Resultados de investigación

Supplementary Table 1. Cardiac function assessed through echocardiography of patients included in the study. Supplementary Table 2. Adjusted means of metabolic phenotypes of fatty liver disease with visceral adipose tissue and visceral adipose tissue/subcutaneous adipose tissue ratio. Supplementary Table 3. Associations between metabolic phenotypes of fatty liver disease and high indexed epicardial adipose tissue (>68.1 mL). Supplementary Table 4. Associations between metabolic phenotypes of fatty liver disease and moderate to severe coronary artery calcification (Agatston CAC score>100)., [Background] To better understand the patient's heterogeneity in fatty liver disease (FLD), metabolic dysfunction–associated fatty liver disease (MAFLD) was proposed by international experts as a new nomenclature for nonalcoholic fatty liver disease (NAFLD). We aimed to evaluate the cardiovascular risk, assessed through coronary artery calcium (CAC) and epicardial adipose tissue (EAT), of patients without FLD and patients with FLD and its different subtypes., [Methods] Cross sectional study of 370 patients. Patients with FLD were divided into 4 groups: FLD without metabolic dysfunction (non-MD FLD), MAFLD and the presence of overweight/obesity (MAFLD-OW), MAFLD and the presence of two metabolic abnormalities (MAFLD-MD) and MAFLD and the presence of T2D (MAFLD-T2D). MAFLD-OW included two subgroups: metabolically healthy obesity (MHO) and metabolically unhealthy obesity (MUHO). The patients without FLD were divided into 2 groups: patients without FLD and without MD (non-FLD nor MD; reference group) and patients without FLD but with MD (non-FLD with MD). EAT and CAC (measured through the Agatston Score) were determined by computed tomography., [Results] Compared with the reference group (non-FLD nor MD), regarding EAT, patients with MAFLD-T2D and MAFLD-MUHO had the highest risk for CVD (OR 15.87, 95% CI 4.26-59.12 and OR 17.60, 95% CI 6.71-46.20, respectively), patients with MAFLD-MHO were also at risk for CVD (OR 3.62, 95% CI 1.83-7.16), and patients with non-MD FLD did not have a significantly increased risk (OR 1.77; 95% CI 0.67-4.73). Regarding CAC, patients with MAFLD-T2D had an increased risk for CVD (OR 6.56, 95% CI 2.18-19.76). Patients with MAFLD-MUHO, MAFLD-MHO and non-MD FLD did not have a significantly increased risk compared with the reference group (OR 2.54, 95% CI 0.90-7.13; OR 1.84, 95% CI 0.67-5.00 and OR 2.11, 95% CI 0.46-9.74, respectively)., [Conclusion] MAFLD–T2D and MAFLD–OW phenotypes had a significant risk for CVD. MAFLD new criteria reinforced the importance of identifying metabolic phenotypes in populations as it may help to identify patients with higher CVD risk and offer a personalized therapeutic management in a primary prevention setting., Peer reviewed

Supplemental Table 1. Fatty acid composition in spleen cells of wild-type C57BL/6J mice at the end of 20-week dietary regimens. Supplemental Figure 1. An example of the gating strategy used to identify CD4+ (CD45+CD3+CD4+CD8–) and CD8+ (CD45+CD3+CD4–CD8+) T cells in spleen or bone marrow of wild-type C57BL/6J mice fed with the LFD and HFDs at the end of 20-week dietary regimens. SSC-A side scatter area, FSC-A forward scatter area, CD cluster of differentiation. Supplemental Figure 2. An example of the gating strategy used to identify CD3+ T cells and CD4+ and CD8+ T cell subsets in spleen cell suspensions of lean wild-type C57BL/6J mice after fatty acid treatment for 18 h. A similar gating strategy was used for bone marrow cell suspensions. SSC-A side scatter area, FSC-A forward scatter area, CD cluster of differentiation, 7-AAD 7-amino-actinomycin D. Supplemental Figure 3. CD3+ and CD4+ T cells in spleen cell suspensions of lean wild-type C57BL/6J mice treated with palmitic acid or oleic acid at indicated concentrations. The percentage relative to the negative control (cells without any treatment) of CD3+ and CD4+ T cells after the treatment of spleen cell suspensions with low-endotoxin fatty acid-free BSA control, BSA-palmitic acid complex (25-300 M) (A, D) or BSA-oleic acid complex (25-300 M) (B, E) for 18 h. The comparative of palmitic and oleic acids at 300 M is also shown (C, F). The data are presented as the mean (bars) and individual points ± SD values (n = 6), and those marked with different letters are significantly different (p<0.05). Differences between groups were assessed by one-way ANOVA with Tukey’s post hoc test or, in the case of variance heterogeneity, by Kruskal-Wallis with Bonferroni correction. CD cluster of differentiation, PA palmitic acid, OA oleic acid. Supplemental Figure 4. CD8+ T cells in spleen and CD3+ T cells in bone marrow (BM) cell suspensions of lean wild-type C57BL/6J mice treated with palmitic acid or oleic acid at indicated concentrations. The percentage relative to the negative control (cells without any treatment) of CD8+ T cells (spleen) and CD3+ T cells (BM) after the treatment of each cell suspension with low-endotoxin fatty acid-free BSA control, BSA-palmitic acid complex (25-300 M) (A, D) or BSA-oleic acid complex (25-300 M) (B, E) for 18 h. The comparative of palmitic and oleic acids at 300 M is also shown (C, F). The data are presented as the mean (bars) and individual points ± SD values (n = 6), and those marked with different letters are significantly different (p<0.05). Differences between groups were assessed by one-way ANOVA with Tukey’s post hoc test or, in the case of variance heterogeneity, by Kruskal-Wallis with Bonferroni correction. CD cluster of differentiation, PA palmitic acid, OA oleic acid. Supplemental Figure 5. CD4+ and CD8+ T cells in bone marrow (BM) cell suspensions of lean wild-type C57BL/6J mice treated with palmitic acid or oleic acid at indicated concentrations. The percentage relative to the negative control (cells without any treatment) of CD4+ and CD8+ T cells after the treatment of BM cell suspensions with low-endotoxin fatty acid-free BSA control, BSA-palmitic acid complex (300 M) (A, D) or BSA-oleic acid complex (300 M) (B, E) for 18 h. The comparative of palmitic and oleic acids at 300 M is also shown (C, F). The data are presented as the mean (bars) and individual points ± SD values (n = 6), and those marked with different letters are significantly different (p<0.05). Differences between groups were assessed by one-way ANOVA with Tukey’s post hoc test or, in the case of variance heterogeneity, by Kruskal-Wallis with Bonferroni correction. CD cluster of differentiation, PA palmitic acid, OA oleic acid., Peer reviewed

Supplementary Movie S1. FRAPexperiment in which the quick recovery of mEGFP-JMJD3 puncta after photobleaching is observed. Supplementary Movie S2. FRAP experiment in which the aggregation of mEGFP-JMJD3 is appreciated by means of a reduced mobility after photobleaching. Supplementary Data file 1. Table depicting the parameters that assess the quality of the Chst8 4C-seq experiments for each sample as previously described. Briefly, the % of reads containing VP sequence should be above 90, the % of fragments that map in cis above 50, and the % of reads that map in unique sites within 1Mb around VP above 60. Supplementary Data file 2 and 3. Tables containing the quality measurements for each sample of the Ldlrad4 4C-seq (2) and Aopep 4C-seq (3) experiments, according to the parameters previously described. Briefly, the % of reads containing VP sequence should be above 90, the % of fragments that map in cis above 50, and the % of reads that map in unique sites within 1Mb around VP above 60., Peer reviewed

A, Predictive models for hospital discharge during the first week in mild patients. B, Predictive models for worsening of clinical status during the first week in patients who were admitted mildly ill. AUC, area under the curve., Peer reviewed

S2 Fig. Migration and activation marker expressing in monocytes subset in mild and several/critical patients (S/C)., Peer reviewed

Quantitative variables are expressing as number (percentage) or median (interquartile range). Pa value for differences between patients who were or not discharged. Pb value for differences between patients who who did and did not get wore. SpO2, peripheral capillary oxygen saturation; CRP, C-reactive protein; LDH, Lactate dehydrogenase; NLR, neutrophil/lymphocyte ratio., Peer reviewed

AUC, area under the curve; SE, sensitivity; S, specificity; PPV, positive predictive value; NPV, negative predictive value. SpO2, peripheral capillary oxygen saturation; CRP, C-reactive protein; LDH, Lactate dehydrogenase; NLR, neutrophil/lymphocyte ratio; TNF-α; tumor necrosis factor α; IL-6, interleukine-6; IL-8, interleukine-8; IL-1β, interleukine-1β; MIP-1β, macrophage inflammatory proteins 1β; sCD25, soluble receptor interleukine-2; IP-10, interferon γ-induced protein 10., Peer reviewed

AUC, area under the curve; SE, sensitivity; S, specificity; PPV, positive predictive value; NPV, negative predictive value. SpO2, peripheral capillary oxygen saturation; CRP, C-reactive protein; LDH, Lactate dehydrogenase; NLR, neutrophil/lymphocyte ratio; TNF-α; tumor necrosis factor α; IL-6, interleukine-6; IL-8, interleukine-8; IL-1β, interleukine-1β; MIP-1β, macrophage inflammatory proteins 1β; sCD25, soluble receptor interleukine-2; IP-10, interferon γ-induced protein 10., Peer reviewed

Data are expressed by percentage and interquartile range. Medians fluorescence intensitive (MFI) were calculated in those markets that have a high rate of expression., Peer reviewed

Supplementary Table 1: Antibodies for Flow Cytometry. Supplementary Figure 1. CD25 and Foxp3 expression, absolute Treg quantification and Tcon:Treg ratio. a. Representative cytometries showing CD25 and Foxp3 staining of CD4+ (blue) and CD8+ (red) cells after anti-CD3/CD28 activation and ruxolitinib treatment. b. Absolute quantification of CD4+CD25+Foxp3high cells. Average and S.E.M. of two independent experiments with three technical replicates each are shown. Cells are normalized to counting beads in the culture (1 bead/50 PBMNCs in the initial culture) c. Quantification of CD4+ or CD8+ to CD4+CD25+Foxp3high cells ratio . Average and S.E.M. of two independent experiments with three technical replicates each are shown. Supplementary Figure 2. Correlation of PD1 and Helios, cytometries of CTLA4 and CD39. a. Freshly isolated huPBMNCs were analyzed for Helios and PD1 expression in CD4+ CD25+ Foxp3high Tregs. One representative cytometry is showing the left, and the quantification of 5 independent experiments is shown in the right panel (mean +/-S.E.M.). b. Dot plot of the percentage of PD1+ cells versus Helios+ cells of CD4+ Foxp3+ gated cells after 2, 5 and 8 days of antiCD3 and antiCD8 stimulation and Ruxolitinib treatments. The linear regression of each day is shown, with the equation, Pearson correlation (R2) and p value. c. Representative cytometry of stimulated huPBMNCs, gated for CD4+, showing CD39 and CTLA4 staining after 2, 5 and 8 days of treatment with 0 or 0.3μM Ruxolitinib. Supplementary Figure 3. Suppression assay after pretreatment of Treg with Ruxolitinib. MACS sorted Treg were preincubated in the presence of ruxolitinib 0.1 and 0.3 μM for 24h and then ruxolitinib was washed. Later, CD4+ cells labeled with cell division tracking dye were activated with anti CD3 and anti CD28 antibodies and the pretreated Treg were added at different proportions. a. CD4+ proliferation was determined by flow cytometry. b. The percentage of suppression was calculated as {1-(%proliferation in the sample/%proliferation in the control)}×100%. Supplementary Figure 4. Cytometry analysis of samples from the GvHD mouse model. Mice were sacrificed 10 weeks after treatment start (14 weeks after BM transplantation) and the cells of different organs were analyzed by cytometry. a. Representative cytometries of peripheral blood from BM only transplanted mice (no GvHD), or spleen and BM transplanted cells treated with vehicle, Tregs, Ruxolitinib or both. CD45 gated cells are shown in the upper row for CD3 and CD19 staining, CD3 gated cells are further gated for CD8 and CD4 staining (middle row) and CD4 gated cells are analyzed for CD25 and GFP expression (lower row). b. Cells from bone marrow are analyzed as in A. c. Cells from BM are stained for Foxp3 expression. Cells are gated for CD4 expression (upper row), and the represented for Foxp3 versus CD25 (middle row) or Foxp3 versus GFP. GFP signal is reduced compared to figure B due to the fixation and permeabilization used for Foxp3 intracellular staining. d. Bone marrow biopsies of mice infused with GFP Tregs at the indicated times post-infusion. CD4 gated cells are analyzed for CD25 and GFP expression. Supplementary figure 5. Quantification of cell populations in the GvHD mouse model. a. Peripheral blood samples were extracted from mice at 2, 4 and 6 weeks after treatment start (6, 8 and 10 weeks post transplantation). Samples of at least 4 mice per group were analyzed following the gating strategy shown in supplemental figure 4A. Mean and S.E.M. is represented. b. Mice were sacrificed 10 weeks after treatment start (14 weeks after BM transplantation). Cell populations from peripheral blood, bone marrow (BM), large intestine (LI), Peyer ́s patches (PP), small intestine (SI), spleen and thymus were analyzed as in A. c. CD25+ Foxp3high Tregs from peripheral blood, bone marrow (BM), spleen and thymus were analyzed following the gating strategy shown in supplementary figure 4C. Mean and S.E.M. is represented. d. Blood samples from mice at 2, 4 and 6 weeks after treatment start were analyzed using an haematocytometer. LYM= lymphocytes (103 /μl), MON=monocytes (103 /μl), GRA=granulocytes (103 /μl), WBC= White blood cells (103 /μl), percent_LYM= percentage of lymphocytes, percent_MON= percentage of monocytes, percent_GRA= percentage of granulocytes. p values of a Student ́s t-test are shown. Significant values are marked with red arrows. Supplementary figure 6. Histopathological analysis of small and large intestine and skin of the GvHD mouse model. Large intestine, small intestine and skin from mice sacrificed after 10 weeks of treatment were fixed in formalin, and included in paraffin. Slices were stained with hematoxylin-eosin and Masson ́s trichrome, and were evaluated by a pathologist for GvHD signs. A histopathological score was assigned according to published scoring system56. Average plus standard error of the mean of four mice per group are shown. LI, Large Intestine. SI, Small Intestine., Peer reviewed