Anti-IL-4 mAb treatment reduces lesion thickness in LTCP393(R)-inoculated BALB/c mice.

<p>The animals were inoculated with 1×10⁶<i>L. braziliensis</i> stationary phase promastigotes, isolates (A) LTCP393(R) or (B) LTCP15171(S), in the right ear dermis, treated with anti-IL-4 monoclonal antibody (black signs) or rat IgG (white signs), and the course of lesion development was monitored for 7 weeks. Lesion thickness was determined as the difference between the infected ear and the contralateral one, noninfected. Results are expressed as mean ± standard error of mean, and are representative of 2 independent experiments. *p<0,05.</p

Anti-IL-4 mAb treatment reduces parasite loads in LTCP393(R)-inoculated BALB/c mice lesions.

<p>The animals were inoculated at right ear dermis with 1×10⁶ stationary phase promastigotes of <i>L. braziliensis</i>, isolates LTCP393(R) (black bars) or LTCP15171(S) (white bars), treated with anti-IL-4 monoclonal antibody (hatched bars) or rat IgG (full bars), and the parasite load estimated at 7 weeks post infection in the ears (A) and draining lymph nodes (B). Results are expressed as mean ± standard error of mean, and are representative of 2 independent experiments. Statistical analysis: ANOVA and Tukey multiple comparision test. *p<0,05.</p

LTCP393(R)-inoculated BALB/c mice present higher parasite loads in the lesion sites.

<p>Parasite load estimate in ears (A) and lymph nodes (B) of BALB/c mice inoculated at right ear dermis with 1×10⁶ stationary phase promastigotes of <i>L. braziliensis</i>, isolates LTCP393(R) (black bars) or LTCP15171(S) (white bars), at 3, 5, 7 and 12 weeks post infection. Results are expressed as mean ± standard error of mean, and are representative of 3 independent experiments. n.d. = non-detected. *p<0,05.</p

Flow cytometry of infected BALB/c mice ears inflammatory infiltrate.

<p>The animals were inoculated with 1×10⁶ stationary phase <i>L. braziliensis</i> promastigotes, isolates LTCP393(R) (black bars) or LTCP15171(S) (white bars). Analyses were conducted at 3, 5, 7 and 12 weeks post infection, FoxP3, CD103, CTLA-4 and GITR expression on CD4⁺CD25⁺ cells were evaluated at 7 and 12 weeks post infection. Results are expressed as total number of cells in the lesion (A, B, C and D), and as percentage of cells inside CD4⁺CD25⁺ gate (E and F). Results are expressed as mean ± standard error of mean, and are representative of 3 independent experiments. *p<0,05.</p

LTCP393(R)-inoculated BALB/c mice present larger lesions.

<p>(A) The animals were inoculated with 1×10⁶<i>Leishmania braziliensis</i> stationary phase promastigotes, isolates LTCP393(R) (black squares) or LTCP15171(S) (white squares) in the right ear dermis and the course of lesion development was monitored for 15 weeks. Lesion thickness was determined as the difference between the infected ear and the contralateral one, noninfected. Results are expressed as mean ± standard error of mean, and are representative of 2–3 independent experiments. (B) Photographs of mouse ears at 3, 5, 7 and 12 weeks post infection (w.p.i.). The arrow indicates ulceration in the ear of an animal infected with LTCP393(R) isolate at 7 weeks post infection, and the arrowhead indicates remaining lesion at 12 weeks post infection in animals infected with LTCP393(R) isolate. *p<0,05.</p

Cytokines and enzymes mRNA expression in the ears from LTCP393(R) and LTCP15171(S)-inoculated mice.

<p>The animals were inoculated with 1×10⁶ stationary phase <i>L. braziliensis</i> promastigotes, isolates LTCP393(R) (black bars) or LTCP15171(S) (white bars). mRNA was extracted from the ears at 3, 5, 7 and 12 weeks post infection, and the specific mRNAs for IFN-γ (A), IL-4 (B), IL-10 (C), TGF-β (D), TNF-α (E), iNOS (F) and Arg I(G) were detected by real time RT PCR. The specific molecules mRNA expression was normalized based in the endogenous expression of the mRNAm for β-actin. Results are listed as the mean of expression in the ears of infected animals in relation to ears of uninfected ones ± standard error of mean, and are representative of 3 independent experiments. *p<0,05.</p

The Severity of Visceral Leishmaniasis Correlates with Elevated Levels of Serum IL-6, IL-27 and sCD14

<div><p>Background</p><p>Visceral leishmaniasis (VL) is a severe disease caused by infection with protozoa of the genus <i>Leishmania</i>. Classic VL is characterized by a systemic infection of phagocytic cells and an intense activation of the inflammatory response. It is unclear why 90% of infected individuals do not develop the disease while a minority develop the classical form. Furthermore, among those that develop disease, a small group progresses to more severe form that is unresponsive to treatment. The presence of inflammatory mediators in serum could theoretically help to control the infection. However, there is also a release of anti-inflammatory mediators that could interfere with the control of parasite multiplication. In this study, we took advantage of the spectrum of outcomes to test the hypothesis that the immune profile of individuals infected with <i>Leishmania (L</i>.<i>) infantum</i> is associated with the development and severity of disease.</p><p>Methodology/Principal Findings</p><p>Sera from patients with confirmed diagnosis of VL were evaluated for the presence of numerous molecules, and levels compared with healthy control and asymptomatic infected individuals.</p><p>Conclusions/Principal Findings</p><p>Although differences were not observed in LPS levels, higher levels of sCD14 were detected in VL patients. Our data suggest that <i>L</i>. <i>infantum</i> may activate the inflammatory response via CD14, stimulating a generalized inflammatory response with production of several cytokines and soluble molecules, including IFN-γ, IL-27, IL-10, IL-6 and sCD14. These molecules were strongly associated with hepatosplenomegaly, neutropenia and thrombocytopenia. We also observed that IL-6 levels greater than 200 pg/ml were strongly associated with death. Together our data reinforce the close relationship of IFN-γ, IL-10, IL-6, TNF-α and IL-27 in the immune dynamics of VL and suggest the direct participation of sCD14 in the activation of the immune response against <i>L</i>. <i>infantum</i>.</p></div

High IL-6 levels in serum is associated with disease severity.

<p>Serum levels of IL-6 and IL-27 measured before treatment by Luminex assay were compared in VL patients. (A) IL-6 levels in patients with classical VL (n = 25) and SVL (n = 12). (B) IL-6 levels in patients with classical VL (n = 25) and SVL that survived (n = 7) and SVL that died (n = 5). (C) IL-27 levels in patients with classical VL (n = 25) and SVL (n = 12).</p

Levels of sCD14 and MIF are elevated in the serum of VL patients.

<p>Serum levels of the LPS (A), FABP2 (B), sCD14 (C) and MIF (D) were measured by ELISA in classical VL patients before (n = 25) and after treatment (n = 17) (D0 and D30, respectively), severe VL patients (n = 12), DTH+ (n = 11) and healthy control (n = 07). * Mann-Whitey test.</p

Serum levels of cytokines.

<p>Cytokines were measured by Luminex assay in sera of VL patients before (n = 25) and after treatment (n = 17) (D0 and D30, respectively), DTH+ (n = 11) and healthy control (n = 7). (A) IFN-γ (B) IL-10, (C) IL-6, (D) IL-27 and (E) TNF-α.</p