Williams & Wilkins: Baltimore. [Google Scholar] 11. isoform (ABC) includes all three of these exons and the smallest isoform (O) lacks all three exons. Five different isoforms of CD45 (ABC, AB, BC, B and O) have been identified on human leukocytes and these can be recognized by antibodies specific to variable exons (A, B or C) or by CD45RO (45). Although the extracellular domains differ among different isoforms, all forms share identical transmembrane and cytoplasmic domains including the phosphatase domains 52, 54. CD45 is one of the most abundantly expressed molecules in lymphocytes (comprising approximately 10% of all surface proteins) and is crucial in lymphocyte development and antigen signaling 2, 12, 23, 54. Consequently, CD45 mutations are associated with severe combined immunodeficiency in mice and humans 5, 28, 51. In lymphocytes, CD45 is expressed in a cell subset\specific and activation\dependent manner. For instance, na?ve T cells express a high molecular weight isoform (RA+/RO?) but upon activation switch to the smallest isoform (RA?/RO+) 16, 31. At the cellular level, the CD45 phosphatase targets several families of proteins, including the Src family tyrosine kinases and Janus kinases (41), resulting in positive or negative signaling 2, 4, 54. In addition to lymphocytes, recent studies demonstrate that CD45 can modulate activation and proliferation of several inflammatory cell types including granulocytes, mast cells and monocyte\lineage cells, broadening its role as a regulator of inflammatory responses 8, 20, 35, 48, 57. In the central nervous system (CNS), microglia constitute a distinct glial cell population that is derived from hematopoietic cells in the bone marrow 17, 29, 42. As resident brain macrophages, microglia function as sentries, but when activated they can mediate tissue damage, a scenario considered for several CNS inflammatory disorders 10, 15, 27. In AIDS dementia and HIV encephalitis (HIVE), microglia and macrophages are productively infected by HIV\1 and show diffuse inflammatory activation, which ultimately leads to neuronal damage and CNS dysfunction 7, 11, 14, 43. Microglia in normal human brain express CD45 and increases in microglial CD45 expression have been detected in Alzheimers disease, graft\versus\host disease, multiple sclerosis, and in HIVE 1, 7, 24, 30, 33, 46. Furthermore, studies in rodent and human cells suggest that CD45 can downregulate microglial activation. For example, murine microglia devoid of CD45 expression demonstrate an over\activated phenotype 49, 50, while in human microglia, CHMFL-BTK-01 an agonist antibody (CD45RO, clone UCHL\1) can stimulate CD45 tyrosine phosphatase activity and suppress granulocyte\macrophage colony\stimulating factor (GM\CSF) signal transduction and cell proliferation (48). CD45 also downregulates HIV\1 replication in microglia, indicating that there might be potential for CHMFL-BTK-01 targeting this phosphatase as a therapy for AIDS dementia (25). Despite these data indicating functional importance of CD45 CHMFL-BTK-01 in microglia, the CD45 isoform expression by microglia and macrophages in HIV\1\infected human brain is not known. Furthermore, the identity of CD45 isoforms other than CD45RO on CNS\infiltrating T cells is unknown. We therefore sought to investigate changes in CD45 isoform expression in the human CNS as it pertains to HIVE and also asked whether there is cell\type or activation\dependent expression of CD45 isoforms. MATERIALS AND METHODS Patient material.? Paraffin\embedded, formalin\fixed brain tissues from 22 patients were obtained from the Manhattan HIV\1 Brain Bank, National NeuroAIDS Tissue Consortium (37). Information regarding the case history and other associated systemic illnesses has been previously reported Rabbit polyclonal to MST1R 6, 7, 58. Our patient material was distributed into three groups: HIVE (n?=?9), HIV\seropositive without HIVE (HIV+, n?=?6) and HIV\seronegative individuals (HIV?, n?=?8). The mean ages were 45.6??3.5 (HIV?), 42.5??2.7 (HIV+) and 38.5??2.5 (HIVE) and were not significantly different (for details. ?AvidinCbiotin complex without TSA. ?Alkaline phosphatase\labeled secondary antibody methods without TSA. Abbreviations: LCA?=?leukocyte common antigen; VWF?=?Von Willebrand Factor. IHC without epitope retrieval or TSA.? CD45RB and CD45RO staining on serial slides of a single paraffin block was compared using three different IHC methods with increasing sensitivity: (i) without antigen retrieval (AR); (ii) with AR; and (iii) with AR and TSA. All other experiments conducted with CD45.