Lymphocytic Choriomeningitis Virus Infection in FVB Mouse Produces Hemorrhagic Disease

The viral family Arenaviridae includes a number of viruses that can cause hemorrhagic fever in humans. Arenavirus infection often involves multiple organs and can lead to capillary instability, impaired hemostasis, and death. Preclinical testing for development of antiviral or therapeutics is in part hampered due to a lack of an immunologically well-defined rodent model that exhibits similar acute hemorrhagic illness or sequelae compared to the human disease. We have identified the FVB mouse strain, which succumbs to a hemorrhagic fever-like illness when infected with lymphocytic choriomeningitis virus (LCMV). FVB mice infected with LCMV demonstrate high mortality associated with thrombocytopenia, hepatocellular and splenic necrosis, and cutaneous hemorrhage. Investigation of inflammatory mediators revealed increased IFN-gamma, IL-6 and IL-17, along with increased chemokine production, at early times after LCMV infection, which suggests that a viral-induced host immune response is the cause of the pathology. Depletion of T cells at time of infection prevented mortality in all treated animals. Antisense-targeted reduction of IL-17 cytokine responsiveness provided significant protection from hemorrhagic pathology. F1 mice derived from FVBxC57BL/6 mating exhibit disease signs and mortality concomitant with the FVB challenged mice, extending this model to more widely available immunological tools. This report offers a novel animal model for arenavirus research and pre-clinical therapeutic testing

Gene-silencing antisense oligomers inhibit Acinetobacter growth in vitro and in vivo

Background: Peptide-conjugated phosphorodiamidate morpholino oligomers (PPMOs) are synthetic DNA/RNA analogs that silence expression of specific genes. We studied whether PPMOs targeted to essential genes in Acinetobacter lwoffii and A. baumannii are active in vitro and in vivo. Methods: PPMOs were evaluated in vitro using MIC and viability assays, and in vivo using murine pulmonary infection models with intranasal PPMO treatment. Results: MICs of PPMOs ranged from 0.1 and 64 μM (~0.6 to 38 μg/ml). The most effective PPMO tested was (RXR)₄-AcpP, which is targeted to acpP. (RXR)₄-AcpP reduced viability of A. lwoffii and A. baumannii by > 10³ cfu/ml at 5 to 8 x MIC. Mice treated with 0.25 mg/kg or more of (RXR)₄-AcpP survived longer and had less inflammation and bacterial lung burden than mice treated with a scrambled-sequence PPMO or PBS. Treatment could be delayed after infection and still increase survival. Conclusions: PPMOs targeted to essential genes of A. lwoffii and A. baumannii were bactericidal and had MICs in a clinically relevant range. (RXR)₄-AcpP increased survival of mice infected with A. lwoffii or A. baumannii, even when initial treatment was delayed after infection. PPMOs could be a viable therapeutic approach in dealing with multidrug resistant Acinetobacter species.This article is published by Oxford University Press on behalf of the Infectious Diseases Society of America. This is a pre-copy-editing, author-produced PDF of an article accepted for publication in the Journal of Infectious Diseases following peer review. The definitive publisher-authenticated version, Geller, B. L., Marshall-Batty, K., Schnell, F. J., McKnight, M. M., Iversen, P. L., & Greenberg, D. E. (2013). Gene-Silencing Antisense Oligomers Inhibit Acinetobacter Growth In Vitro and In Vivo. Journal of Infectious Diseases, 208(10), 1553-1560. doi:10.1093/infdis/jit460, is available online at: http://jid.oxfordjournals.org/content/208/10/1553.full.pdf?keytype=ref&ijkey=qepbqtxt5pt.Keywords: antisense, infection, respiratory infection, phosphorodiamidate morpholino oligomer, lwoffii, baumannii, oligomer, MIC, Acinetobacter, PMO, morpholino, mous

Of Mice and Measures : A Project to Improve How We Advance Duchenne Muscular Dystrophy Therapies to the Clinic.

A new line of dystrophic mdx mice on the DBA/2J (D2) background has emerged as a candidate to study the efficacy of therapeutic approaches for Duchenne muscular dystrophy (DMD). These mice harbor genetic polymorphisms that appear to increase the severity of the dystropathology, with disease modifiers that also occur in DMD patients, making them attractive for efficacy studies and drug development. This workshop aimed at collecting and consolidating available data on the pathological features and the natural history of these new D2/mdx mice, for comparison with classic mdx mice and controls, and to identify gaps in information and their potential value. The overall aim is to establish guidance on how to best use the D2/mdx mouse model in preclinical studies

Viral Escape Mutant Epitope Maintains TCR Affinity for Antigen yet Curtails CD8 T Cell Responses.

T cells have the remarkable ability to recognize antigen with great specificity and in turn mount an appropriate and robust immune response. Critical to this process is the initial T cell antigen recognition and subsequent signal transduction events. This antigen recognition can be modulated at the site of TCR interaction with peptide:major histocompatibility (pMHC) or peptide interaction with the MHC molecule. Both events could have a range of effects on T cell fate. Though responses to antigens that bind sub-optimally to TCR, known as altered peptide ligands (APL), have been studied extensively, the impact of disrupting antigen binding to MHC has been highlighted to a lesser extent and is usually considered to result in complete loss of epitope recognition. Here we present a model of viral evasion from CD8 T cell immuno-surveillance by a lymphocytic choriomeningitis virus (LCMV) escape mutant with an epitope for which TCR affinity for pMHC remains high but where the antigenic peptide binds sub optimally to MHC. Despite high TCR affinity for variant epitope, levels of interferon regulatory factor-4 (IRF4) are not sustained in response to the variant indicating differences in perceived TCR signal strength. The CD8+ T cell response to the variant epitope is characterized by early proliferation and up-regulation of activation markers. Interestingly, this response is not maintained and is characterized by a lack in IL-2 and IFNγ production, increased apoptosis and an abrogated glycolytic response. We show that disrupting the stability of peptide in MHC can effectively disrupt TCR signal strength despite unchanged affinity for TCR and can significantly impact the CD8+ T cell response to a viral escape mutant

Effects of LCMV infection on blood and splenic cellularity in LCMV infected mice.

<p>A) Complete blood count with differential of LCMV-13 infected mice for platelet, neutrophil, lymphocyte, eosinphil and basophil counts. FVB (n = 4) or C57BL/6 (n = 1) mice were infected with 1–2×10⁶ p.f.u. LCMV-13 and blood was taken at day 7 post-infection. Additionally, naïve FVB (n = 2) blood was collected as a reference. Counts are presented as mean +/− standard deviation. Results for FVB mice are combined from two independent experiments. B) Spleen size in FVB mice exhibits an inverse correlation with infections dose of LCMV compared to C57BL/6. C) FVB mice have reduced T cell numbers as assessed by staining with anti-CD3 antibody in spleens after LCMV-13 infection. FVB (n = 10) or C57BL/6 (n = 8) mice were infected with 1–2×10⁶ p.f.u. LCMV-13 and spleens were harvested between day 7–11 post-infection. Results are presented as mean +/− standard deviation (*, p<0.05. t-test). Similar results were found in two additional independent experiments.</p