Journal: PLoS ONE

Article Title: Elevated Serum Levels of Macrophage Migration Inhibitory Factor Are Associated with Progressive Chronic Cardiomyopathy in Patients with Chagas Disease

doi: 10.1371/journal.pone.0057181

Figure Lengend Snippet: C3H/He mice ( n = 5) were infected intraperitoneally with 10 6 blood trypomastigotes of the Sylvio X10/4 clone of T. cruzi (upper panel) or with 50 blood trypomastigotes of the Tulahuén strain of the parasite (central panel). Hearts from infected animals and uninfected controls (lower panel) were removed at 120 days p.i. (A) Immunohistochemical analysis was performed on cardiac muscle sections using murine MIF polyclonal antibodies. Microphotographs of myocardial tissues show a representative experiment of three performed. Bar = 50 µm. (B) MIF overexpression in the heart was closely accompanied by intense inflammatory cell infiltration. The leukocyte population invading the myocardium was isolated from 20 T. cruzi -infected mice at 120 days p.i. and digested with collagenase and hyaluronidase. The mononuclear cell fraction was incubated with anti-mouse CD11b/MAC-1- PerCP-Cy 5.5, CD3-FITC, CD4-PE and CD8-Alexa Fluor 647 antibodies. The labeled cells (at least 5×10 4 ) were fixed with 1% p -formaldehyde and analyzed by flow cytometry. (C), (D) Effect of exogenous MIF on T. cruzi -induced release of TNF-α and ROS in murine J774 macrophages. Adherent cells were infected for 24 h with culture trypomastigotes of T. cruzi (Tulahuén strain) at a 10∶1 parasite/cell ratio, in the presence (+ rMIF) or in the absence (− rMIF) of recombinant mouse MIF (1 µg/ml). TNF-α production was quantified in uninfected (Mock) and parasite-infected cell supernatants using a sandwich ELISA (C). For ROS measurement. J774 macrophages (10 6 ) were incubated with 10 µM DCFH-DA (2′,7′-dichlorodihydrofluorescein diacetate) for 30 min and then infected for 24 h with T. cruzi trypomastigotes with or without addition of rMIF, as described above. Uninfected (Mock) and infected cells were then fixed and analyzed by flow cytometry (D). Data are the means ± S E M of three independent experiments, each performed in triplicate. * P <0.05 and ** P <0.01 versus cells not pre-treated with MIF; # P <0.05 between infected and uninfected MIF-stimulated cells.

Article Snippet: Samples (at least 5×10 4 cells) were acquired in a Partec Past III flow cytometer and selected in the monocyte population gate according to their forward scatter versus side scatter features.

Techniques: Infection, Immunohistochemical staining, Over Expression, Isolation, Incubation, Labeling, Flow Cytometry, Recombinant, Sandwich ELISA