BUSCADOR RECOLECTA

Microglial cells are recognized as very dynamic brain cells, screening the environment and sensitive to signals from all other cell types in health and disease. Apolipoprotein D (ApoD), a lipid-binding protein of the Lipocalin family, is required for nervous system optimal function and proper development and maintenance of key neural structures. ApoD has a cell and state-dependent expression in the healthy nervous system, and increases its expression upon aging, damage or neurodegeneration. An extensive overlap exists between processes where ApoD is involved and those where microglia have an active role. However, no study has analyzed the role of ApoD in microglial responses. In this work, we test the hypothesis that ApoD, as an extracellular signal, participates in the intercellular crosstalk sensed by microglia and impacts their responses upon physiological aging or damaging conditions. We find that a significant proportion of ApoD-dependent aging transcriptome are microglia-specific genes, and show that lack of ApoD in vivo dysregulates microglial density in mouse hippocampus in an age-dependent manner. Murine BV2 and primary microglia do not express ApoD, but it can be internalized and targeted to lysosomes, where unlike other cell types it is transiently present. Cytokine secretion profiles and myelin phagocytosis reveal that ApoD has both long-term pre-conditioning effects on microglia as well as acute effects on these microglial immune functions, without significant modification of cell survival. ApoD-triggered cytokine signatures are stimuli (paraquat vs. Aβ oligomers) and sex-dependent. Acute exposure to ApoD induces microglia to switch from their resting state to a secretory and less phagocytic phenotype, while long-term absence of ApoD leads to attenuated cytokine induction and increased myelin uptake, supporting a role for ApoD as priming or immune training factor. This knowledge should help to advance our understanding of the complex responses of microglia during aging and neurodegeneration, where signals received along our lifespan are combined with damage-triggered acute signals, conditioning both beneficial roles and limitations of microglial functions., Peer reviewed

[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