IL-12 Can Target Human Lung Adenocarcinoma Cells and Normal Bronchial Epithelial Cells Surrounding Tumor Lesions

BACKGROUND: Non small cell lung cancer (NSCLC) is a leading cause of cancer death. We have shown previously that IL-12rb2 KO mice develop spontaneously lung adenocarcinomas or bronchioalveolar carcinomas. Aim of the study was to investigate i) IL-12Rbeta2 expression in human primary lung adenocarcinomas and in their counterparts, i.e. normal bronchial epithelial cells (NBEC), ii) the direct anti-tumor activity of IL-12 on lung adenocarcinoma cells in vitro and vivo, and the mechanisms involved, and iii) IL-12 activity on NBEC. METHODOLOGY/PRINCIPAL FINDINGS: Stage I lung adenocarcinomas showed significantly (P = 0.012) higher frequency of IL-12Rbeta2 expressing samples than stage II/III tumors. IL-12 treatment of IL-12R(+) neoplastic cells isolated from primary adenocarcinoma (n = 6) inhibited angiogenesis in vitro through down-regulation of different pro-angiogenic genes (e.g. IL-6, VEGF-C, VEGF-D, and laminin-5), as assessed by chorioallantoic membrane (CAM) assay and PCR array. In order to perform in vivo studies, the Calu6 NSCLC cell line was transfected with the IL-12RB2 containing plasmid (Calu6/beta2). Similar to that observed in primary tumors, IL-12 treatment of Calu6/beta2(+) cells inhibited angiogenesis in vitro. Tumors formed by Calu6/beta2 cells in SCID/NOD mice, inoculated subcutaneously or orthotopically, were significantly smaller following IL-12 vs PBS treatment due to inhibition of angiogenesis, and of IL-6 and VEGF-C production. Explanted tumors were studied by histology, immuno-histochemistry and PCR array. NBEC cells were isolated and cultured from lung specimens of non neoplastic origin. NBEC expressed IL-12R and released constitutively tumor promoting cytokines (e.g. IL-6 and CCL2). Treatment of NBEC with IL-12 down-regulated production of these cytokines. CONCLUSIONS: This study demonstrates that IL-12 inhibits directly the growth of human lung adenocarcinoma and targets the adjacent NBEC. These novel anti-tumor activities of IL-12 add to the well known immune-modulatory properties of the cytokine and may provide a rational basis for the development of a clinical trial

Integrated clinicopathologic and molecular analysis of endometrial carcinoma: Prognostic impact of the new ESGO-ESTRO-ESP endometrial cancer risk classification and proposal of histopathologic algorithm for its implementation in clinical practice

IntroductionThe European Society of Gynecologic Oncology/European Society of Radiation Therapy and Oncology/European Society of Pathology (ESGO/ESTRO/ESP) committee recently proposed a new risk stratification system for endometrial carcinoma (EC) patients that incorporates clinicopathologic and molecular features. The aim of the study is to compare the new ESGO/ESTRO/ESP risk classification system with the previous 2016 recommendations, evaluating the impact of molecular classification and defining a new algorithm for selecting cases for molecular analysis to assign the appropriate risk class.MethodsThe cohort included 211 consecutive EC patients. Immunohistochemistry and next-generation sequencing were used to assign molecular subgroups of EC: POLE mutant (POLE), mismatch repair deficient (MMRd), p53 mutant (p53abn), and no specific molecular profile (NSMP).ResultsImmuno-molecular analysis was successful in all cases, identifying the four molecular subgroups: 7.6% POLE, 32.2% MMRd, 20.9% p53abn, and 39.3% NSMP. The recent 2020 guidelines showed a 32.7% risk group change compared with the previous 2016 classification system: the reassignment is due to POLE mutations, abnormal p53 expression, and a better definition of lymphovascular space invasion. The 2020 system assigns more patients to lower-risk groups (42.2%) than the 2016 recommendation (25.6%). Considering the 2020 risk classification system that includes the difference between “unknown molecular classification” and “known,” the integration of molecular subgroups allowed 6.6% of patients to be recategorized into a different risk class. In addition, the use of the proposed algorithm based on histopathologic parameters would have resulted in a 62.6% reduction in molecular analysis, compared to applying molecular classification to all patients.ConclusionApplication of the new 2020 risk classification integrating clinicopathologic and molecular parameters provided more accurate identification of low-and high-risk patients, potentially allowing a more specific selection of patients for post-operative adjuvant therapy. The proposed histopathologic algorithm significantly decreases the number of tests needed and could be a promising tool for cost reduction without compromising prognostic stratification

Ottimizzazione di saggi biologici per valutare l'attività mutagena della Bleomicina

La bleomicina è un antibiotico ad azione antitumorale, prodotto dal batterio Streptomyces verticillus. Questo farmaco è utilizzato in ambito chemioterapico per il trattamento di Linfoma di Hodgink e non-Hodgink, cancro dei testicoli, cancro della testa e del collo, cancro alla cervice uterina, cancro ai genitali esterni e carcinomi a cellule squamose. La BLM è in grado di intercalarsi random nel doppio filamento di DNA e formare radicali liberi citotossici che causano Double Strand Break (DSB) in situ. A seguito della formazione di DSB, viene innescata una complessa cascata di eventi finalizzati al blocco del ciclo cellulare e al reclutamento di fattori di riparazione. Uno degli iniziatori del processo è la proteina chinasi ATM (Ataxia Talangiectasia Mutated) che causa, in concerto con la chinasi ATR (Ataxia Talangectasia Related) e la DNA-PKcs (DNA Dependent Protein Kinase catalytic subunit), la fosforilazione delle proteine istoniche attorno alla lesione, fra le quali, l’istone ɤH2AX. Questo viene fosforilato a livello della serina-139 in una regione, che si estende per circa 2Mb di DNA nei pressi del DSB. La lesione può essere successivamente riparata attraverso la ricombinazione non omologa (NHEJ) o la ricombinazione omologa (HR). Nel presente progetto di tesi è stata considerata la proprietà genotossica del farmaco, in particolare, preliminarmente all’utilizzo di BLM-coniugata con un oligonucleotide per verificare se riesca ad indurre un arricchimento di lesioni sito-specifiche nel genoma mimando un Gene-editing locus specifico (simile al ruolo della sg-RNA nel CRISPR-Cas9). Oligonucleotidi a singolo filamento (20bp) sono stati disegnati complementari a sequenze dell’esone 3 del gene ipoxantina fosforibosiltransferasi (HPRT, q26.2-26.3 chrX). L’HPRT assay, un test di mutagenesi in vitro su cellule di mammifero è stato utilizzato per saggiare l’azione mutagena della BLM. L’impiego di una linea cellulare emizigote per il cromosoma X (HCT116 tumorali di cancro al colon retto) assicura al test il vantaggio di poter evidenziare fenotipicamente le mutazioni avvenute sul DNA in seguito al trattamento. Il farmaco, somministrato alle dosi 1µM – 1.4 µM - 2µM per 24h, ha indotto nella linea cellulare un aumento del numero di colonie di 2.14 volte per unità di dose, risultato statisticamente significativo. La “Chromatin immunoprecipitation” (ChIP) con un anticorpo Anti-fosfoɤH2AX è stata effettuata su cellule trattate con BLM non coniugata, e il relativo controllo negativo. La successiva analisi basata su qPCR con sistema EXPRESS SYBR® GreenER ™ usando come gene target HPRT e come geni di controllo GYG2, GSS, OGT, Erα, precedentemente testati, ci consentirà di verificare i DSBs indotti a seguito del trattamento. Il Surveyor Nuclease Assay” è stato utilizzato per individuare le lesioni al DNA non correttamente riparate dal sistema NHEJ che causano mutazioni in/del e polimorfismi. Questa tecnica è basata sulla capacità della surveyor nucleasi di tagliare selettivamente, in prossimità dei “mismatch”, il DNA eteroduplex formatosi per “cross-annealing” della sequenza mutata con la sequenza “wild-type”. I prodotti risultanti sono analizzati successivamente mediante elettroforesi su gel di agarosio. La trasfezione con vettore HR410PA-1, opportunamente costruito, è stata utilizzata per quantificare l’avvenuta HR nel locus HPRT in cellule coltivate e sottoposte a varie tipologie di trattamento. In futuro, questi saggi consentiranno di capire se il locus HPRT sarà soggetto ad attività mutagena sito-specifica indotta dalla BLM quando coniugata con specifici oligonucleotidi

New insights and tools to study innovative delivery strategies of therapeutic peptides: Apolipoprotein A-I Milano, administered orally via genetically modified rice plants, exerts anti-inflammatory effects in vivo.

In the proof-of-concept study published by Romano and others in Int.Cardiology 2018, was shown a new approach for treatment of cardiovascular disease (CVDs) because, despite the high therapeutic potential of Apolipoprotein A-I protein, the practical application is hampered by the low efficiency of purification and delivery. A genetically modified rice plants expressing Apolipoprotein A-I variant Milano (AIM) were created. The product was tested in vitro, and in vivo mouse atherosclerotic model treated with genetically modified rice-milk (in the form of protein extract of rice seed proteins) or wild type rice-milk and it was found to be safe and effective. For subsequent aims, initially, in this context, the APO rice flour (RF) product was better characterized to further understand the potential of transgenic rice seeds as an efficient oral production and delivery system for recombinant AIM protein. WT and APO RF were analysed by ELISA and Western Blot (WB) and APO protein was detected in APO rice as expected. To better understand the biodistribution of the AIM protein administered orally in APO3 pre-clinical study and the molecular mechanisms underlying the athero-protective and anti-inflammatory effects, ELISA, WB and immunohistochemistry (IHC) assays were performed on mice liver and/or other organs. AIM in liver and intestine lysates was not detected by ELISA and WB. By increasing the spatial resolution by IHC, positive spots were detected in the liver sections of the treated mice. These investigations were fundamental to lay the foundations for the main purpose, namely the development of a new system of functional AIM product to improve both in quality and quantity of delivered protein to be able to extend the product for future use in humans. In this order, a set of vectors carrying the coding sequence of the AIM and the respective control vectors was constructed, to be tested at a functional level in vitro and subsequently to be used as source of the AIM sequence to engineer the new rice product

De Novo Genome Assembly at Chromosome-Scale of <i>Hermetia illucens</i> (Diptera Stratiomyidae) via PacBio and Omni-C Proximity Ligation Technology

Hermetia illucens is a species of great interest for numerous industrial applications. A high-quality reference genome is already available for H. illucens. However, the worldwide maintenance of numerous captive populations of H. illucens, each with its own genotypic and phenotypic characteristics, made it of interest to perform a de novo genome assembly on one population of H. illucens to define a chromosome-scale genome assembly. By combining the PacBio and the Omni-C proximity ligation technologies, a new H. illucens chromosome-scale genome of 888.59 Mb, with a scaffold N50 value of 162.19 Mb, was assembled. The final chromosome-scale assembly obtained a BUSCO completeness of 89.1%. By exploiting the Omni-C proximity ligation technology, topologically associated domains and other topological features that play a key role in the regulation of gene expression were identified. Further, 65.62% of genomic sequences were masked as repeated sequences, and 32,516 genes were annotated using the MAKER pipeline. The H. illucens Lsp-2 genes that were annotated were further characterized, and the three-dimensional organization of the encoded proteins was predicted. A new chromosome-scale genome assembly of good quality for H. illucens was assembled, and the genomic annotation phase was initiated. The availability of this new chromosome-scale genome assembly enables the further characterization, both genotypically and phenotypically, of a species of interest for several biotechnological applications

Current Therapeutical Approaches Targeting Lipid Metabolism in NAFLD

Nonalcoholic fatty liver disease (NAFLD, including nonalcoholic fatty liver (NAFL) and nonalcoholic steatohepatitis (NASH)) is a high-prevalence disorder, affecting about 1 billion people, which can evolve to more severe conditions like cirrhosis or hepatocellular carcinoma. NAFLD is often concomitant with conditions of the metabolic syndrome, such as central obesity and insulin-resistance, but a specific drug able to revert NAFL and prevent its evolution towards NASH is still lacking. With the liver being a key organ in metabolic processes, the potential therapeutic strategies are many, and range from directly targeting the lipid metabolism to the prevention of tissue inflammation. However, side effects have been reported for the drugs tested up to now. In this review, different approaches to the treatment of NAFLD are presented, including newer therapies and ongoing clinical trials. Particular focus is placed on the reverse cholesterol transport system and on the agonists for nuclear factors like PPAR and FXR, but also drugs initially developed for other conditions such as incretins and thyromimetics along with validated natural compounds that have anti-inflammatory potential. This work provides an overview of the different therapeutic strategies currently being tested for NAFLD, other than, or along with, the recommendation of weight loss

Pharyngo-esophageal perforations after anterior cervical spine surgery: management and outcomes

To report about the diagnosis, surgical treatment and post-operative management of pharyngo-esophageal perforations (PEP) after anterior cervical spine (ACS) surgery in 17 patients