21 research outputs found
Sensitive quantification of the HIV-1 reservoir in gut-associated lymphoid tissue
Background: The implementation of successful strategies to achieve an HIV cure has become a priority in HIV research. However, the current location and size of HIV reservoirs is still unknown since there are limited tools to evaluate HIV latency in viral sanctuaries such as gut-associated lymphoid tissue (GALT). As reported in the so called "Boston Patients", despite undetectable levels of proviral HIV-1 DNA in blood and GALT, viral rebound happens in just few months after ART interruption. This fact might imply that current methods are not sensitive enough to detect residual reservoirs. Showing that, it is imperative to improve the detection and quantification of HIV-1 reservoir in tissue samples. Herein, we propose a novel non-enzymatic protocol for purification of Lamina Propria Leukocytes (LPL) from gut biopsies combined to viral HIV DNA (vDNA) quantification by droplet digital PCR (ddPCR) to improve the sensitivity and accuracy of viral reservoir measurements (LPL-vDNA assay). Methods: Endoscopic ileum biopsies were sampled from 12 HIV-1-infected cART-suppressed subjects. We performed a DTT/EDTA-based treatment for epithelial layer removal followed by non-enzymatic disruption of the tissue to obtain lamina propria cell suspension (LP). CD45+ cells were subsequently purified by flow sorting and vDNA was determined by ddPCR. Results: vDNA quantification levels were significantly higher in purified LPLs (CD45+) than in bulk LPs (p<0.01). The levels of vDNA were higher in ileum samples than in concurrent PBMC from the same individuals (p = 0.002). As a result of the increased sensitivity of this purification method, the Poisson 95% confidence intervals of the vDNA quantification data from LPLs were narrower than that from bulk LPs. Of note, vDNA was unambiguously quantified above the detection limit in 100% of LPL samples, while only in 58% of bulk LPs. Conclusion: We propose an innovative combined protocol for a more sensitive detection of the HIV reservoir in gut-associated viral sanctuaries, which might be used to evaluate any proposed eradication strategy
Interleukin-10 enhances the intestinal epithelial barrier in the presence of corticosteroids through p38 MAPK activity in Caco-2 monolayers : a possible mechanism for steroid responsiveness in ulcerative colitis
Altres ajuts: 2012 Spanish Gastroenterological Association i CIBER G0034Glucocorticosteroids are the first line therapy for moderate-severe flare-ups of ulcerative colitis. Despite that, up to 60% of patients do not respond adequately to steroid treatment. Previously, we reported that low IL-10 mRNA levels in intestine are associated with a poor response to glucocorticoids in active Crohn's disease. Here, we test whether IL-10 can favour the response to glucocorticoids by improving the TNFα-induced intestinal barrier damage (assessed by transepithelial electrical resistance) in Caco-2 monolayers, and their possible implications on glucocorticoid responsiveness in active ulcerative colitis. We show that the association of IL-10 and glucocorticoids improves the integrity of TNFα-treated Caco-2 cells and that p38 MAPK plays a key role. In vitro, IL-10 facilitates the nuclear translocation of p38 MAPK-phosphorylated thereby modulating glucocorticoids-receptor-α, IL-10-receptor-α and desmoglein-2 expression. In glucocorticoids-refractory patients, p38 MAPK phosphorylation and membrane desmoglein-2 expression are reduced in colonic epithelial cells. These results suggest that p38 MAPK-mediated synergism between IL-10 and glucocorticoids improves desmosome straightness contributing to the recovery of intestinal epithelium and reducing luminal antigens contact with lamina propria in ulcerative colitis. This study highlights the link between the intestinal epithelium in glucocorticoids-response in ulcerative colitis
Steroid-sensitive UC patients show a greater DSG2 staining.
<p>Immunofluorescence staining score (mean±SEM) of DSG2 (Panel A) in colonic biopsies of steroid-sensitive and steroid-refractory UC patients, before and after treatment. Representative DSG2 staining intensity of steroid-sensitive (Panel B) and steroid-refractory (Panel C) patients are also provided. All images are 630 x. *p≤ 0.004 <i>vs</i> steroid-refractory patients, <sup>#</sup>p = 0.023 <i>vs</i> after GC treatment.</p
IL-10 reverts the GC-mediated decrease in phosphorylated-p38 MAPK activity.
<p>Figure shows (median (IQR, limits)) the ratio for total p38 MAPK (Panel A) and phosphorylated p38 MAPK (Panel B) versus control group in TNF-treated Caco-2 cell monolayers. An example of Western blot bands obtained for these proteins and α-tubulin (as housekeeping protein) is provided in Panel C. *p≤ 0.026 <i>vs</i>. all other groups, <sup>#</sup>p = 0.028 <i>vs</i>. TNF_IL10 and TNF_GC_IL10;.</p
Interleukin-10 enhances the intestinal epithelial barrier in the presence of corticosteroids through p38 MAPK activity in Caco-2 monolayers : a possible mechanism for steroid responsiveness in ulcerative colitis
Altres ajuts: 2012 Spanish Gastroenterological Association i CIBER G0034Glucocorticosteroids are the first line therapy for moderate-severe flare-ups of ulcerative colitis. Despite that, up to 60% of patients do not respond adequately to steroid treatment. Previously, we reported that low IL-10 mRNA levels in intestine are associated with a poor response to glucocorticoids in active Crohn's disease. Here, we test whether IL-10 can favour the response to glucocorticoids by improving the TNFα-induced intestinal barrier damage (assessed by transepithelial electrical resistance) in Caco-2 monolayers, and their possible implications on glucocorticoid responsiveness in active ulcerative colitis. We show that the association of IL-10 and glucocorticoids improves the integrity of TNFα-treated Caco-2 cells and that p38 MAPK plays a key role. In vitro, IL-10 facilitates the nuclear translocation of p38 MAPK-phosphorylated thereby modulating glucocorticoids-receptor-α, IL-10-receptor-α and desmoglein-2 expression. In glucocorticoids-refractory patients, p38 MAPK phosphorylation and membrane desmoglein-2 expression are reduced in colonic epithelial cells. These results suggest that p38 MAPK-mediated synergism between IL-10 and glucocorticoids improves desmosome straightness contributing to the recovery of intestinal epithelium and reducing luminal antigens contact with lamina propria in ulcerative colitis. This study highlights the link between the intestinal epithelium in glucocorticoids-response in ulcerative colitis
IL-10 and GC restore control TEER values in Caco-2 cell monolayers.
<p> Figure represents median values (IQR, limits) of percent TEER changes in Caco-2 cell monolayers after 48h of incubation with different stimuli. *p ≤ 0.02 <i>vs</i> control and TNF_GC_IL10.</p
SB203580 reverses the synergistic action of IL-10 and GC on TEER in Caco-2 cell monolayers.
<p>Values are median (IQR, limits) of percent TEER changes in Caco-2 cell monolayers after 48h of incubation with different stimuli. *p≤ 0.042 <i>vs</i> control.</p
IL-10 and GC preserve p38 MAPK-dependent desmosome integrity and strength in Caco-2 monolayers.
<p>Representative transmission electron microscopy images of the paracellular junctions in Caco-2 cell monolayers treated with GC (Panel A), GC plus IL-10 (Panel B) or GC, IL-10 and SB203580 (Panel C) D: desmosomes; TJ: tight junctions. Magnification varies from 25,000 x to 100,000 x.</p
IL-10 plus GC increases GCR-α (p38 MAPK-dependent) and IL-10R-α expression in Caco-2 cell monolayers.
<p>Graphs represent fold change values (2<sup>-AACt</sup>) of IL-10R- β (Panel A), GCR-α (Panel B) and IL-10R-α (Panel C) expression, with respect to their basal condition (control or DMSO group). *p≤ 0.04 <i>vs</i> other groups.</p
p38 MAPK phosphorylation is reduced in colonic epithelial cells from GC-refractory UC patients before treatment.
<p>Panel A shows the immunostaining score (mean±SEM) of phosphorylated p38 MAPK in colonic biopsies of steroid-responsive and steroid-refractory active UC patients, before and after GC treatment. *p = 0.028 <i>vs</i> steroid-sensitive patients before treatment. Panel B provides representative images from steroid-refractory UC biopsies (score value 0–1), and from steroid-sensitive UC biopsies (score value 2–3). Scale bar: 20 μm.</p