Macrophages are phagocytic cells that play a key role in host immune response and clearance of microbial pathogens. Porphyromonas gingivalis is an oral pathogen associated with the development of periodontitis. Escape from macrophage phagocytosis was tested by infecting THP-1-derived human macrophages and RAW 264.7 mouse macrophages with strains of P. gingivalis W83 and 33277 as well as Streptococcus gordonii DL1 and Escherichia coli OP50 at MOI = 100. CFU counts for all intracellular bacteria were determined. Then, infected macrophages were cultured in media without antibiotics to allow for escape and escaping bacteria were quantified by CFU counting. P. gingivalis W83 displayed over 60% of the bacterial escape from the total amount of intracellular CFUs, significantly higher compared to all other bacteria strains. In addition, bacterial escape and re-entry were also tested and P. gingivalis W83, once again, showed the highest numbers of CFUs able to exit and re-enter macrophages. Lastly, the function of the PG0717 gene of P. gingivalis W83 was tested on escape but found not related to this activity. Altogether, our results suggest that P. gingivalis W83 is able to significantly avoid macrophage phagocytosis. We propose this ability is likely linked to the chronic nature of periodontitis.
Neuromyelitis optica (NMO) is an auto-inflammatory demyelinating disease that typically affects optic nerves and spinal cord that is characterized by the presence of serum aquaporin-4 immunoglobulin G antibodies (AQP4-IgG). NMO accounts for >45% of the demyelinating disease in Asians and warrants the development of a suitable therapy other than generalized immunosuppressants. It is thought that autoimmune activated AQP4-specific T cells disrupt the BBB and allow increased entry of AQP4-IgG and other immune effectors into tissues containing astrocytes expressing AQP4 in their membranes. Recently we have established an adeno-associated viral (AAV) gene immunotherapy that effectively prevents and reverses experimental autoimmune encephalomyelitis (EAE) via generation of suppressive antigen-specific regulatory T cells (Tregs). In this report, using a similar antigen-specific approach, we have demonstrated that AAV.AQP4 gene immunotherapy prevented the development of AQP4-mediated neuroinflammation and clinical neurological disability in almost 100% of C57BL/6J mice when AAV.AQP4 vector was administered to mice 2 weeks prior to immunization with an immunogenic epitope of AQP4. Moreover, in contrast to AAV.AQP4 treated mice which remained unremarkable, histological analysis of the spinal cord sections from untreated vehicle only mice showed multiple areas of significant focal inflammation within the spinal cord. Based on our clinical and pathological data, we’ve successfully demonstrated that our novel AAV.AQP4 gene immunotherapy can indeed suppress the induction of AQP4 mediated autoimmune disease. Further evaluation will determine if it can also treat preexisting disease.
To re-establish long-term immune tolerance in Multiple Sclerosis (MS), our lab had previously developed a pre-clinical Adeno-associated virus (AAV) gene immunotherapy that is capable of preventing and reversing Myelin Oligodendrocyte Glycoprotein (MOG) induced Experimental Autoimmune Encephalomyelitis (EAE). However, MS is a disease that involves multiple myelin proteins including Proteolipid Protein (PLP). Therefore we expanded the capability of our gene immunotherapy to restore tolerance and ameliorate disease to multiple major myelin proteins, simultaneously. In this report we demonstrate that a mixture of 2 individual vectors, AAV MOG & AAV.PLP, was capable of preventing, and more importantly reversing, preexisting EAE disease induced with a mixture of MOG35–55 & PLP139–151 peptides in (C57BL/6JxSJL)F1 mice. To minimize the total vector load we further developed this immunotherapy into a novel single AAV-dual transgene expressing vector (AAV.MOG.PLP) using specific gene linkers for independent expression. Following a single peripheral injection, western blot analysis confirmed stable and simultaneous hepatic expression of both neuroprotein transgenes. When injected into mice 2 weeks before induction of EAE with either MOG35–55 or PLP139–151, or combination of both, the dual transgene AAV immunotherapy significantly reduced or prevented disease in mice compared to controls. Overall, these results demonstrate proof of concept that a single AAV vector simultaneously expressing more than one transgene may be an effective therapeutic treatment for restoring tolerance in an autoimmune disease like MS. Supported by NIHSarepta Pharmaceuticals NIH R01Act AI128074Project
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