Anders Permin

In vivo toxicity of cationic micelles and liposomes

UNLABELLED This study investigated toxicity of nanocarriers comprised of cationic polymer and lipid components often used in gene and drug delivery, formulated as cationic micelles and liposomes. Rats were injected intravenously with 10, 25 or 100 mg/kg and sacrificed after 24 or 48 h, or 24 h after the last of three intravenous injections of 100 mg/kg every other day. Histological evaluation of liver, lung and spleen, clinical chemistry parameters, and hematology indicated little effect of treatment. DNA strand breaks were increased in the lung and spleen. Further, in the dose response study we found unaltered expression levels of genes in the antioxidant response (HMOX1) and repair of oxidized nucleobases (OGG1), whereas expression levels of cytokines (IL6, CXCL2 and CCL2) were elevated in lung, spleen or liver. The results indicate that assessment of genotoxicity and gene expression add information on toxicity of nanocarriers, which is not obtained by histology and hematology. FROM THE CLINICAL EDITOR This study investigates the toxicity of cationic micelles and liposomes utilized as nanocarriers in gene and drug delivery, demonstrating its effects on the lungs, spleen and liver.

In vitro inhibition of Clostridium difficile and Clostridium perfringens by commercial probiotic strains

Probiotics have gained importance in human and veterinary medicine to prevent and control clostridial enteric disease. Limited information is available on the ability of different probiotic bacteria used in food products to inhibit Clostridium difficile and Clostridium perfringens. The objective of this study was to examine the in vitro inhibitory effects of selected commercial bacterial strains on pathogenic clostridia and their growth characteristics under simulated gastrointestinal conditions. The inhibitory effects of 17 commercial strains of Lactobacillus (n = 16) and Bifidobacterium (n = 1) on the reference strains of C. difficile and C. perfringens were assessed by an agar well diffusion assay and by a broth culture inhibition assay using cell-free supernatant harvested at different growth phases, with and without pH neutralization. To study growth characteristics, probiotic strains were cultivated in different acid and bile environments, and growth in the modified media was compared to growth in standard medium. In the agar well diffusion assay, supernatant obtained from two probiotic strains inhibited the growth of both reference and clinical strains of C. perfringens. This effect as seen when supernatant was assessed with and without pH neutralization. Supernatants obtained from 10 probiotic strains inhibited C. difficile only when supernatant was added without pH neutralization. In the broth culture inhibition assay, growth of C. perfringens and C. difficile was inhibited by supernatant without pH neutralization from 5 and 10 probiotic strains, respectively. All potential probiotic strains were able to grow at pH 4.0 and in the presence of 0.15% and 0.3% bile but none were able to grow or survive at pH 2.0. Altogether five probiotic strains [Lactobacillus plantarum (n = 2), Lactobacillus rhamnosus (n = 2), Bifidobacterium animalis lactis (n = 1)] were shown to inhibit all strains of C. difficile and C. perfringens. The inhibitory effect was probiotic strain-specific. Two strains showed a pH-independent inhibitory effect likely due to production of either antibiotics or bacteriocins inhibiting C. perfringens only. These strains have favourable growth characteristics for use as probiotics and their efficacy as prophylactic or therapeutic measures against clostridial enteric disease should be further evaluated by clinical trials in animals.

Investigation of the parasitic nematode Ascaridia galli (Shrank 1788) as a potential vector for Salmonella enterica dissemination in poultry.

Abstract During recent years, the level of organically farmed poultry in Denmark has increased. Subsequent investigations have demonstrated an incidence of 64% of Ascaridia galli infections in layers established in organic farming systems. Studies to determine the interaction of Salmonella enterica with the parasitic nematode A. galli associated with poultry were undertaken to establish the significance of A. galli in the dissemination of S. enterica. A. galli was isolated from 40-week-old Lohmann Brown Salmonella-free layers. Worms were subsequently maintained in vitro and exposed to S. e. serovar Typhimurium at concentrations of 105–106 colony forming units/ml for varying times (24–144 h). Eggs were harvested aseptically from the worms and the associations of S. e. Typhimurium in relation both to the eggs and to structures on the surface of the worm were studied, using immunofluorescence, viable counts and in situ hybridisation. Results show attachment of S. e. Typhimurium to the outer coating of the eggs and possible internalisation. Evidence of association of the bacteria with the nematode eggs was further substantiated by establishing Salmonella infection in day-old chicks after dosing them with eggs harvested from parasitic worms infected in vitro with Salmonella.

Differential toxicological response to positively and negatively charged nanoparticles in the rat brain

Abstract We investigated the potential for systemic and local toxicity after administration of empty nanosized anionic and cationic PEGylated-micelles and non-PEGylated liposomes, without a ligand attached, intended for use in drug-delivery systems. The particles were administered to 5–6-week-old male rats by three intravenous (IV) administrations over a period of one week at a dose of 100 mg/kg bodyweight or after a single intracerebroventricular (ICV) injection at a dose of 50 µg. The particles were stable and well characterised with respect to size and zeta potential. ICV administration of cationic particles was associated with histological changes near the injection site (hippocampus). Here, we detected focal infiltration with phagocytic cells, loss of neurons and apoptotic cell death, which were not observed after administration of the vehicle. No significant difference was found after IV or ICV administration of the anionic micelles with regard to haematology, clinical chemistry parameters or at the pathological examinations, as compared to control animals. Our study suggests that ICV delivery of cationic particles to the brain tissue is associated with toxicity at the injection site.

Virulence of Pasteurella multocida subsp. multocida isolated from outbreaks of fowl cholera in wild birds for domestic poultry and game birds

Chickens, turkeys, partridges and pheasants were experimentally infected with Pasteurella multocida subsp. multocida to investigate whether outbreaks of fowl cholera in avifauna might represent a risk for organic, backyard and industrial poultry production. Birds were infected intra-tracheally with a strain of P. multocida subsp. multocida (40605-1) isolated from outbreaks of fowl cholera in wild birds in Denmark. P. multocida subsp. multocida strain P-1059 was included as a reference strain. The outbreak strain was highly virulent for turkeys, partridges and pheasants, while chickens were more resistant. The present findings underline the importance of wild birds as a reservoir for P. multocida. Intratracheal challenge proved useful for studying the virulence of P. multocida.

Comparison of parasite-specific immunoglobulin levels in two chicken lines during sustained infection with Ascaridia galli.

Increasingly large numbers of poultry are held in production systems with access to outdoor areas. In these systems intestinal helminths are found with flock prevalences of up to 100%. Helminth infections influence chicken health negatively, which is why the following investigation has been performed. In the present experiment, 20 chickens of two inbred chicken lines containing the major histocompatibility complex (MHC) haplotypes, B14 and R5, were inoculated with 500 embryonated Ascaridia galli eggs. The A. galli-specific IgG titres of serum samples and the excretion of A. galli eggs in chicken faeces were measured for a period of 81 weeks. The level of excreted A. galli eggs measured as eggs per gram chicken faeces (EPG) varied greatly between chickens in each line. Significant differences were found between the two lines and with the R5 chickens reaching the highest levels. Likewise, the A. galli-specific IgG titres in serum differed significantly between the two lines, and an inverse relationship between infection level (EPG) and antibody titres was found. Although this inverse relationship suggests that humoral immunity may be involved in protection against A. galli infection, the high antibody titres did not prevent continued infection.

Ascaridia galli infection influences the development of both humoral and cell-mediated immunity after Newcastle Disease vaccination in chickens.

Potent vaccine efficiency is crucial for disease control in both human and livestock vaccination programmes. Free range chickens and chickens with access to outdoor areas have a high risk of infection with parasites including Ascaridia galli, a gastrointestinal nematode with a potential influence on the immunological response to vaccination against other infectious diseases. The purpose of this study was to investigate whether A. galli infection influences vaccine-induced immunity to Newcastle Disease (ND) in chickens from an MHC-characterized inbred line. Chickens were experimentally infected with A. galli at 4 weeks of age or left as non-parasitized controls. At 10 and 13 weeks of age half of the chickens were ND-vaccinated and at 16 weeks of age, all chickens were challenged with a lentogenic strain of Newcastle disease virus (NDV). A. galli infection influenced both humoral and cell-mediated immune responses after ND vaccination. Thus, significantly lower NDV serum titres were found in the A. galli-infected group as compared to the non-parasitized group early after vaccination. In addition, the A. galli-infected chickens showed significantly lower frequencies of NDV-specific T cells in peripheral blood three weeks after the first ND vaccination as compared to non-parasitized chickens. Finally, A. galli significantly increased local mRNA expression of IL-4 and IL-13 and significantly decreased TGF-ß4 expression in the jejunum two weeks after infection with A. galli. At the time of vaccination (six and nine weeks after A. galli infection) the local expression in the jejunum of both IFN-? and IL-10 was significantly decreased in A. galli-infected chickens. Upon challenge with the NDV LaSota strain, viral genomes persisted in the oral cavity for a slightly longer period of time in A. galli-infected vaccinees as compared to non-parasitized vaccinees. However, more work is needed in order to determine if vaccine-induced protective immunity is impaired in A. galli-infected chickens.

Immune gene expression in the spleen of chickens experimentally infected with Ascaridia galli.

Ascaridia galli is a gastrointestinal nematode infecting chickens. Chickens kept in alternative rearing systems or at free-range experience increased risk for infection with resulting high prevalences. A. galli infection causes reduced weight gain, decreased egg production and in severe cases increased mortality. More importantly, the parasitised chickens are more susceptible to secondary infections and their ability to develop vaccine-induced protective immunity against other diseases may be compromised. Detailed information about the immune response to the natural infection may be exploited to enable future vaccine development. In the present study, expression of immune genes in the chicken spleen during an experimental infection with A. galli was investigated using the Fluidigm(®) BioMark™ microfluidic qPCR platform which combines automatic high-throughput with attractive low sample and reagent consumption. Spleenic transcription of immunological genes was compared between infected chickens and non-infected controls at week 2, 6, and 9 p.i. corresponding to different stages of parasite development/maturation. At week 2 p.i. increased expression of IL-13 was observed in infected chickens. Increased expression of MBL, CRP, IFN-α, IL-1β, IL-8, IL-12β and IL-18 followed at week 6 p.i. and at both week 6 and 9 p.i. expression of DEFβ1 was highly increased in infected chickens. In summary, apart from also earlier reported increased expression of the Th2 signature cytokine IL-13 we observed only few differentially expressed genes at week 2 p.i. which corresponds to the larvae histotrophic phase. In contrast, we observed increased expression of pro-inflammatory cytokines and acute phase proteins in infected chickens, by week 6 p.i. where the larvae re-enter the intestinal lumen. Increased expression of DEFβ1 was observed in infected chickens at week 6 p.i. but also at week 9 p.i. which corresponds to a matured stage where adult worms are present in the intestinal lumen.

In vitro toxicity of cationic micelles and liposomes in cultured human hepatocyte (HepG2) and lung epithelial (A549) cell lines

The aim of this study was to compare the effects of cationic micelle and liposome drug delivery systems on liver and lung cells in a toxicological in vitro screening model, with observations on cytotoxicity and genotoxicity. A screening battery was established for assessment of a broad range of parameters related to adverse effects. Clear concentration response effects were observed related to impairment of mitochondrial function, membrane integrity and oxidative stress markers, but no effect was observed on genotoxicity. The adverse effects were highest for the liposomes. The High Content Screening seems optimal for initial screening of adverse effects, and combined with standard cytotoxicity measurements initial screening can be performed for predictive toxicological screening.

No protection in chickens immunized by the oral or intra-muscular immunization route with Ascaridia galli soluble antigen

In chickens, the nematode Ascaridia galli is found with prevalences of up to 100% causing economic losses to farmers. No avian nematode vaccines have yet been developed and detailed knowledge about the chicken immune response towards A. galli is therefore of great importance. The objective of this study was to evaluate the induction of protective immune responses to A. galli soluble antigen by different immunization routes. Chickens were immunized with a crude extract of A. galli via an oral or intra-muscular route using cholera toxin B subunit as adjuvant and subsequently challenged with A. galli. Only chickens immunized via the intra-muscular route developed a specific A. galli antibody response. Frequencies of γδ T cells in spleen were higher 7 days after the first immunization in both groups but only significantly so in the intra-muscularly immunized group. In addition, systemic immunization had an effect on both Th1 and Th2 cytokines in caecal tonsils and Meckels diverticulum. Thus both humoral and cellular immune responses are inducible by soluble A. galli antigen, but in this study no protection against the parasite was achieved.