Hoxb8 conditionally immortalised macrophage lines model inflammatory monocytic cells with important similarity to dendritic cells.

We have examined the potential to generate bona fide macrophages (MØ) from conditionally immortalised murine bone marrow precursors. MØ can be derived from Hoxb8 conditionally immortalised macrophage precursor cell lines (MØP) using either M-CSF or GM-CSF. When differentiated in GM-CSF (GM-MØP) the resultant cells resemble GM-CSF bone marrow-derived dendritic cells (BMDC) in morphological phenotype, antigen phenotype and functional responses to microbial stimuli. In spite of this high similarity between the two cell types and the ability of GM-MØP to effectively present antigen to a T-cell hybridoma, these cells are comparatively poor at priming the expansion of IFN-γ responses from naïve CD4(+) T cells. The generation of MØP from transgenic or genetically aberrant mice provides an excellent opportunity to study the inflammatory role of GM-MØP, and reduces the need for mouse colonies in many studies. Hence differentiation of conditionally immortalised MØPs in GM-CSF represents a unique in vitro model of inflammatory monocyte-like cells, with important differences from bone marrow-derived dendritic cells, which will facilitate functional studies relating to the many 'sub-phenotypes' of inflammatory monocytes.

A novel multi-antigen virally vectored vaccine against Mycobacterium avium subspecies paratuberculosis.

BACKGROUND: Mycobacterium avium subspecies paratuberculosis causes systemic infection and chronic intestinal inflammation in many species including primates. Humans are exposed through milk and from sources of environmental contamination. Hitherto, the only vaccines available against Mycobacterium avium subspecies paratuberculosis have been limited to veterinary use and comprised attenuated or killed organisms. METHODS: We developed a vaccine comprising a fusion construct designated HAV, containing components of two secreted and two cell surface Mycobacterium avium subspecies paratuberculosis proteins. HAV was transformed into DNA, human Adenovirus 5 (Ad5) and Modified Vaccinia Ankara (MVA) delivery vectors. Full length expression of the predicted 95 kDa fusion protein was confirmed. PRINCIPAL FINDINGS: Vaccination of naïve and Mycobacterium avium subspecies paratuberculosis infected C57BL/6 mice using DNA-prime/MVA-boost or Ad5-prime/MVA-boost protocols was highly immunogenic resulting in significant IFN-gamma ELISPOT responses by splenocytes against recombinant vaccine antigens and a range of HAV specific peptides. This included strong recognition of a T-cell epitope GFAEINPIA located near the C-terminus of the fusion protein. Antibody responses to recombinant vaccine antigens and HAV specific peptides but not GFAEINPIA, also occurred. No immune recognition of vaccine antigens occurred in any sham vaccinated Mycobacterium avium subspecies paratuberculosis infected mice. Vaccination using either protocol significantly attenuated pre-existing Mycobacterium avium subspecies paratuberculosis infection measured by qPCR in spleen and liver and the Ad5-prime/MVA-boost protocol also conferred some protection against subsequent challenge. No adverse effects of vaccination occurred in any of the mice. CONCLUSIONS/SIGNIFICANCE: A range of modern veterinary and clinical vaccines for the treatment and prevention of disease caused by Mycobacterium avium subspecies paratuberculosis are needed. The present vaccine proved to be highly immunogenic without adverse effect in mice and both attenuated pre-existing Mycobacterium avium subspecies paratuberculosis infection and conferred protection against subsequent challenge. Further studies of the present vaccine in naturally infected animals and humans are indicated.

Identification of differentially expressed genes in scrapie-infected mouse brains by using global gene expression technology.

The pathogenesis of prion diseases, a class of transmissible fatal neurodegenerative diseases in humans and animals, is still unclear. The aim of this study was to identify the differentially regulated genes that correlate with the development of prion diseases for a better understanding of their pathological mechanisms. We employed Affymetrix Mouse Expression Arrays 430A containing >22,000 transcripts and compared the global gene expression profiles from brains of mice who were intracerebrally inoculated with scrapie strains ME7 and RML with those from brains of uninfected and mock-infected mice. The microarray data were analyzed by Significance Analysis of Microarrays, revealing 121 genes whose expression increased at least twofold in both ME7- and RML-infected mouse brains, with an estimated false discovery rate of < or =5%. These genes encode proteins involved in proteolysis, protease inhibition, cell growth and maintenance, the immune response, signal transduction, cell adhesion, and molecular metabolism. The time course of expression generally showed up-regulation of these genes from 120 days postinoculation (dpi) for ME7-inoculated mouse brains and from 90 dpi for RML-inoculated mouse brains. The onset of elevated expression correlated temporally with the onset of PrP(Sc) accumulation and the activation of glia, which may have contributed to neuronal cell death. Among the differentially regulated genes reported in the present study, the emergence of genes for several cathepsins and S100 calcium binding proteins was conspicuous. These and other genes reported here may represent novel potential diagnostic and therapeutic targets for prion disease.