No correlation between secreted levels of α-defensins 1-3 by imMDDC and plasmatic viral load (PVL).

<p>(A) HIV-infected patients (n = 39), (B) patients not receiving HAART (n = 30), (C) controllers (elite and viremic controllers; n = 19), and (D) non-controllers (viremic non-controllers and patients with HAART; n = 20). Spearman correlation test was applied.</p

Levels of α-defensins 1-3 produced by imMDDC from HIV-infected individuals.

<p>(A) Comparison between the secreted levels of α-defensins 1-3 by imMDDC from HIV-controllers (elite controllers and viremic controllers; n = 19) and HIV non-controllers (viremic non-controllers and patients with HAART; n = 20). (B) Secreted levels of α-defensins 1-3 by imMDDC from healthy non-infected (NI; n = 15), elite controllers (ELITE; n = 4), viremic controllers (VC; n = 15), viremic non-controllers (VNC; n = 11) and patients receiving HAART (HAART; n = 9). Dots indicate each patient and lines represent median ± interquartile ranges.</p

Characteristics of the patients included in the study.

<p>Characteristics of the patients included in the study.</p

Positive correlation between secreted levels of α-defensins 1-3 by imMDDC and CD4 T cell counts.

<p>(A) HIV-infected patients (n = 39), (B) patients not receiving HAART (n = 30), (C) controllers (elite and viremic controllers; n = 19), and (D) non-controllers (viremic non-controllers and patients with HAART; n = 20). Spearman correlation test was applied.</p

Levels of α-defensins 1-3 secreted by imMDDC were associated with indicators of disease progression.

<p>Patients without treatment at the moment of blood extraction for the differentiation of MDDC were divided in two groups according to their secreted levels of α-defensins 1-3 by imMDDC. Levels higher than the median (650 pg/ml) of the whole group of patients (black squares; n = 18) and secreted levels lower than the median (grey circles; n = 12). (A) Percentage of patients with a decrease in the number of CD4 T cells below 350 cel/mm³ during follow up time (months). (B) Percentage of patients with an increase in PVL greater than 0.5 logarithms and (C) percentage of patients without HAART at the moment of determination of α-defensins 1-3 levels but started treatment later during the follow up. P values and hazard ratio (HR) with 95% confidence interval values are represented in the figure. Log-Rank test was used to determine statistical differences between curves.</p

Analysis of plasma levels of α-defensins 1-3.

<p>(A) Plasma levels of α-defensins 1-3 detected in the different groups of HIV-infected patients, elite controllers (ELITE; n = 3), viremic controllers (VC; n = 10), viremic non-controllers (VNC; n = 5) and patients receiving HAART (HAART; n = 9). Dots indicate each patient and lines represent median ± interquartile ranges. (B) Correlation between CD4 T cell counts and plasma levels of α-defensins 1-3. (C) Correlation between PVL and plasma levels of α-defensins 1-3. (D) Plasma levels of α-defensins 1-3 in two different determinations over time in patients that remained controllers (VC-VC; white boxes; n = 13), patients that were controllers in the first determination but lost viremic control in the second one (VC-VNC; grey pointed boxes; n = 5), controller patients in the first determination with disease progression and HAART in the second one (VC-ART; grey boxes; n = 8) and patients receiving HAART during both determinations (ART-ART; grey lined boxes; n = 9). Boxes represent interquartile ranges, horizontal lines inside each box indicate the median and whiskers indicate maximum and minimum values. Mann-Whitney test was applied for the statistical analysis of every pair and the differences were not significant (NS).</p

Production of α-defensins 1-3 by immature (im) and mature (m) MDDCs.

<p>(A) Immature and mature MDDC immunophenotype from one representative healthy control individual and one HIV-infected subject. X-axis represents HLA-DR expression and Y-axis represents the surface markers indicated. The percentage of positive cells are shown in each quadrant. (B) Relative expression of α-defensins 1-3 mRNA by real time RT-PCR in healthy control individuals (n = 15; open circles) and HIV-infected subjects (n = 39; open rhombus). Dots represent each patient and horizontal lines represent median ± interquartile ranges. Undetectable samples from mMMDC in HC and HIV-infected patients are not represented in the figure. (C) Levels of α-defensins 1-3 detected in MDDC culture supernatants from healthy non-infected controls (n = 15; white boxes) and HIV-infected subjects (n = 39; grey boxes). Boxes represent interquartile ranges, horizontal lines inside each box indicate the median and whiskers indicate maximum and minimum values. (D) Intracellular staining and flow cytometry analysis of α-defensins 1-3 in immature MDDCs. MDDCs were treated with monensin for 8h and double-stained for CD40 and α-defensins 1-3. CD40 positive cells were selected and the histogram corresponds to positive cells for intracellular α-defensins 1-3. Histogram represents the isotype control (dots line), healthy control (open line) and HIV-infected subject (filled histogram). Numbers indicate the percent of positive cells for healthy control (normal number) or HIV-infected patient (bold numbers). Representative result of four different healthy controls and four HIV-infected patients. (E) Geometric mean fluorescence intensity for intracellular α-defensins 1-3 in non-infected individuals (n = 4; white box) and HIV-infected subjects (n = 4; grey box). Boxes represent interquartile ranges, horizontal lines inside each box indicate the median and whiskers indicate maximum and minimum values.</p

Clinical information of patients with HIV-1.

*<p>VL is expressed as log viral copies/ml.</p><p>NC: Non controller.</p><p>VC: Viremic Controller.</p><p>EC: Elite Controller.</p

Effect of ADA on the iDCs viability.

<p>iDCs, obtained as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051287#s2" target="_blank">Materials and Methods</a>, from healthy or HIV-infected donors were cultured during 48 h in the absence (−ADA) or in the presence (+ADA) of 2 µM ADA. Cell viability was assessed through DIOC₆ and propidium iodide (PI) staining and measured by flow cytometry. In (A), contour plots showing the percentage of viable (bright DIOC₆ and negative propidium iodide staining), apoptotic (low DIOC₆ and negative propidium iodide staining) and necrotic (low DIOC₆ and positive propidium iodide staining) populations from a representative healthy or HIV-infected donor are shown. Percentage of viable (B), apoptotic (C) and necrotic (D) DCs from 7 different healthy and 6 HIV-infected donors are shown.*<i>P</i><0.05.</p

ADA enhances CD83, CD80 and CD86 expression on iDCs.

<p>iDCs, obtained as indicated in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051287#s2" target="_blank">Materials and Methods</a>, were cultured for 48 h in medium in the absence (−ADA) or in the presence (+ADA) of 2 µM ADA or in the presence of maturating cocktail (mDCs). Expression of CD83 (A and B), CD80 (C and D) or CD86 (E and F) in the DCs gate was assessed by flow cytometry. In A, C and E, histogram overlays and the percentage of positive cells (A and B) or geometric mean (C) for a representative healthy donor and HIV-infected subject are shown. In B, D and F, values obtained from 16 to 19 healthy donors (circles) or 12 to 16 HIV-infected individuals (triangles) in the absence (open symbols) or in the presence (filled symbols) of 2 µM ADA are plotted. Each pair of linked symbols represents results from a particular individual. *<i>P</i><0.05; ***<i>P</i><0.001.</p