Temperature is a cue for gene expression in the post-infective L3 of the parasitic nematode Brugia pahangi.

The temporal expression pattern of two genes, Bp-cdd and Bp-S3, was studied at defined points throughout the life cycle of Brugia pahangi. Both mRNAs were up-regulated to coincide with the transition of the L3 from the vector to the mammalian host. Bp-cdd was expressed almost exclusively in the post-infective (p.i.) L3 and L4 stages of the life cycle while Bp-S3 was also expressed in adult worms, but at a much lower level than in the larval stages. Immunogold labelling with an antiserum raised to the recombinant Bp-CDD localised the native antigen to the hypodermis in the p.i. L3 and L4. Specific labelling was not detected in the adult worm. The expression of both mRNAs could be triggered by exposure of the vector-derived L3 to a simple mammalian culture system. Analysis of the factors, which induced expression suggested that the temperature shift which accompanies the transition from mosquito to mammal was the most important cue for expression of both genes.

Developmental expression of a Theileria annulata merozoite surface antigen.

Culture of a lymphoblastoid cell line infected with the macroschizont stage of the protozoan parasite Theileria annulata at 41 degrees C induces differentiation to the next stage, the merozoite. We have demonstrated that this development results in the loss of monoclonal antibody epitopes associated with the macroschizont stage, and the appearance of epitopes associated with the piroplasm (the intra-erythrocytic stages). One of the monoclonals (5E1) was shown by immunoelectron microscopy to bind to the surface of heat-induced culture forms which had size and structural characteristics of the merozoite. The monoclonal was found to detect two polypeptides of 30 kDa and 25 kDa in extracts of piroplasms. The 30-kDa polypeptide was also detected in a merozoite extract, but was not detected in an extract derived from macroschizont-infected lymphoblastoid cells. We conclude that the heat-induced differentiation of T. annulata in vitro results in the expression of a 30-kDa molecule which is located at the surface of the merozoite, and discuss the potential of this molecule as a component in a subunit vaccine.

A major surface antigen of procyclic stage Trypanosoma congolense.

Five monoclonal antibodies (mAb) were raised that bound to the surface of procyclic stage Trypanosoma congolense with high intensity in immunofluorescence. Immunoblot analysis of trypanosome lysates using 3 of these mAb revealed a diffuse SDS-PAGE band of 36-40 kDa. The purified antigen did not react with Coomassie Blue or silver stains, but did stain blue with Stains-all, indicating acidity. For the one mAb tested, the epitope was periodate-sensitive and therefore probably glycan. Although this antigen shares properties with procyclin/PARP, which forms a surface coat on procyclic Trypanosoma brucei, a search in T. congolense for homologues of a procyclin/PARP gene revealed only non-coding sequence of partial similarity. Using a differential screen, a procyclic stage T. congolense cDNA clone was isolated that encoded a putative 256-amino acid protein containing 2 peptides chemically sequenced independently by Beecroft et al. [36]. The protein, termed glutamate and alanine-rich protein (GARP), has potential hydrophobic leader and tail sequences (the latter with potential for replacement by a glycosyl phosphoinositol anchor) and no potential N-linked glycosylation sites. It has no significant sequence homology with known proteins. Antibodies against a translational fusion of GARP bound specifically in Western blots to a band very similar to that detected by the mAb and also to the purified antigen. Immunogold electron microscopy revealed a dense packing of the antigen on the cell surface. It appears that procyclic T. brucei and T. congolense have major surface proteins with structural analogy, but with no sequence homology.

Kinetoplast DNA from Trypanosoma vivax and T. congolense.

We have analysed kinetoplast DNA (kDNA) of the African trypanosomes Trypanosoma vivax and T. congolense. The maxi-circles from these organisms resemble those of T. brucei in size, but only to a limited extent in sequence as judged from restriction enzyme digests and DNA X DNA hybridization. The kDNA networks of T. vivax have three distinguishing features: they contain the highest maxi-circle concentration of any kDNA (at least twice that of T. brucei); they contain the smallest mini-circles (465 bp) yet found thus far and the width of the kDNA nucleoid in thin sections is correspondingly small (55 nm against 91 nm for T. brucei); they contain a substantial fraction of mini-circle dimers.

cut-1-like genes are present in the filarial nematodes, Brugia pahangi and Brugia malayi, and, as in other nematodes, code for components of the cuticle.

A fragment of a cut-1 like gene from the filarial nematode Brugia pahangi (designated Bp-cut-1) was isolated by PCR from genomic DNA. The sequence was used to design primers for use in RT-PCR and resulted in the isolation of a cDNA fragment from larvae in the process of the L3-L4 moult. Screening of a B. malayi genomic library identified a single clone, Bm-cut-1. Using primers designed from the Brugia sequences, semi-quantitative RT-PCR was carried out on 11 different life cycle stages chosen to cover periods around the moult and inter-moult periods. This analysis demonstrated that the cut-1 mRNA was most abundant preceding the moult, consistent with its function as a cuticular protein. Immuno-gold electron microscopy using an affinity purified antiserum raised to the highly conserved region of Ascaris CUT-1 confirmed that the protein was restricted to a tight band in the median layer of the cuticle. Despite the fact that no transcripts could be detected in mature adult worms by RT-PCR, immuno-gold microscopy revealed staining of the microfilarial cuticle within the uterus of the adult female worm, suggesting that other cut-1-like genes are present in Brugia.

Absence of a surface coat from metacyclic Trypanosoma vivax: possible implications for vaccination against vivax trypanosomiasis.

Trypomastigotes attached to the wall of the hypopharynx in tsetse flies infected with Trypanosoma vivax are believed to represent the true metacyclic stage of this trypanosome. Electron microscopy demonstrates that attachment is mediated by hemidesmosome-like junctions along the flagellar membrane and that none of the trypomastigotes, either attached or free in the hypopharynx lumen, possesses a surface coat comparable with that on the metacyclics of T. brucei and T. congolense and on the bloodstream stages of all salivarian trypanosomes. As the variable antigen of bloodstream and metacyclic T. brucei is located in the surface coat, the absence of the coat from metacyclic T. vivax suggests that the mechanism of antigenic variation in this species may be somewhat different from that of antigenic variation in T. brucei, and that vaccination of cattle against T. vivax may prove a simpler proposition than vaccination against T. brucei.

Freeze-fracture studies on the surface membranes of pleomorphic bloodstream and in vitro transformed procyclic Trypanosoma brucei.

The surface membranes of bloodstream long slender, short stumpy and culture procyclic stages of Trypanosoma brucei brucei were compared with respect to freeze-fracture electron microscopy, intramembrane particle (IMP) distribution and beta-hydroxysterol content as shown by the characteristic intramembrane lesions induced by the polyene antibiotic, filipin. Little difference was observed between IMP density of long slender and short stumpy form body membranes: IMP's were more abundant on the protoplasmic face (PF) than on the exoplasmic face (EF). The procyclic culture form body membrane showed an increased density of PF IMPs and a decreased density of EF IMPs over their bloodstream short stumpy form predecessors. Flagellar membrane fracture faces displayed higher IMP densities than body membrane fracture faces of the same trypanosome. The numbers of filipin-induced lesions (FIL) indicated an increased level of beta-hydroxysterols in the short stumpy forms relative to the level in the long slender bloodforms. FIL density was further increased in the body membrane of the procyclic culture form. FIL density was higher in the flagellar membrane than in the corresponding body membrane and FIL were excluded from flagellum to body attachment zones of the flagellar membrane of all stages. The polarity of the FIL in the surface membranes was reversed on transforming from bloodstream to culture procyclic stages. These observations indicate qualitative differences between the surface membranes of the three stages, independent of the presence or absence of the surface coat.

In vitro cultivation of Trypanosoma congolense: the production of infective forms from metacyclic trypanosomes cultured on bovine endothelial cell monolayers.

After transfer to bovine endothelial cell monolayers cultured in Eagle's minimal essential medium at 28 degrees C or 37 degrees C metacyclic trypanosomes of three cloned stocks of Trypanosoma congolense became morphologically similar to parasites found in the bloodstream of the vertebrate host. The trypanosomes resumed division and grew in close association with the mammalian cells, which were essential for growth. These dividing infective forms had the ability to cause local skin reactions and systemic infections when inoculated intradermally into rabbits. Trypanosomes grown in medium supplemented with foetal calf serum (FCS) eventually differentiated into procyclic forms. No such change occurred in medium supplemented with normal bovine serum. If procyclic forms in FCS were allowed to continue their differentiation at 28 degrees C they eventually produced epimastigotes which gave rise to infective metacyclic trypanosomes once more. It was thus possible to grow and maintain several different developmental stages of T. congolense by varying culture conditions.

Liposomes encapsulating polymeric chitosan based vesicles--a vesicle in vesicle system for drug delivery.

Drug delivery systems comprising vesicles prepared from one amphiphile encapsulating vesicles prepared from a second amphiphile have not been prepared previously due to a tendency of the bilayer components of the different vesicles to mix during preparation. Recently we have developed polymeric vesicles using the new polymer-palmitoyl glycol chitosan and cholesterol in a 2:1 weight ratio. These polymeric vesicles have now been encapsulated within egg phosphatidylcholine (egg PC), cholesterol (2:1 weight ratio) liposomes yielding a vesicle in vesicle system. The vesicle in vesicle system was visualised by freeze fracture electron microscopy. The mixing of the different bilayer components was studied by monitoring the excimer fluorescence of pyrene-labelled polymeric vesicles after their encapsulation within egg PC liposomes or hexadecyl diglycerol ether niosomes. A minimum degree of lipid mixing was observed with the polymeric vesicle-egg PC liposome system when compared to the polymeric vesicle-hexadecyl diglycerol ether niosome system. The polymeric vesicle-egg PC vesicle in vesicle system was shown to retard the release of encapsulated solutes. 28% of 5(6)-carboxyfluorescein (CF) encapsulated in the polymeric vesicle compartment of the vesicle in vesicle system was released after 4 h compared to the release of 62% of encapsulated CF from plain polymeric vesicles within the same time period.

Polymeric chitosan-based vesicles for drug delivery.

A simple carbohydrate polymer glycol chitosan (degree of polymerization 800 approx.) has been investigated for its ability to form polymeric vesicle drug carriers. The attachment of hydrophobic groups to glycol chitosan should yield an amphiphilic polymer capable of self-assembly into vesicles. Chitosan is used because the membrane-penetration enhancement of chitosan polymers offers the possibility of fabricating a drug delivery system suitable for the oral and intranasal administration of gut-labile molecules. Glycol chitosan modified by attachment of a strategic number of fatty acid pendant groups (11-16 mol%) assembles into unilamellar polymeric vesicles in the presence of cholesterol. These polymeric vesicles are found to be biocompatible and haemocompatible and capable of entrapping water-soluble drugs. By use of an ammonium sulphate gradient bleomycin (MW 1400), for example, can be efficiently loaded on to these polymeric vesicles to yield a bleomycin-to-polymer ratio of 0.5 units mg(-1). Previously polymers were thought to assemble into vesicles only if the polymer backbone was separated from the membrane-forming amphiphile by a hydrophilic side-arm spacer. The hydrophilic spacer was thought to be necessary to decouple the random motion of the polymer backbone from the ordered amphiphiles that make up the vesicle membrane. However, stable polymeric vesicles for use in drug delivery have been prepared from a modified carbohydrate polymer, palmitoyl glycol chitosan, without this specific architecture. These polymeric vesicles efficiently entrap water-soluble drugs.

The influence of polymer architecture on the protective effect of novel comb shaped amphiphilic poly(allylamine) against in vitro enzymatic degradation of insulin--towards oral insulin delivery.

Nanocomplexes formed between amphiphilic poly(allylamine) (PAA) and insulin were prepared, characterised and the impact of polymer architecture on the protection of insulin against three enzymes was investigated. PAA previously modified with either cetyl or cholesteryl pendant groups at two levels of hydrophobic grafting and its quaternised derivatives were used to produce polymer-insulin nanocomplexes. Transmittance study, differential scanning calorimetry, hydrodynamic size and zeta potential measurement were conducted and the morphology of the complexes were visualised using transmission electron microscopy. All polymers were found to have an optimal polymer to insulin ratio of 0.4:1 mg mL(-1) with particle size ranging from 88 to 154 nm. Polymer architecture has an impact on the morphology of the complexes produced but has little influence on the complexation efficiency (CE). Almost all polymers were unable to produce complexes with a CE of above 50%. Most polymers demonstrated an ability to reduce insulin degradation by trypsin while the polymer architecture plays a pivotal role against alpha-chymotrypsin and pepsin degradation. Quaternised cholesteryl polymers were able to significantly limit insulin degradation by alpha-chymotrypsin while cetyl polymers were particularly effective against pepsin degradation. These results indicated that a combination of polymers might be required to enhance protection against all three proteolytic enzymes for efficacious oral delivery of insulin.

The complexation between novel comb shaped amphiphilic polyallylamine and insulin: towards oral insulin delivery.

Novel amphiphilic polyallylamine (PAA) were previously synthesised by randomly grafting palmitoyl pendant groups and subsequent quaternising with methyl iodide. The ability of these self-assembled polymers to spontaneously form nano-complexes with insulin in pH 7.4 Tris buffer was evaluated by transmittance study, hydrodynamic size and zeta potential measurements. The transmission electron microscopy images showed that non-quaternised polymer complexes appeared to form vesicular structures at low polymer:insulin concentrations. However, at higher concentrations they formed solid dense nanoparticles. The presence of quaternary ammonium moieties resulted in insulin complexing on the surface of aggregates. All polymers exhibited high insulin complexation efficiency between 78 and 93%. Incubation with trypsin, alpha-chymotrypsin and pepsin demonstrated that most polymers were able to protect insulin against enzymatic degradation by trypsin and pepsin. Quaternised polymers appeared to have better protective effect against trypsinisation, possibly due to stronger electrostatic interaction with insulin. Interestingly, non-quaternised polymers significantly enhanced insulin degradation by alpha-chymotrypsin. All polymers were less cytotoxic than PAA, with the quaternised polymers exhibiting up to 15-fold improvement in the IC(50) value. Based on these results, quaternised palmitoyl graft polyallylamine polymers showed promising potential as oral delivery systems for insulin.

Differentiation in Trypanosoma brucei: host-parasite cell junctions and their persistence during acquisition of the variable antigen coat.

Acquisition of the variable antigen-containing surface coat of Trypanosoma brucei occurs at the metacyclic stage in the salivary glands of the tsetse fly vector. The differentiation of the metacyclic trypanosome in the gland has been studied by scanning electron microscopy and by transmission electron microscopy of thin sections and freeze-fracture replicas. The uncoated epimastigote trypanosomes (with a prenuclear kinetoplast) divide while attached to the salivary gland epithelium brush border by elaborate branched flagellar outgrowths, which ramify between the host cell microvilli and form punctate hemidesmosome-like attachment plaques where they are indented by the microvilli. These outgrowths become reduced as the epimastigotes transform to uncoated trypomastigotes (with postnuclear kinetoplast), which remain attached and capable of binary fission. The flagellar outgrowths disappear but the attachment plaques persist as the uncoated trypomastigotes (premetacyclics) stop dividing and acquire the surface coat to become 'nascent metacyclics'. Coat acquisition therefore occurs in the attached trypanosome and not, as previously believed, after detachment. Coating is accompanied by morphological changes in the glycosomes and mitochondrion of the parasite. Freeze-fracture replicas of the host-parasite junctional complexes show membrane particle aggregates on the host membrane but not on the parasite membrane. It is suggested that disruption of the complex occurs when maximum packing of the glycoprotein molecules has been achieved in the trypanosome surface coat, releasing the metacyclic trypanosome into the lumen of the gland.

The glycosomes of trypanosomes: number and distribution as revealed by electron spectroscopic imaging and 3-D reconstruction.

Computer-aided 3-D reconstruction of trypanosomes from 0.35-micron-thick sections imaged on the Zeiss 902 electron microscope are being used to study the dynamics of cell organization. Segregation of glycolytic enzymes into glycosomes raises questions concerning the distribution and biogenesis of these organelles. Direct counts of glycosomes from Trypanosoma evansi indicate 30-40 per cell and for the closely related T. brucei, 65 per cell. These figures contrast with the estimates of others who have used model-based morphometric methods to obtain a value of 230 per cell.

Identification of abundant mRNAs from the third stage larvae of the parasitic nematode, ostertagia ostertagi.

Reverse transcriptase-PCR (RT-PCR) was carried out on total RNA prepared from the third-stage larvae (L3) of Ostertagia ostertagi in order to clone and characterize the major transcripts expressed in this larval stage, as an initial investigation of arrested larval development in the parasite. Distinct bands were visible on an agarose gel and four of these were cloned and sequenced. Three of the bands represented multiple transcripts, while the fourth band encoded the enzyme GTP cyclohydrolase I (GTP-CH), which catalyses the first and rate-limiting step in pteridine biosynthesis. Northern blot analysis and RT-PCR demonstrated that GTP-CH is highly up-regulated in the L3 stage and undetectable in either the L2 or adult stages. Using immunogold electron microscopy, GTP-CH was shown to be predominantly localized to the cell body of the body wall muscles and the cells of the intestine in the L3.

Leishmania mexicana: induction of metacyclogenesis by cultivation of promastigotes at acidic pH.

Cultivation of recently transformed Leishmania mexicana promastigotes at pH 5.5 in Schneider's Drosophila medium supplemented with 20% fetal calf serum produced a homogeneous stationary phase population morphologically similar to metacyclic forms. The cultured forms developed functional characteristics consistent with being metacyclic: they were resistant to complement-mediated lysis, possessed a discernable surface membrane coat in transmission electron micrographs, and were highly infective to peritoneal macrophages in vitro. In contrast, growth of promastigotes at pH 7.0 produced morphologically mixed populations of stationary phase promastigotes, but including a subpopulation with metacyclic-like morphology. These results provide a method for culturing pure populations of L. mexicana metacyclics and provide evidence that metacyclics are biochemically preadapted for survival at acidic pH as amastigotes in macrophage phagolysosomes.

Damage to surface membrane of Schistosoma mansoni by pristane (2, 6, 10, 14 tetramethyl pentadecane) and other hydrophobic compounds.

Intraperitoneal injection of cercariae into pristane (2, 6, 10, 14 tetramethyl pentadecane)-primed Balb/c mice led to greatly diminished numbers of portal and peritoneal worms compared with untreated mice. Schistosomula taken from the peritoneal cavity of pristane-primed mice carried globules of pristane on their surfaces, were contracted and were permeable to Trypan blue. Pristane globules bound also to adult worms in vitro and in vivo causing rapid damage to the surface membrane. Hydrophobic compounds other than hydrocarbons either bound without causing gross damage, or did not bind to the adult worms. 51Cr release studies showed that pristane had no effect on the permeability of human erythrocytes, while causing significant release from both schistosomula and adult worms. The binding of hydrocarbon globules to a variety of other parasites did not occur. The binding of n-[1-14C]hexadecane to adult Schistosoma mansoni was significantly decreased by extraction of the parasite with organic solvents or treatment with staphylococcal delta toxin, which interacts with phospholipids in the membrane. Possible mechanisms of damage of the parasite by the hydrocarbons are discussed.

Leishmania mexicana: amastigote hydrolases in unusual lysosomes.

Leishmania mexicana mexicana (M379) amastigotes were found to contain much higher activities than cultured promastigotes of five putative lysosomal enzymes: cysteine proteinase; arylsulfatase (EC 3.1.6.1); beta-glucuronidase (EC 3.2.1.31); DNase (EC 3.1.22.1), and RNase (EC 3.1.27.1). The release profiles of the first three of these enzymes from digitonin-permeabilized amastigotes suggests that they are located within organelles. Cytochemical staining for cysteine proteinase, using gold labeled antibodies and arylsulfatase, showed that both were present in large organelles previously named "megasomes." Comparative studies with L. mexicana amazonensis (LV78), L. donovani donovani (LV9), and L. major (LV39) revealed that L. mexicana amazonensis was similar to L. mexicana mexicana in possessing both high amastigote cysteine proteinase activity and large numbers of megasome organelles in amastigotes, whereas the other two species lacked both these features. The results suggest that the presence of numerous lysosome-like organelles in the amastigote is a characteristic of the L. mexicana group of parasites.