A multiyear survey of helminths from wild saddleback (Leontocebus weddelli) and emperor (Saguinus imperator) tamarins.

The establishment of baseline data on parasites from wild primates is essential to understand how changes in habitat or climatic disturbances will impact parasite-host relationships. In nature, multiparasitic infections of primates usually fluctuate temporally and seasonally, implying that the acquisition of reliable data must occur over time. Individual parasite infection data from two wild populations of New World primates, the saddleback (Leontocebus weddelli) and emperor (Saguinus imperator) tamarin, were collected over 3 years to establish baseline levels of helminth prevalence and parasite species richness (PSR). Secondarily, we explored variation in parasite prevalence across age and sex classes, test nonrandom associations of parasite co-occurrence, and assess the relationship between group size and PSR. From 288 fecal samples across 105 individuals (71 saddleback and 34 emperor tamarins), 10 parasite taxa were identified by light microscopy following centrifugation and ethyl-acetate sedimentation. Of these taxa, none were host-specific, Dicrocoeliidae and Cestoda prevalences differed between host species, Prosthenorchis and Strongylida were the most prevalent. Host age was positively associated with Prosthenorchis ova and filariform larva, but negatively with cestode and the Rhabditoidea ova. We detected no differences between expected and observed levels of co-infection, nor between group size and parasite species richness over 30 group-years. Logistic models of individual infection status did not identify a sex bias; however, age and species predicted the presence of four and three parasite taxa, respectively, with saddleback tamarins exhibiting higher PSR. Now that we have reliable baseline data for future monitoring of these populations, next steps involve the molecular characterization of these parasites, and exploration of linkages with health parameters.

The effect of poly(ethylene glycol) molecular architecture on cellular interaction and uptake of DNA complexes.

The cellular uptake of plasmid DNA complexes with a series of tertiary amine methacrylate-ethylene glycol (DMAEMA-EG) copolymers with various architectures was studied using flow cytofluorometry and laser confocal microscopy. The complexes displayed different rates and extents of cellular interaction and internalisation, depending on the copolymer molecular architecture. In general, introduction of oligo(ethylene glycol) [OEG] or poly(ethylene glycol) [PEG] chains decreased both the interaction and cellular internalisation of the DNA complexes but subtle differences were observed. Two block copolymers, a 'bottle-brush' type DMAEMA-block-OEGMA and a linear DMAEMA-block-PEG copolymer (each containing a total of 45 EG units), displayed similar uptake profiles. In contrast, only relatively low uptake of complexes formed by a comb-type statistical copolymer, DMAEMA-stat-PEGMA, was observed, despite each PEG chain comprising 45 EG units. Similar trends were observed with three cell lines, A549, HepG2 and COS-7. However, the absolute values were cell-dependent, with COS-7 cells displaying both the highest rate and extent of uptake. Studies of the association and uptake of the complexes demonstrated that cell associations generally increased over time, with the uptake level and the time profile depending on the polymer architecture. Confocal microscopy studies confirmed that, with the exception of the poorly transfecting comb-type copolymer, the association of complexes with cells resulted in endocytosis.

Effect of polymer ionization on the interaction with DNA in nonviral gene delivery systems.

The optimization of DNA-cationic polymer complexation is crucial for nonviral gene delivery. Although physicochemical characterization of the interaction between DNA and cationic polymers has recently attracted more attention in the nonviral DNA delivery field, the literature on the effect of varying polycation charge density on DNA-cationic polymer complexation is still scarce. Thus, the aim of this study was to systematically assess the influence of the degree of ionization of a weak cationic polyelectrolyte (poly[2-(dimethylamino)ethyl methacrylate] or DMAEMA homopolymer) on its ability to form complexes with DNA. This was achieved by varying the solution pH from 4.0 to 8.0 and analyzing the resulting effects on the binding affinity, thermodynamic properties, complex size, and morphology. Lowering the solution pH led to higher degrees of ionization for the cationic polymer and hence greater binding affinities with DNA, as judged by the increased propensity of the former to displace ethidium bromide from DNA and also by relatively low monomer:nucleotide molar ratio (0.8:1) required to retard the migration of free DNA. Isothermal titration microcalorimetry studies further confirmed that a stronger interaction occurred at low pH than at high pH. By decreasing the pH from 8.0 to 6.6, K(obs) increased from 7.8 x 10(5) to 20.4 x 10(5) M(-1). More efficient condensation at low pH was demonstrated by the reduction of ethidium bromide fluorescence in the loading wells from gel electrophoresis, decreased complex sizes without agglomeration occurring at high polymer/DNA ratios, together with discrete and dense spherical complexes observed in TEM studies. This may be attributed to the presence of electrostatic stabilization from excess cationic polymer chains, which provide a repulsive shell around the polymer/DNA complex. The physicochemical data indicate that the increased degree of ionization for the DMAEMA homopolymer at lower pH results in higher binding affinity, smaller and more compact complexes, and more efficient condensation. These findings therefore highlight the importance of the degree of ionization on DNA complex formation for weak cationic polyelectrolytes.

Influence of polymer architecture on the structure of complexes formed by PEG-tertiary amine methacrylate copolymers and phosphorothioate oligonucleotide.

The influence of polymer structure on the characteristics of complexes of a phosphorothioate antisense oligonucleotide (ISIS 5132) was studied, using well-defined cationic copolymers based on 2-(dimethylamino) ethyl methacrylate (DMAEMA) and poly(ethylene glycol) (PEG). The three related copolymer structures were: DMAEMA-PEG (a diblock copolymer) DMAEMA-OEGMA 7 (a brush-type copolymer), DMAEMA-stat-PEGMA (a comb-type copolymer); each of these were examined together with DMAEMA homopolymer, which served as a control. The results revealed that all the polymers exhibited good binding ability with the oligonucleotide (ON). Interestingly, the comb-type polymer DMAEMA-stat-PEGMA demonstrated the highest binding ability and DMAEMA homopolymer the lowest, as judged by a dye displacement assay. DMAEMA homopolymer produced large agglomerates of smaller individual complexes as observed by optical density, photon correlation spectroscopy and transmission electron microscopy studies. In contrast, two PEG-block copolymers, DMAEMA-PEG and DMAEMA-OEGMA 7, formed compact complexes of 80-150 nm which had good long-term colloidal stability. This is attributed to the steric stabilisation effect of the PEG chains on the ON-copolymer complexes. These two copolymers are believed to form complexes with ON that have a micellar structure. Comb-type DMAEMA-stat-PEGMA copolymer formed highly soluble complexes with the ON that did not phase separate from the buffer solution. This study clearly demonstrates that varying the copolymer architecture allows access to a range of ON complexes. In vitro cytotoxicity experiments on HepG2 cells showed that all of the tertiary amine methacrylate copolymers displayed lower cytotoxicity than the control poly(L-lysine).