Additional file 1: of Effects of Trypanosoma cruzi on the phenoloxidase and prophenoloxidase activity in the vector Meccus pallidipennis (Hemiptera: Reduviidae)

Table S1. Activity of PO and proPO in Meccus pallidipennis, according to hemolymph or anterior midgut and group. (DOCX 21 kb

Extracellular eosinophil traps induced by co-incubation with <i>L</i>. <i>mexicana</i>.

A) Extracellular traps (black asterisk) containing MBP-positive granules (black arrow) and parasites within the nets (red arrow). B) Purified eosinophils show their nuclei stained blue with DAPI (green arrow). No nets are formed in the absence of Leishmania. C) Eosinophils co-incubated with L. mexicana promastigotes show the formation of extracellular DNA traps (white arrow). D) The same image (C) was analyzed with light and fluorescence microscopy, showing the parasites (red arrow) in contact with nets (white arrow). Scale bar = 20 μm.</p

Phagocytosed parasites by eosinophils.

A) Parasites stained with CFDA (green color) phagocytosed by eosinophils are observed inside these cells. Black arrows show phagocytized parasites. B) Remains of degraded parasites (black arrows) are observed already inside the eosinophils (green color granules). The co-incubation ratio was 1:10 for 2 hours. Scale bar = 20 μm. (TIF)</p

Anti-<i>Leishmania</i> IgE and IgG in sera from patients with cutaneous leishmaniasis.

A. IgE levels. B. IgG levels. Each symbol represents a patient, and the horizontal line represents the mean of each group. LCL (n = 8) and DCL (n = 8) patients, healthy controls (n = 8). *All symbols represent statistically significant differences (P < 0.05).</p

Eosinophils in peripheral blood of patients with cutaneous leishmaniasis.

A. Percentage of blood eosinophils from patients with LCL (black circles, n = 30) and DCL (blue squares, n = 4). B. Blood eosinophils and disease evolution time according to disease form and gender are shown: LCL male patients are indicated with green filled circles; DCL male patients are indicated by filled red squares. LCL female patients are indicated by empty green circles; DCL female patients are indicated with empty red squares). The horizontal line represents the mean, and each symbol represents a patient (female or male). Bars represent the mean ± SEM. ns: non-significant.</p

Phagocytosis and lysis of <i>Leishmania mexicana</i> by eosinophils.

A. Purified eosinophils in the absence of parasites. B. Eosinophils incubated with L. mexicana promastigotes (Giemsa stained). Close contact between parasites and cells leads to abundant degranulation (green arrow) and damaged parasites (black arrows). C. Leishmania promastigotes stained with CFDA. D. Eosinophils co- incubated with Leishmania promastigotes show phagocytosed parasites (black arrow). Scale bar = 20 μm. E. Percentage of eosinophil degranulation with or without L. mexicana (n = 3). Bars represent the mean ± SD, and the asterisk represents statistically significant differences (P <0.05).</p

Release of cytoplasmic “degranulation sacs” by eosinophils co-incubated with parasites.

A. Eosinophil (green arrow) and release of cytoplasmic “degranulation sacs’’. B. Degranulation sacs (black arrows) and parasites (pink arrow). C and D. Ultramicroscopic images showing released specific or secondary granules (g) or within cytoplasmic pockets limited by an intact membrane (black arrows). These “degranulation sacs” were in close position to distorted L. mexicana (Lm). Parasites, identified by their submembranous microtubule cytoskeleton (arrowheads) show signs of damage as evidenced by osmiophilic areas (white asterisk), nuclear fragmentation (n), and a clear or aqueous cytoplasm denoting an extensive cytoplasmic vacuolization. (D) Some eosinophil specific granules still retain the characteristic electron-dense central crystalline core, surrounded by a clearer peripheral matrix (red arrows). Light microscopy scale bar = 20 μm. TEM scale bar = 500 nm.</p

Eosinophils in lesions of patients with cutaneous leishmaniasis.

A. Eosinophils in the tissue were quantitated and expressed as eosinophils/mm2. LCL patients (black circles, n = 35) and DCL patients (blue squares, n = 5). B. Eosinophils/mm2 in tissue lesions according to disease form and gender. Male LCL patients (n = 25, filled green circles) and female LCL patients (n = 10, empty green circles); Male DCL patients (n = 4, filled red triangles) and female (n = 1, empty red triangle). Bottom panel: Representative H&E staining of biopsies taken from LCL and DCL patients. Black arrows indicate Leishmania amastigotes and blue arrows indicate eosinophils. Scale bar = 20 μm. Bars represent the mean ± SEM, and asterisks represent statistically significant differences (P < 0.05).</p

Cytokine production and oxidative burst were analyzed in eosinophils from patients with LCL and DCL.

Eosinophils from LCL and DCL patients and from healthy controls were incubated with L. mexicana promastigotes. A. IL-6 production. B. IL-8 production. C. IL-13 production. D. ROS production measured by luminol reaction (in mV). Bars represent the mean ± SEM (n = 7 for each group of patients and healthy control subjects). All symbols represent statistically significant differences (P < 0.05).</p

<i>Leishmania mexicana</i> damaged by eosinophil contact.

A) Normal shape and size of a viable Leishmania promastigote. B) Morphological changes in parasite size, damage to membranes, formation of small vacuoles within the parasite, and loss of flagellum were observed in parasites co-incubated with eosinophils in a 1:10 ratio for 1 hour. Black arrows show damaged parasites. Scale bar = 20 μm. (TIF)</p