BUSCADOR RECOLECTA

[Introduction]: The Unfolded Protein Response, a mechanism triggered by the cell in response to Endoplasmic reticulum stress, is linked to inflammatory responses. Our aim was to identify novel Unfolded Protein Response-mechanisms that might be involved in triggering or perpetuating the inflammatory response carried out by the Intestinal Epithelial Cells in the context of Inflammatory Bowel Disease., [Methods]: We analyzed the transcriptional profile of human Intestinal Epithelial Cell lines treated with an Endoplasmic Reticulum stress inducer (thapsigargin) and/or proinflammatory stimuli. Several genes were further analyzed in colonic biopsies from Ulcerative Colitis patients and healthy controls. Lastly, we generated Caco-2 cells lacking HMGCS2 by CRISPR Cas-9 and analyzed the functional implications of its absence in Intestinal Epithelial Cells., [Results]: Exposure to a TLR ligand after thapsigargin treatment resulted in a powerful synergistic modulation of gene expression, which led us to identify new genes and pathways that could be involved in inflammatory responses linked to the Unfolded Protein Response. Key differentially expressed genes in the array also exhibited transcriptional alterations in colonic biopsies from active Ulcerative Colitis patients, including NKG2D ligands and the enzyme HMGCS2. Moreover, functional studies showed altered metabolic responses and epithelial barrier integrity in HMGCS2 deficient cell lines., [Conclusion]: We have identified new genes and pathways that are regulated by the Unfolded Protein Response in the context of Inflammatory Bowel Disease including HMGCS2, a gene involved in the metabolism of Short Chain Fatty Acids that may have an important role in intestinal inflammation linked to Endoplasmic Reticulum stress and the resolution of the epithelial damage., Peer reviewed

Strategic Action in Health of the Institute of Health Carlos III Department of Regional Health of the Castilla y Léon Government Consejería de Educación, Junta de Castilla y León, Peer reviewed

Strategic Action in Health of the Institute of Health Carlos III Department of Regional Health of the Castilla y Léon Government Consejería de Educación, Junta de Castilla y León, Peer reviewed

Additional file 1: Supplementary Fig. 1. A) Box plot showing SLC3A2 gene expression levels in paired normal colon tissue and tumoral CRC tissue, using TNMplot online tool. FC = fold change. P value obtained using Mann Whitney U test. B) Box plot showing SLC3A2 gene expression levels in normal colon tissue and tumoral CRC tissue samples. Data were obtained from the GEPIA2. P value obtained using one-way ANOVA test. C) Box plot showing SLC3A2 gene expression levels in normal colon tissue and tumoral CRC tissue normal, using TNMplot online tool. P value obtained using Mann Whitney U test. D) Box plot showing the CD98hc score analyzed by immunohistochemistry in primary tumor, lymph node and liver metastases. P values were obtained using Student t test (two-sided). E) Immunohistochemical staining of CD98hc in representative samples from the study shown in D, assessed using with anti-CD98hcV509 antibody. Magnification: 40X. F) Box plot showing SLC3A2 gene expression levels in tumor and metastatic tissue of CRC patients. T = tumor, N = normal and M = metastatic. P value obtained using Kruskal–Wallis test. G) Heatmap (top) and boxplot (bottom) representative of the expression of CD98hc in different normal and tumoral tissues, obtained from the TNMplot database. Tissues written in red represent significant differences by the Mann–Whitney test. H) Expression of CD98hc in different normal and tumoral tissues. Data obtained from the GENT2 database., Instituto de Salud Carlos III Consejo Superior de Investigaciones Científicas Junta de Castilla y León ALMOM ACMUMA UCCTA CRIS Cancer Foundation Agencia Estatal de Investigación Consejo Superior de Investigaciones Cientificas (CSIC), Peer reviewed

Additional file 2: Supplementary Fig. 2. A) Internalization of the anti-CD98hcECTO antibody in SW480 cells, analyzed by immunofluorescence. Scale bar = 25 μm. The cells were seeded on coverslips and treated with 10 nM of anti-CD98hcECTO for the times indicated. The images at the right correspond to magnifications of a cell present in the images obtained at 24 h. The colocalization of CD98hc and LAPM1 is show in the merged images. B) Preparation of the antibody–drug conjugate targeting CD98hc. The coupling of DM1 to the anti-CD98hcECTO antibody was evaluated by Western blot using an anti-DM1 antibody. Twenty nanograms of anti-CD98hcECTO-DM1, the nude anti-CD98hcECTO, trastuzumab or T-DM1 were used to detect DM1 (upper panel) and the total amount of protein was evaluated by stain-free blot (lower image). Trastuzumab and T-DM1 were used as a negative and positive controls. C-E) FACS analyses in HT29 (C), cells dissociated from patient PDX BT6224 (D) and human tumoral organoid #48 (E) using anti-CD98hc-DM1 as primary antibody. The red histogram correspond to signals from cells incubated with the secondary antibody alone, whereas the blue histograms represent the fluorescence due to the expression of CD98hc., Instituto de Salud Carlos III Consejo Superior de Investigaciones Científicas Junta de Castilla y León ALMOM ACMUMA UCCTA CRIS Cancer Foundation Agencia Estatal de Investigación Consejo Superior de Investigaciones Cientificas (CSIC), Peer reviewed

Additional file 3: Supplementary Fig. 3. A) Dose–response analyses of the effect of anti-CD98hc-DM1 on the proliferation of parental and CD98hc CRISPR #5 and #11 HT29 cells. Cells were treated with anti-CD98hc-DM1 at the indicated doses for four days. Results are shown as the mean ± SD of quadruplicates of an experiment repeated three times. B) Expression of CD98hc in normal human fibroblasts (NHF) and immortalized keratinocytes (HaCaT), compared to CRC cell lines. Cell extracts (20 µg) were used to identify CD98hc by Western blot with the anti-CD98hcV509 antibody. Calnexin was used as a loading control. C) Dose–response analyses of the anti-CD98hc-DM1 ADC on NHF and HaCaT, compared to HT29 cells. Cells were treated with the ADC for four days at the indicated doses. The data are plotted as the percentage of MTT metabolization with respect to control. Results are shown as the mean ± SD of quadruplicates of an experiment repeated two times. D) Evaluation of the effect of anti-CD98hc-DM1 (10 nM, 48 h) on the distribution of the different cell cycle phases in HT29 and HCT116 cell lines. E) Immunofluorecescence analyses of the action of anti-CD98hc-DM1 on spindle assembly and organization on HCT116 cells treated with CD98hc-DM1 (10 nM) for 48 h. β-Tubulin (green), DAPI (blue). Scale bars = 7.5 µm. F). Detection of giant multinucleated cells or altered nuclear structures after anti-CD98hc-DM1 treatment. HCT116 and HT29 cells were treated with 10 nM anti-CD98hcECTO-DM1 for 72 h, fixed and stained for nucleoporin p62 (red) and DNA (blue). Scale bar = 10 μm. The arrows indicate giant multinucleated cells., Instituto de Salud Carlos III Consejo Superior de Investigaciones Científicas Junta de Castilla y León ALMOM ACMUMA UCCTA CRIS Cancer Foundation Agencia Estatal de Investigación Consejo Superior de Investigaciones Cientificas (CSIC), Peer reviewed

Additional file 4: Supplementary Fig. 4. A) Kaplan–Meier survival curve of mice from the experiment performed in Fig. 6A. The Kaplan–Meier survival plot was created using a tumor volume threshold of 1,000 mm3. P values were calculated using one-sided log-rank tests. B) Effect of the anti-CD98hc ADC on the weight of mice xenografted with HT29 cells. Data are plotted as mean ± SD of six mice/group. C) Analysis of the antitumoral effect of naked anti-CD98hc and DM1 on tumor growth in nude mice implanted with HT29 cells. Arrows indicate days of administration of anti-CD98hc (15 mg/Kg) or DM1 (0.14 mg/Kg). Data are plotted as mean tumor volumes ± SEM. P values were calculated using Student’s t test (two-sided). D) Kaplan–Meier survival curve of mice from the experiment of panel C. The Kaplan–Meier survival plot was created using a tumor volume threshold of 650 mm3. P values were calculated using one-sided log-rank tests. E) Expression levels or phosphorylation of proteins involved in cell cycle and apoptosis in the tumors of the experiment performed in Fig. 6A. Tumor samples were obtained on day 21 after initiation of treatments (seven days after the last treatment). Tissue extracts of the tumors were used to analyze the levels of expression of pH3, PARP, pH2AX, pCDK1 and cleaved Caspase 3. Stain free blot was analyzed to verify equal loading. F) Quantitation of the levels of DM1 (data shown in Fig. 6B), pH3, PARP, pH2AX, pCDK1 and cleaved Caspase 3 of the experiments shown in panel E. The graphs represent the mean intensity (arbitrary units) ± SD of the different proteins present in control (C) or treated (anti-CD98hc-DM1) mice groups. P values were calculated using Student’s t test (two-sided). G) Immunohistochemical detection of CD98hc in PDX BT6224 (P2M1: passage 2, mouse #1) using the anti-CD98hcV509 antibody. H) Kaplan–Meier survival curve of mice from the experiment performed in Fig. 6E. The Kaplan–Meier survival plot was created using a tumor volume threshold of 800 mm3. P values were calculated using one-sided log-rank tests. I) Effect of the anti-CD98hc ADC on the weight of mice xenografted with PDX BT6224. Data are plotted as mean ± SD of four mice/group. J) Quantitation of the levels of DM1 of the experiments performed in Fig. 6F. The graphs represent the mean intensity (arbitrary units) ± SD of IgG Heavy and Light chains coupled to DM1 present in control (C) or treated (anti-CD98hc-DM1) groups. P values were calculated using Student’s t test (two-sided)., Instituto de Salud Carlos III. Consejo Superior de Investigaciones Científicas (España). Junta de Castilla y León. Fundación CRIS contra el Cáncer., Peer reviewed

To describe the incorporation of monoclonal antibodies (mAb) in real-world (RW) practice for the treatment of patients with relapsed refractory multiple myeloma (RRMM) in a setting with other treatment alternatives. This was an observational, multicenter, ambispective study of RRMM treated with or without a mAb. A total of 171 patients were included. For the group treated without mAb, the median (95% CI) progression-free survival (PFS) to relapse was 22.4 (17.8-27.0) months; partial response or better (≥PR) and complete response or better (≥CR) was observed in 74.1% and 24.1% of patients, respectively; and median time to first response in first relapse was 2.0 months and in second relapse was 2.5 months. For the group of patients treated with mAb in first or second relapse, the median PFS was 20.9 (95% CI, could not be evaluated) months; the ≥ PR and ≥ CR rates were 76,2% and 28.6%, respectively; and the median time to first response in first relapse was 1.2 month and in second relapse was 1.0 months. The safety profiles for the combinations were consistent with those expected. The incorporation of mAb in RW practice for the treatment of RRMM has shown good quality and speed of response with a similar safety profile shown in randomized clinical trials., This study was sponsored by Janssen-Cilag. Janssen-Cilag participated in the study design, data analysis and drafting of the manuscript, Peer reviewed

4 files, Supplementary material for the article http://dx.doi.org/10.3390/biology9100337, Doc file: Figure S1: Number of turbot cells and ciliates in the peritoneal cavity; Figure S2: Micrographs of ciliates at different stages of infection; Figure S3: qPCR validation of RNA-seq findings; Table S1: Sample information; Table S2: Primer sequences used in the qPCR analysis.-- File S1: Excel file showing the contigs that were DE in P. dicentrarchi at 1, 2, 4 hpi, the fold change, p-value, and the gene names.-- File S2: Excel file showing the contigs that were DE in turbot peritoneal cells at 1, 2, 4 hpi, the fold change, p-value, and the gene names.-- File S3: Excel file showing the contigs that were DE in turbot peritoneal cells at 12 and 48 hpi, the fold change, p-value, and the gene names, Peer reviewed

Additional file 1: Text S1. Translated questionnaire to include donor in Cohort II. Fig. S1. Impact of genetic background on numbers of circulating immune cells. Each color and symbol indicate members of one family. Fully open symbols represent a centenarian. Semi-open symbol (orange) represents a sibling of a centenarian. Dashed lines indicate the available age-matched reference values produced with flow cytometry panels highly similar to the panels used in this study (earlier or later prototypes of these panels). For B- and T-cell subsets, reference lines indicate a cohort aged 60–79 years. For innate myeloid populations, reference lines indicate a cohort > 55 years old. In plots without dashed lines, no published reference values from highly similar flow cytometry panels were available. Fig. S2. Results of the pilot study to evaluate the calcium flux upon stimulation of the B-cell receptor (BCR) with IgG and IgM Fab fragments. (A) Measurement of calcium release (‘flux’) after B-cell stimulation with IgM Fabs in pre-GC B cells (CD27-IgA-IgG-) or unswitched memory B cells (CD27+IgA-IgG-). (B) Measurement of calcium release (‘flux’) after B-cell stimulation with IgG Fabs in CD27-IgG+ memory B cells (CD27-IgD-IgA-) or CD27+IgG+ memory B cells (CD27+IgD-IgA-). Ionomycin was added to calculate maximum calcium release. N = 14. Pre-GC; pre-Germinal Center, MBC; memory B cell. Fig. S3. Assessment of B-cell activation in all p.P522R-carriers and non-carriers upon BCR stimulation. Measurement of calcium release (‘flux’) after B-cell stimulation with IgM Fabs in pre-GC B cells (CD27-IgG-IgA-) (A) or unswitched memory B cells (CD27+IgG-IgA-) (B) in cohort I. Ionomycin was added to calculate maximum calcium release. Differences between carriers and non-carriers were evaluated by comparing the area under the curve (AUC) of the total Fab stimulation (from stimulation until the moment ionomycin was added, ~ 10 min, flux intensity and duration), the peak of the response after Fab stimulation (the 5 highest points after the Fabs were added to the cells; flux intensity), and after ionomycin was added (to determine the maximum flux). AUC was calculated only for points that were higher than baseline value (unstimulated sample). N = 31 (two samples were lost due to technical failure). No significant differences were observed. Pre-GC; pre-Germinal Center. Fig. S4. Phagocytosis and ROS production in innate immune cell subsets after stimulation with pHRodo™ Green E. coli Bioparticles (FcR/PLCγ2-dependent stimulation). To evaluate the outcome of the phagocytosis assays, three different readouts were used per population: % of cells that were phagocytosing, the average amount of particles phagocytosed per cell, and the ROS production upon phagocytosis. These three readouts were further combined into one value: the normalized ROS. These values are presented for the CD62L+ classical monocyte (cMo) subset (A), intermediate monocytes (iMo) (B), neutrophils (C) CD14- and CD14dim myeloid dendritic cells (mDCs) (D,E), and non-phagocytosing plasmacytoid dendritic cells (pDCs) (F). Lastly, the outcomes for T cells (negative control) are shown (G). Mann-Whitney U test was used to evaluate differences between carriers and non-carriers, but no statistically significant differences were found. N = 14. In two donors, monocytes could not be divided into subsets due to absence of a differentiating antibody in the prepared antibody cocktail, therefore, in panel A and B, only 5 non-carriers and 7 carriers are shown. Dashed lines indicate the background level of ROS (ROS production in negative control population; T cells). All outcomes were corrected for background or baseline activation by subtracting the value of the control (incubated on ice) from the activated (incubated at 37 °C) sample. Negative values (caused by higher background in control samples than activated samples in cell populations that did not perform phagocytosis) were set to 0. Fig. S5. ROS generation in innate immune cell subsets in p.P522R-carriers and non-carriers upon stimulation with PMA (FcR/PLCγ2-independent stimulation). N = 14. In two donors, monocytes could not be divided into subsets due to absence of an antibody in the prepared antibody cocktail, therefore, in panel A-D, only 5 non-carriers and 7 carriers are shown. Dashed lines indicate the background level of ROS (ROS production in negative control population; T cells). All outcomes were corrected for background or baseline activation by subtracting the value of the control (incubated on ice) from the activated (incubated at 37 °C) sample. Table S1. Description of all included families and additional donors. Table S2. Overview of the flow cytometry panels that were used in this study. Table S3. Phenotypic descriptions used to define B-cell subsets stained with EuroFlow PERISCOPE B-cell and plasma cell panel (BIGH) panel by manual analysis. Table S4. Phenotypic descriptions used to define T-cell subsets stained with EuroFlow PERISCOPE CD4 T-cell (TCD4) panel by manual analysis. Table S5. Phenotypic descriptions used to define T-cell and NK-cell subsets stained with the EuroFlow PERISCOPE CD8 cytotoxic T-cell (CYTOX) panel by manual analysis. Table S6. Phenotypic descriptions used to define innate immune cell (sub) sets stained with the EuroFlow PERISCOPE DC-Monocyte panel by manual analysis. Table S7. Polygenic Risk Score and SNP annotation for immune-related SNPs. Table S8. Control and assay tubes measured in the phagocytosis experiment. Table S9. Control and assay tubes measured when detection production of reactive oxygen species (ROS)., ZonMw Health Holland HorstingStuit Foundation, the Hans und Ilse Breuer Foundation and Stichting VUmc Fund H2020 Marie Skłodowska-Curie Actions, Peer reviewed