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.

Characterization of a mitochondrion-like organelle in Cryptosporidium parvum.

Cryptosporidium parvum is a protozoan parasite that causes widespread diarrhoeal disease in humans and other animals and is responsible for large waterborne outbreaks of cryptosporidiosis. Unlike many organisms belonging to the phylum Apicomplexa, such as Plasmodium spp. and Toxoplasma gondii, there is no clinically proven drug treatment against this parasite. Aspects of the basic biology of C. parvum remain poorly understood, including a detailed knowledge of key metabolic pathways, its genome organization and organellar complement. Previous studies have proposed that C. parvum lacks a relic plastid organelle, or 'apicoplast', but that it may possess a mitochondrion. Here we characterize a mitochondrion-like organelle in C. parvum by (i) ultrastructural and morphological description (ii) localization of heterologous mitochondrial chaperonin antibody probes (iii) phylogenetic analysis of genes encoding mitochondrial transport proteins (iv) identification and analysis of mitochondrion-associated gene sequences. Our descriptive morphological analysis was performed by energy-filtering transmission electron microscopy (EFTEM) of C. hominis and C. parvum. The 'mitochondrion-like' organelle was characterized by labelling the structure with a heterologous mitochondrial chaperonin probe (hsp60) both in immunoelectron microscopy (IMEM) and immunofluorescence (IMF). Phylogenetic analysis of the mitochondrial import system and housekeeping components (hsp60 and hsp70-dnaK) suggested that the C. parvum mitochondrion-like organelle is likely to have descended from a common ancestral apicomplexan mitochondrion. We also identified a partial cDNA sequence coding for an alternative oxidase (AOX) gene, a component of the electron transport chain which can act as an alternative to the terminal mitochondrial respiratory complexes III and IV, which has not yet been reported in any other member of this phylum. Degenerate primers developed to identify selected mitochondrial genes failed to identify either cytochrome oxidase subunit I, or cytochrome b. Taken together, our data aim to provide new insights into the characterization of this Cryptosporidium organelle and a logical framework for future functional investigation.

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.

Processing and trafficking of cysteine proteases in Leishmania mexicana.

Removal of the pro-domain of a cysteine protease is essential for activation of the enzyme. We have engineered a cysteine protease (CPB2.8) of the protozoan parasite Leishmania mexicana by site-directed mutagenesis to remove the active site cysteine (to produce CPB(C25G)). When CPB(C25G) was expressed in a L. mexicana mutant lacking all CPB genes, the inactive pro-enzyme was processed to the mature protein and trafficked to the lysosome. These results show that auto-activation is not required for correct processing of CPB in vivo. When CPB(C25G) was expressed in a L. mexicana mutant lacking both CPA and CPB genes, the majority of the pro-enzyme remained unprocessed and accumulated in the flagellar pocket. These data reveal that CPA can directly or indirectly process CPB(C25G) and suggest that cysteine proteases are targeted to lysosomes via the flagellar pocket. Moreover, they show that another protease can process CPB in the absence of either CPA or CPB, albeit less efficiently. Abolition of the glycosylation site in the mature domain of CPB did not affect enzyme processing, targeting or in vitro activity towards gelatin. This indicates that glycosylation is not required for trafficking. Together these findings provide evidence that the major route of trafficking of Leishmania cysteine proteases to lysosomes is via the flagellar pocket and therefore differs significantly from cysteine protease trafficking in mammalian cells.

Preliminary characterization of novel amino acid based polymeric vesicles as gene and drug delivery agents.

The amino acid homopolymers, poly-L-lysine and poly-L-ornithine, have been modified by the covalent attachment of palmitoyl and methoxypoly(ethylene glycol) (mPEG) residues to produce a new class of amphiphilic polymers-PLP and POP, respectively. These amphiphilic amino acid based polymers have been found to assemble into polymeric vesicles in the presence of cholesterol. Representatives of this new class of polymeric vesicles have been evaluated in vitro as nonviral gene delivery systems with a view to finding delivery systems that combine effective gene expression with low toxicity in vivo. In addition, the drug-carrying capacity of these polymeric vesicles was evaluated with the model drug doxorubicin. Chemical characterization of the modified polymers was carried out using (1)H NMR spectroscopy and the trinitrobenzene sulfonic acid (TNBS) assay for amino groups. The amphiphilic polymers were found to have an unreacted amino acid, palmitoyl, mPEG ratio of 11:5:1, and polymeric vesicle formation was confirmed by freeze-fracture electron microscopy and drug encapsulation studies. The resulting polymeric vesicles, by virtue of the mPEG groups, bear a near neutral zeta-potential. In vitro biological testing revealed that POP and PLP vesicle-DNA complexes are about one to 2 orders of magnitude less cytotoxic than the parent polymer-DNA complexes although more haemolytic than the parent polymer-DNA complexes. The polymeric vesicles condense DNA at a polymer:DNA weight ratio of 5:1 or greater and the polymeric vesicle-DNA complexes improved gene transfer to human tumor cell lines in comparison to the parent homopolymers despite the absence of receptor specific ligands and lysosomotropic agents such as chloroquine.

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.

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.

Niosomes and polymeric chitosan based vesicles bearing transferrin and glucose ligands for drug targeting.

PURPOSE: To prepare polymeric vesicles and niosomes bearing glucose or transferrin ligands for drug targeting. METHODS: A glucose-palmitoyl glycol chitosan (PGC) conjugate was synthesised and glucose-PGC polymeric vesicles prepared by sonication of glucose-PGC/cholesterol. N-palmitoylglucosamine (NPG) was synthesised and NPG niosomes also prepared by sonication of NPG/ sorbitan monostearate/ cholesterol/ cholesteryl poly-24-oxyethylene ether. These 2 glucose vesicles were incubated with colloidal concanavalin A gold (Con-A gold), washed and visualised by transmission electron microscopy (TEM). Transferrin was also conjugated to the surface of PGC vesicles and the uptake of these vesicles investigated in the A431 cell line (over expressing the transferrin receptor) by fluorescent activated cell sorter analysis. RESULTS: TEM imaging confirmed the presence of glucose units on the surface of PGC polymeric vesicles and NPG niosomes. Transferrin was coupled to PGC vesicles at a level of 0.60+/-0.18 g of transferrin per g polymer. The proportion of FITC-dextran positive A431 cells was 42% (FITC-dextran solution), 74% (plain vesicles) and 90% (transferrin vesicles). CONCLUSIONS: Glucose and transferrin bearing chitosan based vesicles and glucose niosomes have been prepared. Glucose bearing vesicles bind Con-A to their surface. Chitosan based vesicles are taken up by A431 cells and transferrin enhances this uptake.

The isolation of differentially expressed cDNA clones from the filarial nematode Brugia pahangi.

A cDNA library constructed from 3 day post-infective L3 of the filarial nematode Brugia pahangi was screened by differential hybridization with cDNA probes prepared from different life-cycle stages. Five cDNA clones hybridizing selectively to the mosquito-derived L3 probe were isolated and characterized. Northern blot analysis of 4 of the clones confirmed that each was most highly expressed in the mosquito-derived L3. The expression of each mRNA during parasite development in the mosquito vector was investigated using RT-PCR, and all were shown to be abundant in the immature L3. Four of the 5 cDNAs cloned coded for structural proteins: 2 cuticular collagens, and the muscle proteins tropomyosin and troponin. Further studies on troponin using an antiserum raised to the recombinant protein demonstrated that the protein, unlike the mRNA, was present in all life-cycle stages examined, while immunogold labelling demonstrated that it was localized to the muscle blocks.

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.

Developmental activation of infective Trichinella spiralis larvae.

The initiation of further development is fundamental to the infectious processes of parasitic nematodes. We have examined early developmental activation of Trichinella spiralis larvae during host invasion, with particular emphasis on the timing of events. Using a novel approach, we have observed changes in tissue-specific transcriptional activity in live larvae during the infectious process with the fluorescent nucleic acid dyes SYTO12 and acridine orange. Simultaneously, the metabolic switch from anaerobic metabolism, characteristics of the infective stage, to aerobic metabolism, as found in the enteral stages, was tracked by measuring activities of the key regulatory enzymes phosphoenolpyruvate carboxykinase and pyruvate kinase, as well as isocitrate dehydrogenase (NADP) activity, and used as a co-indicator for developmental activation. Both metabolic enzyme activities and transcription patterns were found to change in response to host death, liberation from the nurse cell, and exposure to components of the host stomach environment. The results give a clear indication that the activation processes of T. spiralis infective larvae occur at a much earlier time than previously thought, and are stimulated upon liberation of the larvae from the nurse cell inside the host stomach.

Lipid vesicle size determines the Th1 or Th2 response to entrapped antigen.

Understanding the factors that control the differential induction of Th1 and Th2 responses is a key immunologic objective with profound implications for vaccination and immunotherapy of infectious and autoimmune diseases. Using Ag formulated in lipid vesicles prepared from nonionic surfactants, we describe a novel mechanism influencing the balance of the Th1 or Th2 response. Our results indicate that inoculation of BALB/c mice with vesicles with a mean diameter > or = 225 nm preferentially induces Th1 responses, as characterized by increased titers of IgG2a in plasma and elevated IFN-gamma production by lymph node cells. However, preparation of the same quantity of Ag in vesicles with mean diameter of < or = 155 nm induces a Th2 response, as identified by IgG1 in the absence of IgG2a production and increased lymph node IL-5 production. Although large (> or = 225 nm) vesicles could induce IL-12 production, smaller vesicles (< or = 155 nm) could not. However, small vesicles did induce higher levels of IL-1beta production by macrophages than larger vesicles. The role of IL-12 in this response was confirmed in IL-12-deficient mice, whose spleen cells failed to produce IFN-gamma following in vivo priming with Ag prepared in large vesicles. Our results therefore indicate that macrophages respond to endocytosis of large or small vesicles by producing different patterns of cytokines that can subsequently direct the immune response toward a Th1 or a Th2 phenotype.

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.

Protective effect on Leishmania major infection of migration inhibitory factor, TNF-alpha, and IFN-gamma administered orally via attenuated Salmonella typhimurium.

The genes encoding murine macrophage migration inhibitory factor (MIF), IL-2, IFN-gamma or TNF-alpha were cloned individually into an expression plasmid under the control of the inducible promoter nirB and transfected into the aroA- aroD- deletion mutant strain of Salmonella typhimurium (BRD509). These S. typhimurium derivatives (henceforward called constructs and termed GIDMIF, GIDIL2, GIDIFN and GIDTNF) expressed their respective cytokines in vitro under anaerobic conditions and stably colonized BALB/c mice up to 14 days after oral administration. The highly susceptible BALB/c mice that had received the constructs orally and that had been subsequently infected via the footpad with Leishmania major, developed significantly reduced disease compared with control mice administered the untransfected Salmonella strain (BRD509). Importantly, a combination of GIDMIF, GIDIFN, and GIDTNF administered orally after L. major infection was able to significantly limit lesion development and reduced parasite loads by up to three orders of magnitude. Spleen and lymph node cells of mice administered this combination expressed markedly higher levels of inducible nitric oxide synthase (iNOS) compared with those from mice receiving an equivalent dose of the control strain of Salmonella (BRD509). These data therefore demonstrate the feasibility of therapeutic treatment in an infectious disease model using cytokines delivered by attenuated Salmonella. The protective effect observed correlates with the induction of inducible nitric oxide synthase in vivo.

The multiple cpb cysteine proteinase genes of Leishmania mexicana encode isoenzymes that differ in their stage regulation and substrate preferences.

The cpb genes of Leishmania mexicana encode stage-regulated, cathepsin L-like cysteine proteinases that are leishmanial virulence factors. Field inversion gel electrophoresis and genomic mapping indicate that there are 19 cpb genes arranged in a tandem array. Five genes from the array have been sequenced and their expression analyzed. The first two genes, cpb1 and cpb2, differ significantly from the remaining 17 copies (cpb3-cpb19) in that: 1) they are expressed predominantly in metacyclic promastigotes (the form in the insect vector which is infective to mammalian macrophages) rather than amastigotes (the form that parasitizes mammals); 2) they encode enzymes with a truncation in the COOH-terminal extension, an unusual feature of these cysteine proteinases of trypanosomatids. Transfection of cpb1 into a cpb null mutant resulted in expression of an active enzyme that was shown by immunogold labeling with anti-CPB antibodies to be targeted to large lysosomes. This demonstrates that the 100-amino acid COOH-terminal extension is not essential for the activation or activity of the enzyme or for its correct intracellular trafficking. Transfection into the cpb null mutant of different copies of cpb and analysis of the phenotype of the lines showed that individual isoenzymes differ in their substrate preferences and ability to restore the loss of virulence associated with the null mutant. Comparison of the predicted amino acid sequences of the isoenzymes implicates five residues located in the mature domain (Asn18, Asp60, Asn61, Ser64, and Tyr84) with differences in the activities of the encoded isoenzymes. The results suggest that the individual isoenzymes have distinct roles in the parasite's interaction with its host. This complexity reflects the adaptation of cathepsin L-like cysteine proteinases to diverse functions in parasitic protozoa.

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.

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.