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The focus of this study is the expression of Toll-like receptor-3 (TLR-3), a receptor for double-stranded RNA, in human brains affected by Alzheimer's disease (AD) pathology. Toll-like receptors are a family of pattern recognition molecules primarily involved in host defenses to microbial pathogens, but roles in neurodegenerative disease have also been shown, as amyloid beta (Aβ) can be a ligand for TLR-2 and -4 and α-synuclein for TLR-1 and TLR-2, while TLR-9 activation promotes Aβ removal. However, involvement of TLR-3 in AD has not been rigorously studied. Immunohistochemical analyses in human temporal cortical sections with a validated antibody for TLR-3 predominantly identified microglia, particularly strongly in cells associated with amyloid plaques, also brain vascular endothelial cells and subsets of astrocytes, but not neurons or p62-immunoreactive structures. Microglial TLR-3 colocalized with the endosomal/lysosomal marker CD68, which identifies phagocytic cells. Quantitative analyses of neuropathologically-staged human brain middle temporal gyrus samples using immunohistochemistry and mRNA expression methods demonstrated increased TLR-3 immunoreactivity and increased TLR-3 mRNA in AD compared to non-demented cases. There were significant positive correlations between TLR-3 mRNA levels and plaque or tangle loads in both series of samples. Increased expression of interferon beta (IFN-β) and interferon regulatory factor (IRF)-3 mRNA, two factors induced by TLR-3 signaling, were detected in the AD cases. Increased expression of TLR-4 and TLR-9 mRNA was also observed in these same samples, but not TLR-2. In vitro cultured human brain microglia responses to Aβ inflammatory activation were not altered by TLR-3 activation with activator polyinosinic;polycytidylic acid (poly I:C), while human brain endothelial cells showed reduction in responses when stimulated with both agents. Treatment of microglia with poly I:C did not increase their uptake and breakdown of Aβ.Increased TLR-3 immunoreactivity and mRNA in middle temporal gyrus of Alzheimer's disease cases.TLR-3 was localized to most microglia and strongest in microglia associated with amyloid plaques.Microglia TLR-3 immunoreactivity colocalized with endosomal/lysosomal marker CD68.TLR-3, IRF-3 and TLR-9 mRNA expression positively correlated with AD plaque and tangle pathology.Treatment of cultured human microglia with Aβ 1–42 did not increase expression of TLR-3.