Colonization and development of Oribatida mite communities (Acari: Oribatida) in the forest reclaimed limestone mine dumps

In order to study the colonization and development of moss mites (Oribatida) communities in a Scots pine forest of a reclaimed limestone mine dump in Northern Poland, 3 plots from the dump were chosen. The selected plots differed in age, 5 years old, 35 and 50 years old. From a total of 30 samples 499 mites (Acari) were extracted in Tullgren funnel from which 262 were Oribatida. Abundance (N) was analyzed in all mites and after determining the species of both, juvenile and adult stages of oribatids, the following indices were analyzed: Abundance (N), Dominance (D), Species diversity (S), Species richness (s) and Shannon’s diversity index (H). Regarding to the results obtained; oribatid mites were dominant with the highest abundance in all assemblages (Plot 1: 139 Oribatida /299 Acari. Plot 2: 40/55 and Plot 3: 83/145). Tectocepheus velatus showed a very high dominance (45,99%) in plot 1; the highest value for Shannon’s diversity index belonged to plot 3. On the other hand, juvenile’s percentage was significantly higher than adult’s percentage, especially at plot 2 (95,02%). These results made us to conclude that the high abundance of oribatids in the youngest forest is due to T. velatus’s high abundance and that plot 3 is the best habitat for mites. Finally, the high occurrence of juvenile stages requires keeping on studying the area

Colonization and development of Oribatida mite communities (Acari: Oribatida) in the forest reclaimed limestone mine dumps

In order to study the colonization and development of moss mites (Oribatida) communities in a Scots pine forest of a reclaimed limestone mine dump in Northern Poland, 3 plots from the dump were chosen. The selected plots differed in age, 5 years old, 35 and 50 years old. From a total of 30 samples 499 mites (Acari) were extracted in Tullgren funnel from which 262 were Oribatida. Abundance (N) was analyzed in all mites and after determining the species of both, juvenile and adult stages of oribatids, the following indices were analyzed: Abundance (N), Dominance (D), Species diversity (S), Species richness (s) and Shannon’s diversity index (H). Regarding to the results obtained; oribatid mites were dominant with the highest abundance in all assemblages (Plot 1: 139 Oribatida /299 Acari. Plot 2: 40/55 and Plot 3: 83/145). Tectocepheus velatus showed a very high dominance (45,99%) in plot 1; the highest value for Shannon’s diversity index belonged to plot 3. On the other hand, juvenile’s percentage was significantly higher than adult’s percentage, especially at plot 2 (95,02%). These results made us to conclude that the high abundance of oribatids in the youngest forest is due to T. velatus’s high abundance and that plot 3 is the best habitat for mites. Finally, the high occurrence of juvenile stages requires keeping on studying the area

Expresión del CD146 en el AAA e identificación de su forma circulante como biomarcador diagnóstico de la enfermedad

L’aneurisma d’aorta abdominal és una malaltia vascular caracteritzada per una dilatació local de l’aorta abdominal superior al 50% del diàmetre normal i que afecta el 5-8% de la població masculina major de 65 anys. És un procés degeneratiu que pot culminar en el trencament de la paret vascular fet que s’associa al 75% de mortalitat. No existeix cap tractament farmacològic efectiu per a limitar la seva progressió o evitar el seu trencament sent la cirurgia l’única teràpia disponible. És una malaltia asimptomàtica el que fa necessari la identificació de biomarcadors circulants que permetin el diagnòstic i/o pronòstic de la malaltia. Histológicamente es caracteritza per una degeneració dels elements estructurals de la paret de l’aorta i un remodelat de la mitjana, amb evidència d’inflamació crònica, hipervascularización, destrucció de la làmina elàstica i depleció del múscul llis. En aquest remodelat de la paret vascular associat al desenvolupament del AAA les CMVL juguen un paper fonamental. El CD146 és una glicoproteïna de membrana implicada en la unió de les cèl·lules vasculars a la MB. És conegut com a receptor de la cadena α-4 de la laminina i correceptor del VEGFR2. Està implicat en processos com l’adhesió, diferenciació, migració i proliferació cel·lular, angiogénesis i inhibició de l’apoptosi i la seva expressió i la de la seva forma circulant, sCD146, s’ha associat amb patologies inflamatòries que presenten mal vascular. L’objectiu principal d’aquesta tesi és comparar l’estructura de la paret de l’aorta del AAA humana amb la de la AN i estudiar l’expressió i activitat del CD146 en el AAA, així com valorar la seva forma soluble com a possible biomarcador diagnòstic de la malaltia. Hem observat que els teixits de AAA humana presenten alteracions en l’estructura de la paret acompanyades de canvis dels nivells d’expressió del ARNm del DAG-1 i de les lamininas de la MB. de lLas cadenes α-2, 3, 4 de la laminina i del DAG-1 que es troben incrementats en el AAA mentre que la expresióny de la cadena α5 que està disminuïda. També hem demostrat que l’expressió de CD146 a nivell de ARNm i proteïna disminueix en el teixit patològic degut majoritàriament a la disminució de la seva expressió en les CMVL de la capa mitjana. La pèrdua d’expressió de CD146 en les CMVL s’associa amb la disminució de l’expressió de les proteïnes del fenotip contràctil, Calponina-1, TAGLN i SM-MHC i l’increment de l’expressió del marcador de fenotip desdiferenciado S100A4. Utilitzant cultius primaris de CMVL tractats amb TGF-β1 vam demostrar que l’expressió de CD146 es correlaciona amb el fenotip contràctil de les CMVL però la sobreexpresión de CD146 no regula el fenotip de les CMVL ni modifica la capacitat de migració i proliferació de les CMVL en cultiu. Per contra, la sobreexpresión de CD146 augmenta la capacitat d’adhesió de les cèl·lules al substrat i disminueix l’apoptosi induïda per citocines proinflamatorias. Finalment, els pacients amb AAA presenten nivells circulants de sCD146 més baixos que els controls sans. En els pacients de AAA els nivells de sCD146 es correlacionen positivament amb els nivells de ARNm de CD146 en teixit i negativament amb el diàmetre de l’aorta. L’anàlisi estadística indica que els nivells de sCD146 prediuen la presència de AAA amb alta especificitat i sensibilitat. En conjunt, aquest treball demostra la importància de la interacció de les CMVL amb la MB en el desenvolupament del AAA i que es produeix un canvi fenotípic de les CMVL que passen del fenotip contràctil de l’aorta sana a un fenotip desdiferenciado desdiferenciado de l’aorta patològica. D’altra banda, proposa un biomarcador diagnòstic amb possible utilitat clínica per a detectar la presència de AAA des de fases primerenques de la malaltia.El aneurisma de aorta abdominal (AAA) es una enfermedad vascular caracterizada por una dilatación local de la aorta abdominal superior al 50% del diámetro normal que afecta al 5-8% de la población masculina mayor de 65 años. Es un proceso degenerativo que puede culminar en la rotura de la pared vascular hecho que se asocia al 75% de mortalidad. No existe ningún tratamiento farmacológico efectivo para limitar su progresión o evitar su rotura siendo la cirugía la única terapia disponible. Es una enfermedad asintomática lo que hace necesario la identificación de biomarcadores circulantes que permitan el diagnóstico y/o pronóstico de la enfermedad. Histológicamente se caracteriza por una degeneración de los elementos estructurales de la pared de la aorta y un remodelado de la media, con evidencia de inflamación crónica, hipervascularización, destrucción de la lámina elástica y depleción del músculo liso. En este remodelado de la pared vascular las CMVL juegan un papel fundamental. El CD146 es una glicoproteína de membrana implicada en la unión de las células vasculares a la MB. Es conocido como receptor de la cadena α-4 de la laminina y correceptor del VEGFR2. Está implicado en procesos como la adhesión, diferenciación, migración y proliferación celular, angiogénesis e inhibición de la apoptosis y su expresión y la de su forma circulante, sCD146, está asociada con patologías inflamatorias que presentan daño vascular. El objetivo principal de esta tesis es comparar la estructura de la pared de la aorta del AAA humana con la de la AN y estudiar la expresión y actividad del CD146 en el AAA, así como valorar su forma soluble como posible biomarcador diagnóstico de la enfermedad. Hemos observado que los tejidos de AAA presentan alteraciones en la estructura de la pared acompañadas de cambios de los niveles de expresión del ARNm de las cadenas α-2, 3, 4 de la laminina y del DAG-1 que se encuentran incrementados en el AAA y de la cadena α5 que está disminuida. También hemos demostrado que la expresión de CD146 a nivel de ARNm y proteína disminuye en el tejido patológico debido mayoritariamente a la disminución de su expresión en las CMVL de la capa media. La pérdida de expresión de CD146 en las CMVL se asocia con la disminución de la expresión de las proteínas del fenotipo contráctil, Calponina-1, TAGLN y SM-MHC y el incremento de la expresión del marcador de fenotipo desdiferenciado S100A4. Utilizando cultivos primarios de CMVL tratados con TGF-β1 demostramos que la expresión de CD146 se correlaciona con el fenotipo contráctil de las CMVL pero la sobreexpresión de CD146 no regula el fenotipo de las CMVL ni modifica la capacidad de migración y proliferación de las CMVL en cultivo. Por el contrario, la sobreexpresión de CD146 aumenta la capacidad de adhesión de las células al sustrato y disminuye la apoptosis inducida por citoquinas proinflamatorias. Finalmente, los pacientes con AAA presentan niveles circulantes de sCD146 más bajos que los controles. En los pacientes de AAA los niveles de sCD146 se correlacionan positivamente con los niveles de ARNm de CD146 en tejido y negativamente con el diámetro de la aorta. El análisis estadístico indica que los niveles de sCD146 predicen la presencia de AAA con alta especificidad y sensibilidad. En conjunto, este trabajo demuestra la importancia de la interacción de las CMVL con la MB en el desarrollo del AAA y que se produce un cambio fenotípico de las CMVL que pasan del fenotipo contráctil de la aorta sana a un fenotipo desdiferenciado de la aorta patológica. Por otro lado, propone un biomarcador diagnóstico con posible utilidad clínica para detectar la presencia de AAA desde fases tempranas de la enfermedad.Abdominal aortic aneurysm (AAA) is a vascular disease characterized by a focal dilatation of the abdominal aorta larger than 50% of the normal diameter, and affects 5-8% of male over 65 years old. It is a degenerative process and it can culminate in a vascular wall’s rupture, which is associated with 75% of death. To date, there is no pharmacological treatment to limit an aneurysm progression or to prevent its rupture, and surgery is the only available therapy. AAA is usually asymptomatic until it ruptures, which emphasizes the need to identify new circulating biomarkers that facilitate the diagnosis and/or the prognosis of the disease. Histologically, AAAs are characterized by destruction of the structural elements of the aortic wall and remodeling of the media, along with a chronic inflammatory process, hypervascularization, destruction of the elastic lamina and smooth muscle depletion. In this remodeling of the vascular wall related to AAA development, vascular smooth muscle cells (VSMC) play a crucial role. CD146 is a membrane glycoprotein implicated in the adhesion of vascular cells to the basal membrane (BM). It is known to act as a receptor of the α-4 laminin subunit as well as cor-receptor of VEGFR2. Moreover, it is involved in processes such as adhesion, cellular differentiation, migration and proliferation, angiogenesis, and apoptosis inhibition. Besides, CD146 expression and the expression of its circulating form, sCD146, have been associated with inflammatory pathologies that present vascular remodeling. The aim of the present work is to compare the aortic wall structure of human AAA and NA tissues and to study CD146 expression and activity in the AAA tissue, as well as to analyze its circulating form as a putative diagnostic biomarker of the disease. We have observed that human AAA tissues present structural alterations in the aortic wall accompanied with changes in BM’s DAG-1 and laminin mRNA expression levels. α-2, 3 and 4 subunits of laminins and DAG-1 are overexpressed in AAA, while α-5 subunit is downregulated. Comparing human AAA and NA tissues, we have also demonstrated that CD146 mRNA and protein expression is downregulated in the pathological tissue, mostly due to the loss of its expression in VSMC from the media layer. Likewise, the loss of CD146 expression in VSMC is associated with downregulation of the VSMC’s contractile phenotype’s markers Calponina-1, TAGLN and SM-MHC, while it associates with upregulation of the de-differentiated phenotype’s marker S100A4. By using primary culture of VSMCs treated with TGF-β1, we have demonstrated that CD146 expression correlates with VSMC’s contractile phenotype. However, CD146 upregulation does not regulate cell phenotype or cell migration and proliferation capability in vitro. Conversely, CD146 upregulation increases cell adhesion to the substrate and decreases pro-inflammatory cytokine’s induced cell apoptosis. Finally, AAA patients present lower circulating sCD146 expression levels than healthy controls. In AAA patients, sCD146 levels positively correlate with CD146 mRNA tissue levels and show an inverse correlation with the diameter of the aorta. Statistical analyses indicate that sCD146 levels predict AAA presence with a high specificity and sensitivity. Overall, this work demonstrates the importance of VSMC and BM interaction in AAA development and suggests that VSMC modulate their phenotype during the disease process, from a contractile phenotype of the healthy aorta to a de-differentiated or synthetic phenotype of the pathologic aorta. On the other hand, it proposes a diagnostic biomarker with a possible clinical use to detect AAA presence at early stages.Universitat Autònoma de Barcelona. Programa de Doctorat en Bioquímica, Biologia Molecular i Biomedicin

Circulating CCL20 as a New Biomarker of Abdominal Aortic Aneurysm

Autoimmunity appears to play a role in abdominal aortic aneurysm (AAA) pathology. Although the chemokine CCL20 has been involved in autoimmune diseases, its relationship with the pathogenesis of AAA is unclear. We investigated CCL20 expression in AAA and evaluated it as a potential biomarker for AAA. CCL20 was measured in plasma of AAA patients (n = 96), atherosclerotic disease (AD) patients (n = 28) and controls (n = 45). AAA presence was associated with higher plasma levels of CCL20 after adjustments for confounders in the linear regression analysis. Diagnostic performance of plasma CCL20 was assessed by ROC curve analysis, AUC 0.768 (CI:0.678-0.858; p<0.001). Classification and regression tree analysis classified patients into two CCL20 plasma level groups. The high-CCL20 group had a higher number of AAA than the low-CCL20 group (91% vs 54.3%, p< 0.001). mRNA of CCL20 and its receptor CCR6 were higher in AAA (n = 89) than in control aortas (n = 17, p<0.001). A positive correlation was found between both mRNA in controls (R = 0674; p = 0.003), but not in AAA. Immunohistochemistry showed that CCR6 and CCL20 colocalized in the media and endothelial cells. Infiltrating leukocytes immunostained for both proteins but only colocalized in some of them. Our data shows that CCL20 is increased in AAA and circulating CCL20 is a high sensitive biomarker of AA

Targeting the extra domain A of fibronectin for cancer therapy with CAR-T cells

Background One of the main difficulties of adoptive cell therapies with chimeric antigen receptor (CAR)-T cells in solid tumors is the identification of specific target antigens. The tumor microenvironment can present suitable antigens for CAR design, even though they are not expressed by the tumor cells. We have generated a CAR specific for the splice variant extra domain A (EDA) of fibronectin, which is highly expressed in the tumor stroma of many types of tumors but not in healthy tissues. Methods EDA expression was explored in RNA-seq data from different human tumor types and by immunohistochemistry in paraffin-embedded tumor biopsies. Murine and human anti-EDA CAR-T cells were prepared using recombinant retro/lentiviruses, respectively. The functionality of EDA CAR-T cells was measured in vitro in response to antigen stimulation. The antitumor activity of EDA CAR-T cells was measured in vivo in C57BL/6 mice challenged with PM299L-EDA hepatocarcinoma cell line, in 129Sv mice-bearing F9 teratocarcinoma and in NSG mice injected with the human hepatocarcinoma cell line PLC. Results EDA CAR-T cells recognized and killed EDA-expressing tumor cell lines in vitro and rejected EDA-expressing tumors in immunocompetent mice. Notably, EDA CAR-T cells showed an antitumor effect in mice injected with EDA-negative tumor cells lines when the tumor stroma or the basement membrane of tumor endothelial cells express EDA. Thus, EDA CAR-T administration delayed tumor growth in immunocompetent 129Sv mice challenged with teratocarcinoma cell line F9. EDA CAR-T treatment exerted an antiangiogenic effect and significantly reduced gene signatures associated with epithelial-mesenchymal transition, collagen synthesis, extracellular matrix organization as well as IL-6-STAT5 and KRAS pathways. Importantly, the human version of EDA CAR, that includes the human 41BB and CD3 zeta endodomains, exerted strong antitumor activity in NSG mice challenged with the human hepatocarcinoma cell line PLC, which expresses EDA in the tumor stroma and the endothelial vasculature. EDA CAR-T cells exhibited a tropism for EDA-expressing tumor tissue and no toxicity was observed in tumor bearing or in healthy mice. Conclusions These results suggest that targeting the tumor-specific fibronectin splice variant EDA with CAR-T cells is feasible and offers a therapeutic option that is applicable to different types of cancer

Targeting the extra domain A of fibronectin for cancer therapy with CAR-T cells

Background One of the main difficulties of adoptive cell therapies with chimeric antigen receptor (CAR)-T cells in solid tumors is the identification of specific target antigens. The tumor microenvironment can present suitable antigens for CAR design, even though they are not expressed by the tumor cells. We have generated a CAR specific for the splice variant extra domain A (EDA) of fibronectin, which is highly expressed in the tumor stroma of many types of tumors but not in healthy tissues. Methods EDA expression was explored in RNA-seq data from different human tumor types and by immunohistochemistry in paraffin-embedded tumor biopsies. Murine and human anti-EDA CAR-T cells were prepared using recombinant retro/lentiviruses, respectively. The functionality of EDA CAR-T cells was measured in vitro in response to antigen stimulation. The antitumor activity of EDA CAR-T cells was measured in vivo in C57BL/6 mice challenged with PM299L-EDA hepatocarcinoma cell line, in 129Sv mice-bearing F9 teratocarcinoma and in NSG mice injected with the human hepatocarcinoma cell line PLC. Results EDA CAR-T cells recognized and killed EDA-expressing tumor cell lines in vitro and rejected EDA-expressing tumors in immunocompetent mice. Notably, EDA CAR-T cells showed an antitumor effect in mice injected with EDA-negative tumor cells lines when the tumor stroma or the basement membrane of tumor endothelial cells express EDA. Thus, EDA CAR-T administration delayed tumor growth in immunocompetent 129Sv mice challenged with teratocarcinoma cell line F9. EDA CAR-T treatment exerted an antiangiogenic effect and significantly reduced gene signatures associated with epithelial-mesenchymal transition, collagen synthesis, extracellular matrix organization as well as IL-6-STAT5 and KRAS pathways. Importantly, the human version of EDA CAR, that includes the human 41BB and CD3 zeta endodomains, exerted strong antitumor activity in NSG mice challenged with the human hepatocarcinoma cell line PLC, which expresses EDA in the tumor stroma and the endothelial vasculature. EDA CAR-T cells exhibited a tropism for EDA-expressing tumor tissue and no toxicity was observed in tumor bearing or in healthy mice. Conclusions These results suggest that targeting the tumor-specific fibronectin splice variant EDA with CAR-T cells is feasible and offers a therapeutic option that is applicable to different types of cancer