Vies de senyalització regulades per les Presenilines en càncer de pell : paper del receptor d'EGF /

Als preliminars: Projecte subvencionat per la Fundació Marató de TV3 (Nº ID: 050710; Regulació de les vies de transducció de senyals depenents de les presenilines en el càncer de pell)...Consultable des del TDXTítol obtingut de la portada digitalitzadaMutacions autosòmiques dominants en els gens de presenilines (PS) són la principal causa d'Alzheimer familiar hereditari. Aquestes mutacions alteren el metabolisme de l'APP i la generació del pèptid -amiloide, el qual s'acumula en forma de plaques en el cervell dels malalts d'Alzheimer. Les PS són les unitats catalítiques del complex enzimàtic -secretasa, responsable de la proteòlisi de proteïnes tipus I, entre elles l'APP i Notch. A més del seu important paper en la patologia de la malaltia d'Alzheimer, les PS regulen processos de proliferació ja que la seva inactivació produeix tumors epitelials en ratolins a través de mecanismes moleculars poc coneguts. Degut a l'important paper del receptor d'EGF (EGFR) en processos oncogènics, varem hipotetitzar que les PS podien alterar la transformació cel·lular en part mitjançant la regulació de la via de senyalització de l'EGFR. Els resultats obtinguts indiquen que la inactivació de les PS en fibroblasts causa transformació cel·lular que és específicament bloquejada amb un inhibidor de l'EGFR. Observarem un increment en l'expressió i senyalització de l'EGFR degut a un retard en la degradació a causa d'alteracions en els processos d'ubiquitinització/deubiquitinització d'aquest receptor. La regulació de PS sobre EGFR depèn de l'activitat de la E3 ubiquitina lligasa Fbw7. En aquest treball hem descrit per primera vegada que la pèrdua de funció de PS ocasiona un excés en la transcripció gènica de Fbw7, el qual regula positivament els nivells d'EGFR. Aquests resultats són innovadors ja que, tot i que Fbw7 actua com gen supressor de tumors en altres teixits, en aquest cas està promovent transformació cel·lular sent la primera vegada que es descriu un increment en els nivells de Fbw7 en un procés tumoral epitelial. Aquests resultats són corroborats en un model de ratolí induïble amb deficiència específica d'ambdues PS en pell (ePS cDKO), que ha estat generat en el desenvolupament d'aquest treball. Els ratolins ePS cDKO desenvolupen espontàniament tumors escatosos (SCC) a la pell del coll i presenten susceptibilitat a desenvolupar tumors en front a l'exposició a agents citotòxics. En aquesta tesi doctoral s'ha descrit que la pèrdua de funció de PS en pell té greus conseqüències en l'homeòstasi epitelial ja que l'absència de PS produeix el desenvolupament de SCC que porta a la mort dels animals a una edat màxima de 2.5-3 mesos d'edat. Aquest fenotip és degut a la desregulació de la via de senyalització de Notch, -catenina i EGFR, tenint aquest últim un paper cabdal segons els resultats obtinguts en aquest treball. El paper central de PS en la regulació de les diferents vies de senyalització esmentades suggereix que PS és un important gen supressor de tumors.Dominant autosomic mutations of presenilin (PS) genes are the most important cause of familiar Alzheimer disease. These mutations modify APP metabolism and -amiloid peptide generation, which is accumulated forming plaques on the brain of Alzheimer disease patients. Presenilins are the catalytic unit of -secretase enzymatic complex, responsible of proteins type I proteolysis, such as APP and Notch. Besides their important role on Alzheimer disease, PS regulate proliferation processes since its inactivation on mice leads to development of epithelial tumor through poor understood molecular mechanisms. Due to the important role of EGF receptor (EGFR) on tumoral processes we hypothesized that presenilin could be involved on cellular transformation in part by regulating EGFR signaling pathway. Results obtained in this work indicate that inactivation of PS in fibroblasts cause cellular transformation that is specifically blocked by using an EGFR inhibitor. We observed an increase of EGFR expression and signaling due to a delayed degradation caused by an alteration of ubiquitination and de-ubiquitination processes. Regulation of EGFR by PS is depending of E3 ubiquitin ligase Fbw7 activity. This work describes for first time that lost of PS function leads to increased transcription of Fbw7, which positively regulates EGFR levels. These results are pioneering since, whereas Fbw7 is a tumor suppressor gene in other tissues, in skin it promotes cellular transformation being the first time that is described an increase of Fbw7 levels in skin tumor. These results are corroborated in a mice model inducible and conditional with PS deletion in skin (ePS cDKO), which has been generated during the development of this study. ePS cDKO mice develop spontaneously squamous cell carcinoma (SCC) in neck skin area and show increased tumor susceptibility in front of cytotoxic agents. This thesis has described that lost of PS function in skin has drastic consequences on epithelial homeostasis since PS deficiency leads to development of SCC and causes death of animals at 2.5-3 moths of age. This phenotype is due to a deregulation of Notch, -catenin and EGFR signaling pathways, where the last one has the most important role in accordance to the results obtained in this study. Key role of PS in regulation of several pathways suggest PS as an important tumor suppressor gene

Lactate Regulates Metabolic and Proinflammatory Circuits in Control of T Cell Migration and Effector Functions

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Interleukin-27 inhibits ectopic lymphoid-like structure development in early inflammatory arthritis

Ectopic lymphoid-like structures (ELSs) reminiscent of secondary lymphoid organs often develop at sites of chronic inflammation where they contribute to immune-mediated pathology. Through evaluation of synovial tissues from rheumatoid arthritis (RA) patients, we now show that low interleukin-27 (IL-27) expression corresponds with an increased incidence of ELS and gene signatures associated with their development and activity. The presence of synovial ELS was also noted in mice deficient in the IL-27 receptor (IL-27R) after the onset of inflammatory arthritis. Here, pathology was associated with increased synovial expression of pro-inflammatory cytokines, homeostatic chemokines, and transcriptional regulators linked with lymphoid neogenesis. In both clinical and experimental RA, synovial ELS coincided with the heightened local expression of cytokines and transcription factors of the Th17 and T follicular helper (Tfh) cell lineages, and included podoplanin-expressing T cells within lymphoid aggregates. IL-27 inhibited the differentiation of podoplanin-expressing Th17 cells, and an increased number of these cells were observed in IL-27R–deficient mice with inflammatory arthritis. Thus, IL-27 appears to negatively regulate ELS development in RA through control of effector T cells. These studies open new opportunities for patient stratification and treatment

Vies de senyalització regulades per les Presenilines en càncer de pell: paper del receptor d'EGF

Mutacions autosòmiques dominants en els gens de presenilines (PS) són la principal causa d'Alzheimer familiar hereditari. Aquestes mutacions alteren el metabolisme de l'APP i la generació del pèptid β-amiloide, el qual s'acumula en forma de plaques en el cervell dels malalts d´Alzheimer. Les PS són les unitats catalítiques del complex enzimàtic γ-secretasa, responsable de la proteòlisi de proteïnes tipus I, entre elles l'APP i Notch. A més del seu important paper en la patologia de la malaltia d´Alzheimer, les PS regulen processos de proliferació ja que la seva inactivació produeix tumors epitelials en ratolins a través de mecanismes moleculars poc coneguts. Degut a l'important paper del receptor d'EGF (EGFR) en processos oncogènics, varem hipotetitzar que les PS podien alterar la transformació cel·lular en part mitjançant la regulació de la via de senyalització de l´EGFR.Els resultats obtinguts indiquen que la inactivació de les PS en fibroblasts causa transformació cel·lular que és específicament bloquejada amb un inhibidor de l'EGFR. Observarem un increment en l'expressió i senyalització de l'EGFR degut a un retard en la degradació a causa d'alteracions en els processos d'ubiquitinització/deubiquitinització d'aquest receptor. La regulació de PS sobre EGFR depèn de l'activitat de la E3 ubiquitina lligasa Fbw7. En aquest treball hem descrit per primera vegada que la pèrdua de funció de PS ocasiona un excés en la transcripció gènica de Fbw7, el qual regula positivament els nivells d'EGFR. Aquests resultats són innovadors ja que, tot i que Fbw7 actua com gen supressor de tumors en altres teixits, en aquest cas està promovent transformació cel·lular sent la primera vegada que es descriu un increment en els nivells de Fbw7 en un procés tumoral epitelial. Aquests resultats són corroborats en un model de ratolí induïble amb deficiència específica d'ambdues PS en pell (ePS cDKO), que ha estat generat en el desenvolupament d'aquest treball. Els ratolins ePS cDKO desenvolupen espontàniament tumors escatosos (SCC) a la pell del coll i presenten susceptibilitat a desenvolupar tumors en front a l'exposició a agents citotòxics.En aquesta tesi doctoral s'ha descrit que la pèrdua de funció de PS en pell té greus conseqüències en l'homeòstasi epitelial ja que l'absència de PS produeix el desenvolupament de SCC que porta a la mort dels animals a una edat màxima de 2.5-3 mesos d'edat. Aquest fenotip és degut a la desregulació de la via de senyalització de Notch, β-catenina i EGFR, tenint aquest últim un paper cabdal segons els resultats obtinguts en aquest treball. El paper central de PS en la regulació de les diferents vies de senyalització esmentades suggereix que PS és un important gen supressor de tumors.Dominant autosomic mutations of presenilin (PS) genes are the most important cause of familiar Alzheimer disease. These mutations modify APP metabolism and β-amiloid peptide generation, which is accumulated forming plaques on the brain of Alzheimer disease patients. Presenilins are the catalytic unit of γ-secretase enzymatic complex, responsible of proteins type I proteolysis, such as APP and Notch. Besides their important role on Alzheimer disease, PS regulate proliferation processes since its inactivation on mice leads to development of epithelial tumor through poor understood molecular mechanisms. Due to the important role of EGF receptor (EGFR) on tumoral processes we hypothesized that presenilin could be involved on cellular transformation in part by regulating EGFR signaling pathway. Results obtained in this work indicate that inactivation of PS in fibroblasts cause cellular transformation that is specifically blocked by using an EGFR inhibitor. We observed an increase of EGFR expression and signaling due to a delayed degradation caused by an alteration of ubiquitination and de-ubiquitination processes. Regulation of EGFR by PS is depending of E3 ubiquitin ligase Fbw7 activity. This work describes for first time that lost of PS function leads to increased transcription of Fbw7, which positively regulates EGFR levels. These results are pioneering since, whereas Fbw7 is a tumor suppressor gene in other tissues, in skin it promotes cellular transformation being the first time that is described an increase of Fbw7 levels in skin tumor. These results are corroborated in a mice model inducible and conditional with PS deletion in skin (ePS cDKO), which has been generated during the development of this study. ePS cDKO mice develop spontaneously squamous cell carcinoma (SCC) in neck skin area and show increased tumor susceptibility in front of cytotoxic agents. This thesis has described that lost of PS function in skin has drastic consequences on epithelial homeostasis since PS deficiency leads to development of SCC and causes death of animals at 2.5-3 moths of age. This phenotype is due to a deregulation of Notch, β-catenin and EGFR signaling pathways, where the last one has the most important role in accordance to the results obtained in this study. Key role of PS in regulation of several pathways suggest PS as an important tumor suppressor gene

Treatment-resistant synovitis and radiographic progression are increased in elderly-onset rheumatoid arthritis patients : findings from a prospective observational longitudinal early arthritis cohort study

© 2020 Elsevier Inc. All rights reservedBackground: Clinical outcomes in elderly-onset rheumatoid arthritis (EORA), starting after the age of 60, are conflicting. Thus, we aimed to investigate in a unique biopsy-driven, treatment-naïve early arthritis cohort, the relationship between synovial pathobiology of elderly- (EORA) and younger-onset rheumatoid arthritis (YORA) patients through clinical, imaging and treatment response outcome-measures. Methods: Patients (n = 140) with early RA (<12months) starting before (YORA, n = 99) or after (EORA, n = 41) age 60 had an ultrasound-guided synovial biopsy prior to conventional immunosuppressive therapy and after 6 months. Clinical, ultrasound and radiographic data were collected prospectively and compared between groups and against immunohistological features. Using multivariate logistic regression, we determined predictors of clinical response (disease activity score-28-erythrocyte sedimentation rate [DAS28-ESR]<3.2) at 6 months and radiographic progression ( 1-unit-increase in Sharp van der Heijde [SvdH] score) at 12 months. Results: EORA patients were more frequently male and presented most commonly with an abrupt, polymyalgia rheumatica-like onset and extra-articular features. Both before and after treatment, DAS28-ESR was similar but ultrasound synovial-thickening (p<0.05) and power-Doppler (p<0.01) synovitis and SvdH (p<0.001) scores were higher in EORA patients. EORA was independently associated with poor treatment response at 6 months (OR=0.28, p = 0.047) and radiographic progression at 12 months (OR=4.08, p = 0.029). Synovial pathotype, synovitis scores and cellular infiltration were similar before treatment, but a pauci-immune-fibroid pathotype tended to be more common in YORA at 6 months (p = 0.093). Moreover, YORA patients had a marked improvement of all synovitis parameters (p<0.001), whereas EORA presented only mild decreases in synovitis (p<0.05), sublining macrophage (p<0.05) and T cell scores (p<0.05), with no significant changes in lining macrophages, B cells or plasma cells. Conclusion: Early EORA presents differently and has a worse overall prognosis than YORA, with poorer clinical, histological, ultrasonographic and radiographic outcomes.This work was supported by the Medical Research Council (grant number 36661, to PEAC); Arthritis Research UK (now Versus Arthritis; Experimental Arthritis Treatment Centre Grant, number 20022, to Centre for Experimental Medicine and Rheumatology); Fundação para a Ciência e Tecnologia (Interno Doutorando Bursary reference SFRH/SINTD/95030/2013, to VCR); European League Against Rheumatism (EULAR Scientific Training Bursary 2014, to VCR); and Sociedade Portuguesa de Reumatologia (Fundo de Apoio à Investigação da SPR 2015, to VCR).info:eu-repo/semantics/publishedVersio

Proliferation Tumour Marker Network (PTM-NET) for the identification of tumour region in Ki67 stained breast cancer whole slide images

Uncontrolled proliferation is a hallmark of cancer and can be assessed by labelling breast tissue using immunohistochemistry for Ki67, a protein associated with cell proliferation. Accurate measurement of Ki67-positive tumour nuclei is of critical importance, but requires annotation of the tumour regions by a pathologist. This manual annotation process is highly subjective, time-consuming and subject to inter- and intra-annotator experience. To address this challenge, we have developed Proliferation Tumour Marker Network (PTM-NET), a deep learning model that objectively annotates the tumour regions in Ki67-labelled breast cancer digital pathology images using a convolution neural network. Our custom designed deep learning model was trained on 45 immunohistochemical Ki67-labelled whole slide images to classify tumour and non-tumour regions and was validated on 45 whole slide images from two different sources that were stained using different protocols. Our results show a Dice coefficient of 0.74, positive predictive value of 70% and negative predictive value of 88.3% against the manual ground truth annotation for the combined dataset. There were minimal differences between the images from different sources and the model was further tested in oestrogen receptor and progesterone receptor-labelled images. Finally, using an extension of the model, we could identify possible hotspot regions of high proliferation within the tumour. In the future, this approach could be useful in identifying tumour regions in biopsy samples and tissue microarray images

Schematic of the proposed mechanism of lactate effects on T cells in the inflammatory site.

<p>(<b>A</b>) The motility of CD4⁺ and CD8⁺ T cells is blocked once they get exposed to elevated levels of lactate in the inflammatory site. Lactic acid also causes loss of cytolytic activity by CD8⁺ T cells, and sodium lactate promotes the production of IL-17 by CD4⁺ T cells. (<b>B</b>) Pharmacologic targeting of lactate transporters re-establish T cell migration away from the inflammatory site and block the production of high amounts of IL-17.</p

Inhibition of lactate transporters promotes the release of T-cells from the inflamed site in zymosan-induced peritonitis.

<p>(A) Lactate levels in the peritoneum of zymosan-treated mice. (B) Number of CD4⁺ and CD8⁺ T cells, respectively, in the peritoneal lavage of C57BL/6 mice injected i.p. with zymosan (1 mg/mouse) to induce peritonitis, and 5 d later, i.p. treated with phloretin (50 μM), an anti-Slc5a12 antibody (5 μg/ml) or an isotype control antibody. (C) Number of carboxyfluorescein succinimidyl ester (CFSE)-labeled activated CD4⁺ T cells in the peritoneal lavage (left panel) or spleen (right panel), respectively, of C57BL/6 mice injected i.p. with zymosan (1 mg/mouse), then i.p. treated with phloretin (50 μM), an anti-Slc5a12 specific antibody (5μg/ml) or an isotype control antibody. (A–C) <i>n</i> = 3 or more. Underlying numerical data and statistical analysis can be found in the supporting file, <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002202#pbio.1002202.s001" target="_blank">S1 Data</a>, Fig 6A–6C. Values denote mean ± SD. <i>*p <</i> 0.05; <i>**p <</i> 0.01; <i>***p <</i> 0.001.</p

Sodium lactate and lactic acid act on CD4⁺ and CD8⁺ T cell subsets, respectively, through specific cell membrane transporters.

<p>(A) Total protein levels of the transporters Slc16a1 and Slc5a12 as assessed by western blot in activated CD4⁺ and CD8⁺ T cell subsets. (B–D) In vitro chemotaxis (4 h time point) of activated CD8⁺ T cells towards CXCL10 (300 ng/ml) in the presence of lactic acid (10 mM) alone, or in combination with α-cyano-4-hydroxycinnamate (CHC) (425 μM), phloretin (25 μM), or anti-Slc16a1 antibody (2.5 μg/ml) (B), or increasing concentrations of AR-C155858 as indicated in the figure (C), and activated CD4⁺ T cells towards CXCL10 (300 ng/ml) in the presence of sodium lactate (10 mM) alone, or in combination with an anti-Slc5a12 antibody (2.5 μg/ml) or two specific short hairpin RNAs (shRNAs) (D). An isotype control antibody has been included to control for antibody specificity (B, D), and a nonspecific shRNA has been included to control for gene knockdown specificity (D). (B–D) <i>n</i> = 3. Underlying numerical data and statistical analysis can be found in the supporting file, <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002202#pbio.1002202.s001" target="_blank">S1 Data</a>, Fig 2B–2D. Values denote mean ± SD. <i>*p <</i> 0.05; <i>**p <</i> 0.01; <i>***p <</i> 0.001.</p