Project Summary Our group has developed specialized techniques to determine the microbial content of formalin-fixed paraffin-embedded pathologic specimens. This innovative proposal seeks to identify infectious agents that are associated with mediastinal lymph nodes affected by sarcoidosis in comparison to a set of matched normal control specimens. Some preliminary data is presented from a similar project involving sequencing and pathogen identification in demyelinating (MS) brain lesions. There is immunohistochemical demonstration of bacterial antigen within a representative sarcoidosis lymph node specimen. The specific aims of the project are: 1) Define the microbial sequence profile in each diseased subject?s mediastinal lymph node biopsy (N = 6) that are significantly different than controls (N = 10), as determined by the false-discovery rate method where q < 0.05, 2) Determine significant differences in human gene expression between the sarcoidosis group and controls. These objectives will be accomplished by using RNA sequencing with metagenomic analysis of the sequencing data. The approach will be validated in two positive control specimens, one infected with Mycobacterium tuberculosis, the other with Mycobacterium avium-intracellulare. The project is innovative because it applies advanced techniques to the microbiology of sarcoidosis lesions, and because the design allows for the detection of different microbial triggers in each diseased specimen compared to a set of controls. The proposed study is intended as a highly focused proof-of-principle that might lead to a more definitive study in a larger number of sarcoidosis patients and controls. This study could plausibly lead to significant advances in sarcoidosis diagnostics and therapeutics.