interacting with the ligand and was inhibited by incubation with sodium fluoride, 2-deoxy-D-glucose, cytochalasin B, cytochalasin D, caffeine, or at 4 degrees C. These data indicate a sophisticated control mechanism exercised by the parasite over its interface with the host and may indicate a complexity of the host-parasite relationship not heretofore recognized.

Evidence for adsorption of heterospecific host immunoglobulin on the tegument of Schistosoma mansoni.

Mice infected with Schistosoma mansoni were immunized against human type B Rh-positive (B+) RBC, bovine serum albumin, or horseradish peroxidase. Adult parasites, recovered by perfusion, extensively washed, and incubated in their respective antigens, selectively bound to their tegumental surfaces only those antigens to which their murine host had been immunized. All controls supported the specificity of those reactions leading to the conclusion that adult S. mansoni in mice have the ability to adsorb heterospecific antibody onto their tegumental surfaces. These surface immunoglobulins were lost within 10 min when complexed with theri antigens or within 2.5 hr when incubated at 37 degrees C. Parasites that had lost their tegumental immunoglobulins regained them when incubated in normal mouse or rat anti-human type B Rh-negative (B-) RBC serum. Those parasites that had their surface immunoglobulins reconstituted with rat anti-human B- serum specifically bound human B- RBC, suggesting the possible presence of Fc receptors on adult S. mansoni.