A. Chediak Higashi syndrome (CHS), a rare autosomal recessive disease (mutations in the lysosomal trafficking regulator gene (LYST/CHS1)) characterized by partial oculocutaneous albinism (OCA), immunodeficiency, mild bleeding tendency, and varying neurologic problems. Furthermore, considered to be related to the immune-comprised situation, several case reports indicate severe periodontal disease. We hypothesized that primary skin fibroblasts obtained from CHS patients would exhibit increased expression of cytokines and immune regulatory factors, as well as a hypersensitive response to immunogenic challenge, compared to control fibroblasts. Cells were provided by Dr. Introne, previously obtained from enzymatic digestion of skin biopsies and frozen until use. PCR-arrays were used to profile the expression of inflammation-related genes in fibroblasts of healthy people as control cells and CHS patients cultured with or without Escherichia coli LPS (10ng/mL for 3 h) (n=3). Of the 84 genes evaluated by PCR-array, at baseline 15 were up-regulated (include IL-1β, IL-6, and COX2) and 6 down-regulated (include TLR-2 and -4) with more than 2-fold change in CHS cells compared to control cells. With LPS challenge, CHS cells had only 9.5% of the 84 immune responsive genes significantly affected compared to 33.3% of the genes affected in control cells. Furthermore, control cells presented a more robust response (4,000-fold change) than CHS cells (30-fold change). Protein expression evaluated by Western blot shows that at baseline, cell membrane associated TLR-2 and -4 were significantly lower in CHS versus control cells;while cytosolic TLR-2 protein level was significantly lower in control cells than CHS cells, cytosolic TLR-4 protein was expressed at the same level in CHS cells as in control cells, suggesting that exportation of TLR-4 to CHS cell membrane was not efficient and may be affected by malfunction of lysosomal trafficking regulator encoded by mutant LYST. Furthermore, with LPS treatment, CHS cells presented a blunted response due to unchanged expression of TLRs compared to robust elevation of TLRs on control cells. The Toll-like receptor pathway is consider highly responsive to LPS challenge and can induce production of cytokines such as IL-6 as a mechanism to initiate immune responses. Thus, down-regulated gene expression of TLRs together with less expression of TLR protein on CHS cell membrane may lead to the significant less IL-6 protein levels in CHS cell culture medium both at baseline and with LSP treatment compared to the culture medium of control cells, providing a mechanism in which CHS patients are more susceptible to infection. Although the function of LYST remains incompletely understood, these findings support an important role in modulating key factors directing inflammatory responses. B. Disorders of mineralization: In collaboration with Michael Collins, a NIDCR clinical researcher and endocrinologist, we have been examining individuals under his care with disorders of mineralized tissues metabolism for alterations in tissues/cells of the DOC complex. To date we have obtained tissues and samples from one individual and currently processing tissues for evaluation. C. Idiopathic tooth root resorption: During our research on the Bsp KO mice we identified an idiopathic tooth root resportption phenotype. Based on this finding we are obtaining saliva samples from individuals with idiopathic root resorption (and appropriate controls) for genome wide association analysis.

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National Institute of Arthritis and Musculoskeletal and Skin Diseases
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