1,721,233 research outputs found
Proceedings from the 2009 genetic syndromes of the Ras/MAPK pathway: from bedside to bench and back
No full textOvergrowth syndromes: A classification. In Endocrine Involvement in Developmental Syndromes
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Simpson–Golabi–Behmel syndrome
No full textThe Simpson–Golabi–Behmel syndrome (SGBS; OMIM 312870) is an overgrowth/multiple congenital anomalies/dysplasia condition, inherited as an X-linked semi-dominant trait, with variable expressivity in males and reduced penetrance and expressivity in females. The clinical spectrum is broad, ranging from mild manifestations in both males and females to multiple malformations and neonatal death in the more severely affected cases. An increased risk of neoplasia is reported, requiring periodical surveillance. Intellectual development is normal in most cases. SGBS is caused by a loss-of-function mutation of the GPC3 gene, either deletions or point mutations, distributed all over the gene. Notably, GPC3 deletion/point mutations are not found in a significant proportion of clinically diagnosed SGBS cases. The protein product GPC3 is a glypican functioning as a receptor for Hh at the cell surface, involved in the Hh-Ptc-Smo signaling pathway, a regulator of cellular growth
Studio e utilizzo di linfociti anti-GD2 CAR T per il trattamento del carcinoma polmonare a piccole cellule (SCLC)
No full textIl carcinoma polmonare a piccole cellule o microcitoma (SCLC) è un tumore neuroendocrino che rappresenta circa il 15% di tutte le neoplasie polmonari. È caratterizzato da una sopravvivenza media a 5 anni del 7%. I linfociti Chimeric Antigen Receptor (CAR) T sono linfociti T geneticamente modificati per esprimere un recettore chimerico in grado di attivare il linfocita contro l’antigene tumorale che si desidera. Questo nuovo approccio ha prodotto risultati significativi nelle neoplasie del sangue a cellule B. Il disialoganglioside GD2 è un ganglioside di membrana noto per essere espresso su numerosi tumori di origine neuroectodermica, tra cui il microcitoma. Per questo motivo abbiamo deciso di testare un CAR T anti-GD2 In Vitro e In Vivo in modelli di SCLC con lo scopo di portare una nuova e promettente alternativa terapeutica per questa malattia.
In questo lavoro di tesi è stata valutata l’espressione di GD2 su due linee commerciali di SCLC H69 (GD2+ > 90%) e H209 (GD2+ < 6%). Abbiamo in seguito isolato i linfociti T e ingegnerizzati per esprimere il nostro CAR anti-GD2. È stata condotta un’analisi immunofenotipica sulle cellule CAR T e sui linfociti non trasdotti, per quantificare la presenza di cellule CD8+, NK, NKT e gamma delta (GD). Abbiamo valutato la presenza di cellule di memoria Naïve/stem, centrale, effettrice, terminale e la presenza di marker di esaustione come PD1, LAG3 e TIM3. I linfociti trasdotti con GFP e quelli trasdotti con il CAR presentano un aumentato numero di cellule di memoria Naive/Stem. I marker di esaustione non cambiano con il processo di trasduzione ad eccezione del TIM3 nei linfociti CD8+ la cui espressione aumenta significativamente. I CAR T e le cellule GFP sono stati utilizzati per co-colture In Vitro 2D e 3D con le cellule tumorali di H69 e H209. Entrambi questi setting hanno dimostrato come i linfociti CAR T siano stati in grado di produrre un significativa citotossicità sulla linea GD2+ a diversi rapporti effetore:target come il 2:1 e 4:1. Basandoci su questi risultati è stato impostato un esperimento In Vivo su un modello murino sottocute di SCLC utilizzando le cellule H69. I linfociti CAR T sono riusciti a bloccare e invertire il processo di crescita della massa tumorale (p<0,001 CAR T vs Controllo PBS, p<0,01 CAR T vs GFP). Si è evidenziato un effetto anche dei linfociti GFP, da attribuire al contesto allogenico dell’esperimento dove non c’è compatibilità di HLA tra donatore dei linfociti e linea tumorale. Per avvicinarci ad un contesto autologo che rispecchia l’utilizzo dei CAR T in clinica, abbiamo avviato un comitato etico per la raccolta di campioni di tumore e di linfociti da paziente affetto da SCLC.
Abbiamo inoltre deciso di sviluppare un protocollo di espansione per linfociti GD e trasdurli con il nostro CAR per creare delle cellule anti-GD2 GD T. Il protocollo di espansione sviluppato porta ad avere una popolazione di linfociti che è più del 90% GD+ mentre quello di trasduzione porta ad avere più del 65% di linfociti CAR GD. I primi studi In Vitro utilizzando CAR GD T hanno dimostrato una forte citotossicità verso SCLC. Per questo motivo abbiamo testato i linfociti CAR GD T, sullo stesso modello sottocute menzionato precedentemente. Durante questa sperimentazione In Vivo non abbiamo constato alcun effetto dei CAR GD T.
Questi risultati valorizzano l’uso della terapia a base di cellule CAR T contro il microcitoma, confermando il ruolo di GD2 come bersaglio. Ulteriori esperimenti dovranno essere condotti per poter traslare questa tecnologia in un contesto allogenico utilizzando cellule GD, che hanno dimostrato la loro efficacia In Vitro, ma non In Vivo.Small Cell Lung Cancer (SCLC) is a neuroendocrine tumor which accounts for 15% of all lung cancers. It is characterized by a 5-year general survival rate of only 7%. Chimeric Antigen Receptor (CAR) T cells are T lymphocytes genetically modified to express a synthetic modular protein capable of redirecting immune cell reactivity toward a target of interest. This new immunotherapeutic approach demonstrated substantial clinical effects in the treatment of B cell and plasma cell malignancies. The disialoganglioside GD2 is a ganglioside known to be expressed by neuroectoderm-derived tumors such as SCLC with a highly restricted expression on healthy tissues. Therefore, we sought to exploit an anti-GD2 CAR T cell approach against SCLC, aiming to provide a new promising strategy to treat the extensive-stage SCLC.
We assessed the GD2 expression on two commercial SCLC human cell lines H69 (GD2+ >90%) and H209 (GD2+ <6%). We isolated the T cell lymphocytes from healthy donors' peripheral blood and engineered them to express our anti-GD2 CAR. The CAR T cells were characterized via flow-cytometry to assess the presence of cytotoxic population as CD8, NK and gamma delta (GD) lymphocytes. We also evaluated the presence of memory populations as: Naïve/stem cell, central, effector and terminal effector and the presence of exhaustion markers such as PD1, LAG3 and TIM3. GFP T and CAR T cells had a Naïve/stem Cell memory enriched phenotype. Exhaustion markers did not change after the transduction process except TIM3 expression, which is increased in CD8+ transduced cells. In vitro 2D and 3D spheroid co-cultures were set up to assess the anti-GD2 CAR T cytotoxic efficiency. We compared the activity of GFP T and CAR T lymphocytes to different effector target (E:T) ratios and different time points (24h,48h,72h). Both In Vitro settings showed CAR T cells efficiency against H69 (GD2high) cell line at different effector:target (E:T) ratios.
According to these results, an In Vivo subcutaneous model of SCLC was challenged with anti-GD2 CAR T cells. We managed to revert the tumor masses growth in the mice group treated with CAR T cells, achieving a significant reduction of volumes compared to the control group treated with PBS (p<0.001) and the group treated with lymphocytes expressing the GFP only (p<0.01). During this experiment, we experienced a significant activity of GFP T lymphocytes due to the allogenic setting of the experiment. Hence, we started developing a protocol to isolate patient-derived SCLC cell lines and T lymphocytes to set up the same experiments In Vitro and In Vivo in an autologous setting.
A non-trivial topic within the cell therapy field is to move from an autologous to an allogenic source of cells to diminish costs, intra-donor variability and to enhance therapeutic outcomes. Thus, we focused on developing an expansion protocol for GD T cells and transducing them with the anti-GD2 CAR. Our expansion protocol led to a yield of >90% of GD T cells (CD3+/TCR GD+) and the transduction efficiency achieved to obtain CAR GD T cells was above 65%. In Vitro cytotoxicity of CAR GD T cells was tested on SCLC cell line H69LUC. CAR GD T cells were then used In Vivo in a subcutaneous SCLC model. Contrarily to their AB counterpart, they did not manage to control the tumor mass growth probably due to lack of persistence.
These encouraging results pave the way to the CAR T approach against SCLC, demonstrating that GD2 is a valuable target for CAR T therapy and providing an efficient alternative to the currently available treatments. Further experiments on GD T cells must be discharged to enhance their anti-tumor activity In Vivo
Fragile X syndrome: causes, diagnosis, mechanisms, and theraupetics.
No full textFragile X syndrome (FXS) is the most frequent form of inherited intellectual disability and is also linked to other neurologic and psychiatric disorders. FXS is caused by a triplet expansion that inhibits expression of the FMR1 gene; the gene product, FMRP, regulates mRNA metabolism in the brain and thus controls the expression of key molecules involved in receptor signaling and spine morphology. While there is no definitive cure for FXS, the understanding of FMRP function has paved the way for rational treatment designs that could potentially reverse many of the neurobiological changes observed in FXS. Additionally, behavioral, pharmacological, and cognitive interventions can raise the quality of life for both patients and their families
The CFC syndrome. Report of the first two cases outside the United States
No full textWe report on two additional patients with the cardiofaciocutaneous (CFC) syndrome, the first to be reported outside the United States. They have several of the characteristic manifestations of this new multiple congenital anomalies/mental retardation syndrome, namely, mental retardation, growth retardation, relative macrocephaly, unusual face, abnormal hair, skin involvement, heart defect, hernias, and splenomegaly. Similar to all previously reported cases, these also were sporadic and had normal chromosomes
Proceeding from the 2009 genetic syndromes of the Ras/MAPK pathway: from bedside to bench and back
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