Characterization of bromadiolone resistance in a danish strain of Norway rats, Rattus norvegicus, by hepatic gene expression profiling of genes involved in vitamin K-dependent gamma-carboxylation

The present study characterizes the anticoagulant resistance mechanism in a Danish bromadiolone-resistant strain of Norway rats (Rattus norvegicus), with a Y139C VKORC1 mutation. We compared liver expression of the VKORC1 gene, which encodes a protein of the vitamin K 2,3-epoxide reductase complex, the NQO1 gene, which encodes a NAD(P)H quinone dehydrogenase and the Calumenin gene between bromadiolone-resistant and anticoagulant-susceptible rats upon saline and bromadiolone administration. Additionally, we established the effect of bromadiolone on the gene expression in the resistant and susceptible phenotype. Bromadiolone had no effect on VKORC1 and NQO1 expression in resistant rats, but induced significantly Calumenin expression in the susceptible rats. Calumenin expression was similar between the resistant and the susceptible rats upon saline administration but twofold lower in resistant rats after bromadiolone treatment. These results indicate that Danish bromadiolone resistance does not involve an overexpression of calumenin. Independent of the treatment, we observed a low VKORC1 expression in resistant rats, which in conjugation with the Y139C polymorphism most likely explains the low VKOR activity and the enhanced need for vitamin K observed in Danish resistant rats. Furthermore the bromadiolone resistance was found to be associated with a low expression of the NQO1 gene.     

Ratiometric Imaging of Extracellular pH in Bacterial Biofilms with C-SNARF-4

pH in the extracellular matrix of bacterial biofilms is of central importance for microbial metabolism. Biofilms possess a complex three-dimensional architecture characterized by chemically different microenvironments in close proximity. For decades, pH measurements in biofilms have been limited to monitoring bulk pH with electrodes. Although pH microelectrodes with a better spatial resolution have been developed, they do not permit the monitoring of horizontal pH gradients in biofilms in real time. Quantitative fluorescence microscopy can overcome these problems, but none of the hitherto employed methods differentiated accurately between extracellular and intracellular microbial pH and visualized extracellular pH in all areas of the biofilms. Here, we developed a method to reliably monitor extracellular biofilm pH microscopically with the ratiometric pH-sensitive dye C-SNARF-4, choosing dental biofilms as an example. Fluorescent emissions of C-SNARF-4 can be used to calculate extracellular pH irrespective of the dye concentration. We showed that at pH values of <6, C-SNARF-4 stained 15 bacterial species frequently isolated from dental biofilm and visualized the entire bacterial biomass in in vivo-grown dental biofilms with unknown species composition. We then employed digital image analysis to remove the bacterial biomass from the microscopic images and adequately calculate extracellular pH values. As a proof of concept, we monitored the extracellular pH drop in in vivo-grown dental biofilms fermenting glucose. The combination of pH ratiometry with C-SNARF-4 and digital image analysis allows the accurate monitoring of extracellular pH in bacterial biofilms in three dimensions in real time and represents a significant improvement to previously employed methods of biofilm pH measurement.     

Binding of human milk to pathogen receptor DC-SIGN varies with bile salt-stimulated lipase (BSSL) gene polymorphism

Objective

Dendritic cells bind an array of antigens and DC-SIGN has been postulated to act as a receptor for mucosal pathogen transmission. Bile salt-stimulated lipase (BSSL) from human milk potently binds DC-SIGN and blocks DC-SIGN mediated trans-infection of CD4⁺ T-lymphocytes with HIV-1. Objective was to study variation in DC-SIGN binding properties and the relation between DC-SIGN binding capacity of milk and BSSL gene polymorphisms.

Study Design

ELISA and PCR were used to study DC-SIGN binding properties and BSSL exon 11 size variation for human milk derived from 269 different mothers distributed over 4 geographical regions.

Results

DC-SIGN binding properties were highly variable for milks derived from different mothers and between samplings from different geographical regions. Differences in DC-SIGN binding were correlated with a genetic polymorphism in BSSL which is related to the number of 11 amino acid repeats at the C-terminus of the protein.

Conclusion

The observed variation in DC-SIGN binding properties among milk samples may have implications for the risk of mucosal transmission of pathogens during breastfeeding.     

CAF01 potentiates immune responses and efficacy of an inactivated influenza vaccine in ferrets

Trivalent inactivated vaccines (TIV) against influenza are given to 350 million people every year. Most of these are non-adjuvanted vaccines whose immunogenicity and protective efficacy are considered suboptimal. Commercially available non-adjuvanted TIV are known to elicit mainly a humoral immune response, whereas the induction of cell-mediated immune responses is negligible. Recently, a cationic liposomal adjuvant (dimethyldioctadecylammonium/trehalose 6,6'-dibehenate, CAF01) was developed. CAF01 has proven to enhance both humoral and cell-mediated immune responses to a number of different experimental vaccine candidates. In this study, we compared the immune responses in ferrets to a commercially available TIV with the responses to the same vaccine mixed with the CAF01 adjuvant. Two recently circulating H1N1 viruses were used as challenge to test the vaccine efficacy. CAF01 improved the immunogenicity of the vaccine, with increased influenza-specific IgA and IgG levels. Additionally, CAF01 promoted cellular-mediated immunity as indicated by interferon-gamma expressing lymphocytes, measured by flow cytometry. CAF01 also enhanced the protection conferred by the vaccine by reducing the viral load measured in nasal washes by RT-PCR. Finally, CAF01 allowed for dose-reduction and led to higher levels of protection compared to TIV adjuvanted with a squalene emulsion. The data obtained in this human-relevant challenge model supports the potential of CAF01 in future influenza vaccines.     

A novel human recombinant single-chain antibody targeting CD166/ALCAM inhibits cancer cell invasion in vitro and in vivo tumour growth

Screening a phage-display single-chain antibody library for binding to the breast cancer cell line PM-1 an antibody, scFv173, recognising activated leukocyte cell adhesion molecule (ALCAM, CD166) was isolated and its binding profile was characterized. Positive ALCAM immunohistochemical staining of frozen human tumour sections was observed. No ALCAM staining was observed in the majority of tested normal human tissues (nine of ten). Flow cytometry analyses revealed binding to 22 of 26 cancer cell lines of various origins and no binding to normal blood and bone marrow cells. Antibody binding inhibited invasion of the breast cancer cell line MDA-MB-231 by 50% in an in vitro Matrigel-coated membrane invasion assay. Reduced growth of tumours in nude mice was observed in an in vivo model in which the mice were injected subcutaneously with colorectal carcinoma HCT 116 cells and treated with scFv173 when compared to control. In summary, we have characterized a novel fully human scFv antibody recognising ALCAM on cancer cells and in tumour tissues that reduces cancer cell invasion and tumour growth in accordance with the hypothesised role for ALCAM in cell growth and migration control.     

Viral and bacterial diseases of Atlantic cod Gadus morhua, their prophylaxis and treatment: a review

This review summarises the state of knowledge of both viral and bacterial diseases of Atlantic cod Gadus morhua, and their diagnosis, prophylaxis and treatment. The most important losses have been at the larval and juvenile stages, and vibriosis has long been the most important bacterial disease in cod, with Listonella (Vibrio) anguillarum dominant among pathogenic isolates. Vaccination of cod against pathogens such as L. anguillarum and Aeromonas salmonicida clearly demonstrates that the cod immune system possesses an effective memory and appropriate mechanisms sufficient for protection, at least against some diseases. Well-known viruses such as the nodavirus that causes viral encephalopathy and retinopathy (VER), infectious pancreatic necrosis virus (IPNV) and viral haemorrhagic septicaemia virus (VHSV) have been isolated from Atlantic cod and can be a potential problem under intensive rearing conditions. No commercial vaccines against nodavirus are currently available, whereas vaccines against IPNV infections based upon inactivated virus as well as IPNV recombinant antigens are available. A number of investigations of the pharmacokinetic properties of antibacterial agents in cod and their efficacy in treating bacterial infections have been reviewed.     

Respiratory burst activity of Atlantic cod (Gadus morhua L.) blood phagocytes differs markedly from that of rainbow trout

In the present study we investigated the respiratory burst (RB) activity of Atlantic cod (Gadus morhua L.) blood phagocytes and we evaluated how the RB activity of cod blood cells differ from that of trout. The RB activities were measured directly from highly diluted whole blood as luminol-amplified chemiluminescence (CL) under various conditions. Studies regarding the blood dilutions for cod whole blood chemiluminescence measurements (WBCL) revealed that at a final blood dilution of 1.5 microl ml(-1) or less the CL response was strictly proportional to the number of phagocytes. This range of blood dilution did not markedly differ from that of trout. However, the opsonisation capacity of cod plasma was markedly poorer. The RB activity of phagocytes was most active at 15 degrees C when heterologous cod serum was used as a source of opsonin, whereas at final blood dilution of 8.0 microl ml(-1) (when homologous cod plasma was at a higher concentration) the highest RB activity was observed at 10 degrees C. Aeromonas salmonicida strain MT004 (As MT004) induced higher RB activity than the two known pathogens for cod, atypical A. salmonicida and Vibrio anguillarum. Cod blood phagocytes were more responsive to plastic surfaces and the adhesion response of phagocytes was partly inhibited but did not totally vanish even at a final gelatin concentration of 0.4%. Moreover, cod serum enhanced the adherence of phagocytes and cod blood phagocytes also showed slow spontaneous degranulation. Finally, within the tested anticoagulants (heparin, Na-citrate, EDTA) heparin treated blood phagocytes generated the highest RB activity.