Pulmonary sarcoidosis is associated with exosomal vitamin D-binding protein and inflammatory molecules

BACKGROUND: Sarcoidosis is an inflammatory granulomatous disorder characterized by accumulation of TH1-type CD4+T cells and immune effector cells within affected organs, most frequently the lungs. Exosomes are extracellular vesicles conveying intercellular communication with possible diagnostic and therapeutic applications.OBJECTIVES: Weaimed to provide an understanding of the proinflammatory role of bronchoalveolar lavage fluid (BALF) exosomes in patients with sarcoidosis and to find candidates for disease biomarkers.METHODS: Weperformed a mass spectrometric proteomics characterization of BALF exosomes from 15 patients with sarcoidosis and 5 healthy control subjects and verified the most interesting results with flow cytometry, ELISA, and Western blot analyses in an additional 39 patients and 22 control subjects.RESULTS: Morethan 690 proteins were identified in the BALF exosomes, several of which displayed significant upregulation in patients, including inflammation-associated proteins, such as leukotriene A4 hydrolase. Most of the complement-activating factors were upregulated, whereas the complement regulator CD55 was seen less in patients comparedwith healthy control subjects. In addition, for the first time, we detected vitamin D-binding protein in BALF exosomes, which was more abundant in patients. To evaluate exosome-associated vitamin D-binding protein as a biomarker for sarcoidosis, we investigated plasma exosomes from 23 patients and 11 healthy control subjects and found significantlyhigher expression in patients.CONCLUSION: Together,these data contribute to understanding the role of exosomes in lung disease and provide suggestions for highly warranted sarcoidosis biomarkers. Furthermore, the validation of an exosome-associated biomarker in the blood of patients provides novel, and less invasive, opportunities for disease diagnosis.</h4

Determination of the physical environment within the Chlamydia trachomatis inclusion using ion-selective ratiometric probes

Chlamydia trachomatis is an obligate intracellular bacterium with a biphasic life cycle that takes place entirely within a membrane-bound vacuole termed an inclusion. The chlamydial inclusion is non-fusogenic with endosomal or lysosomal compartments but intersects a pathway involved in transport of sphingomyelin from the Golgi apparatus to the plasma membrane. The physical conditions within the mature chlamydial inclusion are unknown. We used ratiometric imaging with membrane-permeant, ion-selective fluorescent dyes for microanalyis of the physical environment within the inclusion. Determination of H + , Na + , K + and Ca 2 + concentrations using CFDA (carboxy fluorescein diacetate) or BCECF-AM (2 ′ ,7 ′ -bis (2-carboxyethyl)-5,6-carboxyfluorescein acetoxymethyl ester, SBFI-AM, PBFI-AM and fura-PE3-acetomethoxyester (Fura-PE3-AM), respectively, indicated that all ions assayed within the lumenal space of the inclusion approximated the concentrations within the cytoplasm. Stimulation of purinergic receptors by addition of extracellular ATP triggered a dynamic Ca 2 + response that occurred simultaneously within the cytoplasm and interior of the inclusion. The chlamydial inclusion thus appears to be freely permeable to cytoplasmic ions. These results have implications for nutrient acquisition by chlamydiae and may contribute to the non-fusogenicity of the inclusion with endocytic compartments.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/72253/1/j.1462-5822.2002.00191.x.pd