This study aimed to detect intracellular trehalose in boar sperm that were cryopreserved with liposomes and conduct an analysis of its effects on some characteristics of thawed sperm, including rheological properties. First, soybean lecithin cholesterol-based liposomes were produced and characterized in the presence of 300 mM trehalose. Next, semen samples were frozen in two freezing media: a control medium with 300 mM trehalose and an experimental medium supplemented with 300 mM trehalose and 10% liposomes, both of which were thawed and then studied to ascertain their integrity, motility, rheological response, and trehalose quantities by testing two methods of spermatic lysis via high-performance liquid chromatography with an evaporative light-scattering detector (HPLC-ELSD). The results found spherical liposomes measuring 357 nm that were relatively stable in an aqueous medium and had an entrapment efficiency of 73%. An analysis of the cryopreserved ejaculates showed that their viability and motility did not significantly differ between groups (P > 0.05). The viscous response of the samples was influenced by the extracellular medium rather than by the freezing–thawing process, which resulted in a loss of interaction between the cells and cryoprotectants. Finally, intracellular trehalose levels were determined using HPLC-ELSD, with no differences observed (P > 0.05) when comparing both sperm lysis methods. The use of liposomes with trehalose appears to be a promising option for boar semen cryopreservation, with a marked effect on rheological properties. The proposed HPLC-ELSD method was effective for measuring trehalose in cryopreserved cell samples.

Understanding the fundamental properties of macromolecules has enhanced the development of emerging technologies used to improve biomedical research. Currently, there is a critical need for innovative platforms that can illuminate the function of biomedical reagents in a native environment. To address this need, we have developed an in situ approach to visualize the dynamic behavior of biomedically relevant macromolecules at the nanoscale. Newly designed silicon nitride devices containing integrated “microwells” were used to enclose active macromolecular specimens in liquid for transmission electron microscopy imaging purposes.We were able to successfully examine novel magnetic resonance imaging contrast reagents, micelle suspensions, liposome carrier vehicles, and transcribing viral assemblies. With each specimen tested, the integrated microwells adequately maintained macromolecules in discrete local environments while enabling thin liquid layers to be produced.

Rabbits were vaccinated once, by subcutaneous and intradermal injection, with sonicates of oncospheres (TpO) or conditioned media from in vitro maintained mature metacestodes (TpMcES) of Taenia pisiformis. Extracts were either incorporated into or mixed with unilammelar liposomes (reverse phase evaporative vesicles) or emulsified in Freund's Incomplete Adjuvant (FIA). Control groups received liposomes or FIA without antigen, or antigen preparation without adjuvant. Rabbits were challenged orally two weeks after vaccination with approximately 1500 eggs of T. pisiformis and necropsied eight weeks after challenge. A mean of 155 cysts was recovered from seven control rabbits. A 67% reduction in peritoneal cyst numbers was obtained in TpO–IFA vaccinated rabbits compared to 75% for the TpO–liposome entrapped group. The highest level of protection (86%) was obtained when TpO was mixed with but not entrapped in liposomes. Only 32% and 39% reduction in peritoneal cyst numbers was obtained after immunizing with the TpMcES preparation in liposomes or IFA respectively, however >85% of peritoneal metacestodes were dead (necrotic or calcified) and suggests a different immune response than occurs after vaccination with oncosphere extracts. Specific anti-oncospheral or anti-metacestode ES antibody (IgG) responses at two weeks post vaccination were similar in rabbits immunized with liposome or IFA associated extracts.

The pathomorphological effects of daily and weekly doses of liposomized albendazole on Trichinella spiralis larvae have been examined and compared during the course of muscle infections in mice. Treatment with three and six daily doses of the drug given by intraperitoneal injection to mice resulted in marked pathological alterations to encapsulated larvae, mainly in the walls of the capsules. There was a 5% efficacy against T. spiralis in mice given three daily doses of albendazole, and 38% efficacy in mice given six daily doses of the drug. The same doses of drug, when administered weekly, did not reduce the numbers of parasite larvae, and the application of drug at 6-weekly intervals resulted in a 5% reduction in numbers.

Anhydrobiosis, or life without water, is the remarkable ability of certain types of plants and animals to survive almost total dehydration. This phenomenon requires a coordinated series of events within the cells of anhydrobiotes that protect their cellular components, particularly proteins and lipid membranes, from damage caused by the removal of water. Much of what is now understood about preserving biological samples during drying was learned by studying naturally desiccation-tolerant organisms and extended using model systems such as phospholipid vesicles. Most anhydrobiotic organisms accumulate disaccharides in their cells and tissues during the dehydration process. These carbohydrates, usually sucrose or trehalose, satisfy two criteria that appear to be necessary for protecting membranes during desiccation and during storage in the dry state. These requirements include: (1) depression of the gel-to-liquid crystalline phase transition temperature (Tm) in the dehydrated lipid to a temperature at or near that of the hydrated lipid, a process that appears to require a direct interaction between the carbohydrates and the lipid molecules of the membrane; and (2) formation of a carbohydrate glass with a relatively high glass transition temperature, leading to inhibition of fusion between the vesicles.

The effects of a liposomal preparation of lipophilic immunomodulator β-D-GlcNstearoyl-(1-4)-norMurNAc-L-Abu-D-isoGln (N-L18-norAbu-GMDP) were investigated on resistance to Cryptosporidium parvum infection in neonatal kids. The liposomal preparation was administered subcutaneously or intranasally/orally (i.n./p.o.) twice at doses of 100 μg, 200 μg, or 1000 μg per kid pre-infection challenge. The treatment schemes were (i) 72 and 24 h pre-infection challenge, (ii) 24 h pre-infection challenge and 24 h post-infection challenge (oral inoculation with 1×107 oocysts of C. parvum in 5 ml of PBS). Administration of liposomal N-L18-norAbu-GMDP by i.n./p.o. route at the cumulative dose of 2000 μg per kid 72 and 24 h pre-infection challenge, lead to substantially increased clearance of coccidian parasites from various parts of the intestine. On the basis of histological examination, the distribution of cryptosporidia in the intestine and the severity of the infection, treated kids were classified on day 5 as having a strong reduction in infection in comparison to the control group (P<0·05). No cryptosporidia were found on the mucosal surface of treated kids by day 10, while the intestines of the control kids were still infected. All doses and routes of administration were judged effective with respect to suppression of cryptosporidia infections.

We had previously developed a methodology to introduce foreign DNA into mouse eggs and embryos using cationic lipids as vectors. In this report we use this technique to produce transgenic animals. Mouse embryos at the pronuclear stage were transfected using a mixture of a plasmid DNA, encoding for a nuclear form of β-galactosidase, and a commercial lipid transfection reagent. Embryos were washed and incubated overnight. Those that cleaved and develop to the 2-cell stage of normal appearance were transferred to the Fallopian tubes of pseudopregnant foster mothers. We analysed a total of 158 offspring and found two, a female and a male, to be transgenic (1.27% of the total). Integration of the foreign DNA in the female was showed by Southern blot. Both animals expressed the lacz in several organs, but none of them either displayed expression in the germ cells or transmitted the transgene to their offspring. Taken together our results show that lipid transfection can generate transgenic mice, but the efficiency needs to be improved for this method to be widely applied.

The present study shows that treatment with recombinant human tumour necrosis factor-α (rhTNF-α) can suppress parasitaemia and prevents development of experimental cerebral malaria (ECM) in Plasmodium berghei K173-infected mice. Mice received rhTNF-α treatment either by subcutaneous injection of free or liposome-encapsulated rhTNF-α or sustained intraperitoneal administration of rhTNF-α given via mini-osmotic pumps. Low-dose treatment with a subcutaneous bolus injection of rhTNF-α protected against ECM when treatment was started on day 5 or 6 after infection. The same protective efficacy was obtained either by subcutaneous injection of liposome-encapsulated rhTNF-α or by sustained release from osmotic pumps, but in the latter case a 10-fold lower daily dose of rhTNF-α was sufficient. Treatment with rhTNF-α substantially suppressed parasitaemia in ECM-protected mice, but not in mice developing ECM. Thus, the rhTNF-α mediated suppression of parasitaemia is directly or indirectly involved in protection against ECM. Sustained delivery of rhTNF-α through osmotic pumps, but not by subcutaneous injection of liposome-encapsulated rhTNF-α, resulted in increased concentrations of soluble mouse TNF receptor R75 (sTNFR75) in plasma at day 9 after infection when non-treated mice die of ECM. Thus, protection against ECM is not directly correlated with the sTNFR75 concentrations at day 9 after infection.

The activation of peritoneal macrophage effector functions after therapy with free PZQ and PZQ incorporated in liposomes (lip.PZQ) was studied in the Mesocestoides corti–mouse model system. Each drug formulation was administered to an infected group of mice in 6 daily doses from day 14 p.i. Phagocytic activity of macrophages increased significantly after the administration of both drug formulations, more after lip.PZQ with an earlier peak observed for PZQ (day 3) than for lip.PZQ (day 6). Empty liposomes had no significant effect. The average counts of ingested particles in phagocytosing cells were significantly higher only after lip.PZQ administration. The pattern of changes in phagocytic activity correlated with the reduction of parasite numbers in the peritoneal cavity, with the highest observed on day 6 after therapy with lip.PZQ. Phagocytosis of lip.PZQ in vivo stimulated significantly the respiratory burst in peritoneal macrophages, with the highest concentration of superoxide anions recorded on day 1 after the last dose, whereas therapy with PZQ itself did not increase this process significantly. The capacity for the respiratory burst declined in all groups with progressing infection. It is proposed that the phagocytic activity of peritoneal macrophages after therapy was stimulated indirectly as a consequence of activation of the specific immune response. The larvicidal effect of lip.PZQ on the tetrathyridia in the peritoneal cavity was synergistic with the phagocytic activity and might be the result of double action of drug and superoxide anions generated during the respiratory burst stimulated by this drug formulation.