The twitcher mouse is a murine model of globoid cell leukodystropy, a genetic demyelinating disease caused by a mutation of the galactosylceramidase gene. Demyelination of the central nervous system commences around 20 postnatal days. Using GFP-transgenic mice as donors, the distribution of hematogenous cells after bone marrow transplantation was investigated in the twitcher mice. Bone marrow transplantation was carried out at 8 postnatal days. In twitcher chimeric mice examined before 30 postnatal days, numerous GFP(+) cells were detected in spleen and peripheral nerve but only a few were detected in the liver, lung, and spinal white matter. In contrast, at 35 to 40 postnatal days when demyelination is evident, many GFP(+) cells with ameboid form were detected in the white matter of the spinal cord, brainstem, and cerebrum. Approximately half of these GFP(+) cells were co-labeled with Mac-1. In twitcher chimeric mice examined after 100 postnatal days, the majority of GFP/Mac-1 double-positive cells displayed the morphological features of ramified microglia with fine delicate processes and was distributed diffusely in both gray and white matter. These results suggest that a significant number of donor hematogenous cells are able to infiltrate into the brain parenchyma, repositioning themselves into areas previously occupied by microglia, and to ameliorate lethality.
The bacterium Francisella tularensis is the causative agent of tularemia. F. tularensis induces a host response that is dependent on the route of infection. Intranasal (i.n.) inoculations are more virulent and require fewer bacteria to produce a lethal infection than intradermal (i.d.) inoculations (10^3 organisms i.n. versus 10^6 i.d.). Interestingly, one day post infection, the bacterial loads are similar in the spleen and lung regardless of the route of infection. We also found that i.d. inoculation resulted in IFN-γ+ T cells in the lung whereas i.n. inoculation produced very few IFN-γ+ T cells and instead many IL-17+ T cells in the lung. Due to the similar bacterial loads systemically after 1 day, but very different host responses, we hypothesize that the adaptive immune response is influenced by local events at the site of infection immediately following inoculation. To test this hypothesis, we infected C57Bl/6 mice i.n. or i.d. and identified cytokine production in the lung and tail at four hours post-infection. Additionally, we have identified alveolar macrophages as the primary cell type infected four hours post-infection, to the exclusion of other myeloid cells and lung parenchyma. Furthermore, we have identified cytokines produced by alveolar macrophages when cultured ex vivo and infected with F. tularensis. These data will help us further understand how the adaptive immune response to F. tularensis is shaped early after infection.
The binding of a peptide to a major histocompatibility (MHC) class I allele depends upon a few key residues within the peptide sequence, which is present only in ~1-2% of peptides in an average protein. Identification of this motif allows one to select candidate epitopes from a protein that may be recognized by cytotoxic T lymphocytes (CTL). While several studies have determined the binding specificities of numerous class I alleles in both humans and mice, no such study has been performed in the domestic dog. The principal aim of this study was to determine the peptide binding motif of a prevalent MHC class I allele, Dog Leukocyte Antigen (DLA) -88*50801. We hypothesized that a conserved binding motif could be identified through sequencing of endogenously presented peptides eluted from DLA-88*50801. To accomplish this goal, a plasmid encoding the DLA-88*50801 gene with an attached flag tag was stably transfected into a canine cell line (DH82). DLA-88*50801 protein was obtained through affinity purification of lysate from ~8 x 109 cells. Bound peptides were eluted from the MHC binding groove utilizing low pH and heat, isolated by size exclusion and affinity purification, and subjected to tandem mass spectrometry analysis to obtain individual peptide sequences. Sequences were manually aligned to determine the binding motif. This study provides essential information to study CTL responses in dogs.
Francisella tularensis is highly virulent and infectious bacterial pathogen and a category A select agent. Many aspects of immunity to the infection are not well understood. This study is part of ongoing work to understand the immune response following cutaneous exposure. The aim of the study was to determine cytokines/chemokines induced in response to intradermal infection with F. tularensis subspp novicida. Further aim was to determine the correlation between induction of cytokines/chemokines with bacterial load. C57BL/6J mice were intradermally infected with F. novicida U112 WT and DotU. DotU mutants do not grow inside macrophages and are greatly attenuated. The results investigated wide array of induced anti-and pro-inflammatory cytokines/chemokines in spleen. Higher levels of INF-γ and P-10 were expressed at 24hrs post-infection in spleen of mice infected with WT compared to DotU or mock control. The recovered number of viable bacteria from the spleen peaked at 24hrs and correlated with increased induction of INF-γ. Studies are underway to elucidate the role of cytokines/chemokines in shaping adaptive immunity.
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