Phagocytosis and the killing of <i>L</i>. <i>braziliensis</i> by monocytes from CL patients.

<p>Monocytes from CL patients (n = 9) and HS individuals (n = 6) were infected with <i>L</i>. <i>braziliensis</i> promastigotes at a 5:1 ratio for 2, 24, 48 and 72 hours. The number of infected cells (A) and the number of intracellular parasites (B) were determined by microscopic evaluation after May-Grunwald-Giemsa staining from cytocentrifuge preparations. Monocytes were preincubated with either DPI (10mM) or L-NMMA (1mM), for 10 minutes and were infected with <i>L</i>. <i>braziliensis</i> promastigotes at a 5:1 ratio for 72 hours. (C) The number of infected cells. (D) The number of intracellular parasites. Statistical analysis was performed using the Kruskal-Wallis test (* p < 0.05, ** p < 0.01).</p

Inhibition of NADPH oxidase decreases the oxidative burst.

<p>Monocytes from CL patients (n = 15) and HS individuals (n = 7) were preincubated with either DPI (10mM), an inhibitor of the NADPH oxidase, or L-NMMA (1mM), an iNOS inhibitor, for 10 minutes. The monocytes were pre-incubated with DHR (10 minutes) and infected with <i>L</i>.<i>braziliensis</i> promastigotes (5:1cells) or stimulated with PMA (1 ug/mL) for 25 minutes. Cells were stained with anti-CD14. Data were collected using flow cytometry and analyzed using FLOWJO software. (A) Representative contour plots. (B) The data represent the median of mean intensity of fluorescence (MIF) of DHR expression by monocytes from CL patients and HS individuals (C). Statistical analysis was performing using ANOVA with Bonferoni´s pos-test and Manny Whitney test. The results were considered significant with a p< 0.05 (** p<0.01; ***p<0.001).</p

Oxidative burst production before and after therapy and cure of CL patients.

<p>Production of burst oxidative, NO and ROS by monocytes from CL patients (n = 6) after infection with <i>L</i>.<i>braziliensis</i> promastigotes or upon PMA stimulus, were determined before and after therapy (i.v. pentavalent antimonial, 20mg/kg body weight daily for 20 days) and cure of cutaneous leishmaniasis. The data represent the median of mean intensity of fluorescence (MIF) of oxidative burst production (A), frequency of NO production (B) and frequency of ROS production (C). Statistical analysis was performed using Wilcoxon test and results were considered significant (p<0.05).</p

Clinical and epidemiological characteristics of cutaneous leishmaniasis patients (CL) and Healthy Subjects (HS).

<p>Clinical and epidemiological characteristics of cutaneous leishmaniasis patients (CL) and Healthy Subjects (HS).</p

The role of NO and NO in the control of <i>L</i>.<i>braziliensis</i> infection by monocytes from CL patients.

<p>Monocytes from CL patients (n = 9) and HS individuals (n = 6) were treated with inhibitor of the NADPH oxidase (DPI-10mM) or with an iNOS inhibitor (L-NMMA-1mM) for 10 minutes and infected with <i>L</i>.<i>braziliensis</i> at a 5:1. After 72 hours, the medium of monocytes culture was replaced by Schneider’s medium and after 5 days the number of viable promastigotes was estimated. Statistical analysis was performed using the Manny Whitney test. (*** p < 0.001).</p

Monocytes from CL patients produced high levels of reactive oxygen species after infection with <i>L</i>.<i>braziliensis</i>.

<p>Monocytes from CL patients (n = 13) and HS individuals (n = 7) were stained with DAF-FM diacetate (NO probe, 10mM) and CMH-2DCFDA (ROS probe, 1 μM) for 10 minutes, infected with <i>L</i>.<i>braziliensis</i> promastigotes for 25 minutes at a ratio of 5:1cells, and stained with anti-CD14. PMA was used as positive control. Data were collected using flow cytometry and analyzed using FLOWJO software (A). Representative histograms of ROS production, (B) Frequency of <i>L</i>.<i>braziliensis</i>-infected monocytes expressing ROS, (C) Representative histograms of NO production, (D) Frequency of <i>L</i>.<i>braziliensis</i>-infected monocytes expressing ROS. Statistical analysis was performing using ANOVA with Bonferoni´s pos-test and Manny Whitney test. The results were considered significant with a p< 0.05 (*p<0.05).</p

Correlation between NO and ROS production by monocytes and lesion size of CL patients.

<p>Monocytes from CL patients (n = 8) were treated with DAF-FM diacetate (NO probe, 10mM) or CMH-2DCFDA (ROS probe, 1 μM) for 10 minutes and infected with <i>L</i>.<i>braziliensis</i> promastigotes for 25 minutes at a ratio of 5:1cells as described in materials and methods. Production of NO and ROS was evaluated by flow cytometry. (A) Correlation between NO production (%) and lesion size (mm). (B) Correlation between ROS production (%) and lesion size (mm). Statistical analysis was performed using the Pearson correlation.</p

Atypical Manifestations of Cutaneous Leishmaniasis in a Region Endemic for <i>Leishmania braziliensis</i>: Clinical, Immunological and Parasitological Aspects

<div><p>Background</p><p>Atypical cutaneous leishmaniasis (ACL) has become progressively more frequent in Corte de Pedra, Northeast Brazil. Herein we characterize clinical presentation, antimony response, cytokine production and parasite strains prevailing in ACL.</p><p>Methodology/Principal Findings</p><p>Between 2005 and 2012, 51 ACL (cases) and 51 temporally matched cutaneous leishmaniasis (CL) subjects (controls) were enrolled and followed over time in Corte de Pedra. Clinical and therapeutic data were recorded for all subjects. Cytokine secretion by patients’ peripheral blood mononuclear cells (PBMC) stimulated with soluble parasite antigen in vitro, and genotypes in a 600 base-pair locus in chromosome 28 (CHR28/425451) of the infecting <i>L</i>. <i>(V</i>.<i>) braziliensis</i> were compared between the two groups. ACL presented significantly more lesions in head and neck, and higher rate of antimony failure than CL. Cytosine–Adenine substitutions at CHR28/425451 positions 254 and 321 were highly associated with ACL (p<0.0001). In vitro stimulated ACL PBMCs produced lower levels of IFN-γ (p = 0.0002) and TNF (p <0.0001), and higher levels of IL-10 (p = 0.0006) and IL-17 (p = 0.0008) than CL PBMCs.</p><p>Conclusions/Significance</p><p>ACL found in Northeast Brazil is caused by distinct genotypes of <i>L</i>. <i>(V</i>.<i>) braziliensis</i> and presents a cytokine profile that departs from that in classical CL patients. We think that differences in antigenic contents among parasites may be in part responsible for the variation in cytokine responses and possibly immunopathology between CL and ACL.</p></div

Comparison of SNP allele frequencies in locus CHR28/425451^* between <i>L. (V.) braziliensis</i> isolated from 16 atypical (ACL) and 38 localized cutaneous leishmaniasis (CL) temporally matched patients from Corte de Pedra, Brazil.

<p>SNPs in this table correspond to individual SNPs in haplotypes delineated in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005100#pntd.0005100.t002" target="_blank">Table 2</a>.</p

Clinical variables compared between 51 atypical (ACL) and 51 localized cutaneous leishmaniasis (CL) temporally matched patients from Corte de Pedra, Brazil.

<p>Clinical variables compared between 51 atypical (ACL) and 51 localized cutaneous leishmaniasis (CL) temporally matched patients from Corte de Pedra, Brazil.</p