PBMCs from subjects with HAM/TSP demonstrate increased NF-κB activation in association with viral protein expression. NF-κB activation was measured by an NF-κB DNA-binding ELISA (TransAM NF-κB transcription factor ELISA; Active Motif) on nuclear extracts from PBMCs of subjects with HAM/TSP and healthy donors to detect nuclear translocation of NF-κB subunits. (A) Nuclear RelA (p65) levels in PBMCs from subjects with HAM/TSP compared with healthy donors (HD), measured directly ex vivo and after 20 hours (20h) of culture. RLU, relative LIGHT units. (B) RelA, RelB, and c-Rel levels in nuclear extracts from healthy donor (HD) PBMCs (n = 6) compared with HAM/TSP PBMCs (n = 6). *P < .05, unpaired t test. (C) Immunofluorescence analysis of PBMCs from a subject with HAM/TSP showing HTLV-I Tax protein expression (left; Alexa Fluor 488), RelA expression (center; Alexa Fluor 594), and merged image (right), with DAPI nuclear counterstain. One representative image from 2 subjects is shown. Note the nuclear translocation of RelA in cells expressing Tax (arrow) compared with the intracytoplasmic sequestration of RelA in cells not expressing Tax. Observation was performed with a Zeiss Axiovert 200M equipped with mercury lamp house HBO-100 and appropriate filters for detecting DAPI and the 2 secondary antibodies conjugated to Alexa Fluor 488 and Alexa Fluor 594, using a 63× oil objective.