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Terapia di precisione per malattie autoimmuni mediate da chinasi: sviluppo di un sistema in vitro per la diagnosi e il monitoraggio clinico.
No full textQuesto progetto si concentra sulle tirosin chinasi della famiglia JAK e sui suoi inibitori, JAKi, e ha l'obiettivo di sviluppare strumenti in vitro per la personalizzazione nelle malattie oncoematologiche e autoimmuni, come la sindrome di Aicardi-Goutieres (AGS). Il primo tool è un saggio ELISA in vitro basato su biosensori peptidici (PJAK2-L e PJAK2-S) per misurare l'attività aberrante di JAK2. Il secondo invece è la creazione di neuroni derivati da cellule staminali pluripotenti indotte (iPSC) per testare la sicurezza e l'efficacia dei JAKi in modelli paziente-specifici di AGS.
Le linee cellulari oncoematologiche (HEL, SET-2 e MHH-CALL-4) caratterizzate da alterazioni genetiche che portano all’attivazione della pathway JAK2-STAT5, hanno mostrato che HEL e SET-2 erano sensibili ai JAKi ruxolitinib, baricitinib, tofacitinib e pacritinib. Abbiamo ottimizzato il protocollo ELISA lavorando prima sulla tirosina chinasi ABL1 come pubblicato (Montecchini, Braidotti et. al, 2021, Front Pharmacol) e applicando poi la metodologia ai peptidi PJAK2-L e PJAK2-S, ma senza successo dal momento che i peptidi non sono stati fosforilati dopo l'incubazione dei lisati ottenuti dalle linee cellulari. Il saggio è stato quindi eseguito sulla chinasi JAK2 immunoprecipitata a partire da lisati della linea HEL; emerge un segnale di fosforilazione rilevante per PJAK2-L rispetto al background (intensità di fluorescenza: HEL lisato, 2510,26±931,01; HEL lisato + PJAK2-L, 87710,99± 3278,33, P<0.0001), ma non per PJAK2-S (HEL lisato + PJAK2- S, 2074,34±964,74). Si sono susseguiti tentativi di ottimizzare le condizioni del saggio con PJAK2-L, tra cui l'aumento della quantità di lisato (4 vs 40 μg), la temperatura di incubazione (25°C vs 37°C) e il tempo (1h vs 2h), ma senza miglioramento del segnale di fosforilazione di PJAK-L. Il set-up sperimentale ideale per il saggio ELISA non è ancora stato definito.
Le iPSC sono state ottenute riprogrammando i fibroblasti di 3 pazienti con AGS (AGS1, AGS2 e AGS7, che ospitano rispettivamente mutazioni nei geni TREX1, RNASEH2B, IFIH1) e 1 donatore sano (BJ) e differenziati in cellule staminali neurali (NSC) per studiare la citotossicità in vitro di JAKi. Solo pacritinib era citotossico per iPSC e NSC (intervallo IC50: 0,67-1,44 μM, mediante test MTT). Un'esposizione di 3 giorni a un'alta concentrazione di ruxolitinib (> 2, 5 μM) ha aumentato la vitalità cellulare in AGS2-iPSC rispetto ai controlli BJ-iPSC ( P <0, 05); un risultato simile è stato osservato per AGS7-iPSC e ruxolitinib o baricitinib a 2,5 μM (P <0,05). L'esposizione ad alte concentrazioni di ruxolitinib, baricitinib e tofacitinib ha aumentato la vitalità cellulare in AGS7-NSC rispetto a BJ-NSC (P<0,05); allo stesso modo, AGS2-NSC e baricitinib a 0,6 μM e 2,5 μM (P <0,001) o tofacitinib a 2,5 μM (p <0,05) e per AGS1-NSC trattato con tofacitinib a 10 μM (P <0,05). L'espressione dei geni coinvolti nella farmacodinamica JAKi (JAK1/STAT1, TYK2/STAT2) era paragonabile tra iPSC e NSC e tra NSC e iPSC. In conclusione, la maggior parte dei JAKi è sicura per NSC. Come prospettiva futura, potrebbe essere interessante ampliare il panel di farmaci testati su NSC, per differenziare NSC in neuroni e studiare gli effetti citotossici di tutti i farmaci usati per trattare l'AGS nelle cliniche, anche nelle cellule neuronali derivate da NSC.This project focuses on the JAK tyrosine kinase and their inhibitors, JAKi, and has the aim of developing in vitro tools to personalize in oncohematological and autoimmune diseases, such as Aicardi-Goutieres syndrome (AGS). The first tool is an in vitro ELISA assay based on peptide biosensors (PJAK2-L and PJAK2-S) to measure aberrant JAK2 activity. The second tool is the setting up of induced pluripotent stem cells (iPSCs) derived neurons for testing JAKi safety and efficacy in AGS- patient specific models.
Oncohematological cell lines (HEL, SET-2 and MHH-CALL-4) harboring genetic alterations leading to an hyperactivated JAK2-STAT5 pathway showed that HEL and SET-2 were sensitive to JAKi ruxolitinib, baricitinib, tofacitinib and pacritinib. We optimize the ELISA protocol working on the ABL1 tyrosine kinase as published (Montecchini, Braidotti et. al, 2021, Front Pharmacol) and applied to PJAK2-L and PJAK2-S, without success because peptides were not phosphorylated after whole cell lysates incubation. The assay was then performed on JAK2 immunoprecipitated from HEL lysates; a relevant phosphorylation signal emerges for PJAK2-Lcompared to background (fluorescence intensity: HEL lysate, 2510.26±931.01; HEL lysate + PJAK2-L, 87710.99± 3278.33, P<0.0001), but not for PJAK2-S (HEL lysate + PJAK2-S, 2074.34±964.74). Attempts to optimize the conditions of the assay with PJAK2-L followed, including the increase in lysate amount (4 vs 40 μg), the incubation temperature (25°C vs 37°C) and time (1h vs 2h), however with no improvement in the phosphorylation signal of PJAK-L. The ideal experimental settings for the ELISA assay still need to be defined.
iPSCs were obtained by reprogramming fibroblasts of 3 AGS patients (AGS1, AGS2 and AGS7, harboring mutations in TREX1, RNASEH2B, IFIH1 genes respectively), and 1 healthy donor (BJ) and differentiated into neural stem cells (NSC) to investigate in vitro cytotoxicity by JAKi. Only pacritinib was cytotoxic to iPSC and NSC (IC50 range: 0.67-1.44 μM, by MTT assay). A 3-day exposure to high concentration of ruxolitinib (>2.5 μM) increased cell viability in AGS2-iPSC compared to controls BJ-iPSC (P<0.05); a similar result was observed for AGS7-iPSC and ruxolitinib or baricitinib at 2.5 μM (P<0.05). The exposure to high concentrations of ruxolitinib, baricitinib and tofacitinib increased cell viability in AGS7- NSC compared to BJ-NSC (P<0.05); similarly, AGS2-NSC and baricitinib at 0.6 μM and 2.5 μM (P<0.001) or tofacitinib at 2.5 μM (p<0.05), and for AGS1-NSC treated with tofacitinib at 10 μM (P<0.05). Expression of genes involved in JAKi pharmacodynamics (JAK1/STAT1, TYK2/STAT2) were comparable between iPSC and NSC, and among NSC and iPSC. In conclusion, the majority of JAKi are safe for NSC. As a future prospective, It may be interesting to broaden the panel of drugs tested on NSC, to differentiate NSC into neurons and investigate the cytotoxic effects of all drugs used to treat AGS in clinics, also in NSC-derived neuronal cells
Effect of early post-hematopoietic stem cell transplant tacrolimus concentration on transplant outcomes in pediatric recipients: One facility’s ten-year experience of immunosuppression with tacrolimus
Full text linkAcute graft-versus-host disease (GVHD) is a common life-threatening complication of allogeneic hematopoietic stem cell transplantation (allo-HSCT), ranking as the second leading cause of death among recipients, surpassed only by disease relapse. Tacrolimus is commonly used for GVHD prophylaxis, but achieving therapeutic blood levels is challenging, particularly in pediatrics, due to the narrow therapeutic window and the high interindividual variability. The retrospective study conducted at IRCCS “Burlo Garofolo” in Italy aimed to assess the impact of early post-HSCT tacrolimus levels on transplant-related outcomes in pediatric recipients. The population pharmacokinetic model (POP/PK) was set up to describe tacrolimus pharmacokinetics. Elevated tacrolimus (>12–15 ng/ml) levels within the initial weeks post-HSCT are associated with reduced post-transplant infections (p < 0.0001) and decreased incidence of early transplant-related events (p < 0.01), including a lower incidence of acute GVHD (p <0.05 on day 0). High tacrolimus exposure can lead to an increased risk of chronic GVHD (p < 0.0001) and reduced overall survival (p < 0.01). Personalized dosing and therapeutic monitoring of tacrolimus are crucial to ensure optimal outcomes. POP/PK could help achieve this goal, giving us a model by which we can balance immunosuppression while looking at the patient’s general well-being and providing the necessary treatment
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Full text linkWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Iron Bioavailability in the Extracellular Environment Is More Relevant Than the Intracellular One in Viability and Gene Expression: A Lesson from Oligodendroglioma Cells
Full text linkOligodendroglioma (OG) is a brain tumor that contributes to <1% of brain tumor diagnoses in the pediatric population. Unfortunately, pediatric OG remains without definitive molecular characteristics to aid in diagnosis, and little is known about the tumor microenvironment. Tumor cells’ metabolism and proliferation rate are generally higher than those of healthy cells, so their iron demand is also significantly higher. This consideration underlines the great importance of iron for tumor development and progression. In this context, this study aims to evaluate the effect of iron in a cellular in vitro model of human oligodendroglioma brain tumor. Cell morphology, the effect of siderotic medium on cell growth, iron uptake, and the expression of iron-metabolism-related genes were evaluated via optic microscopy, ICP-MS, confocal microscopy, and real-time PCR, respectively. This study underlines the great importance of iron for tumor development and progression and also the possibility of reducing the available iron concentration to determine an antiproliferative effect on OG. Therefore, every attempt can be promising to defeat OG for which there are currently no long-term curative therapies
Dispelling the Myths Behind First-author Citation Counts
Full text linkWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
koamabayili/VECTRON-author-checklist: VECTRON author checklist
No full textWe have done our best to complete the author checklist relating to the use of animals in the hut study. Note that the objective for the hut study was to evaluate the IRS treatment applications for residual efficacy against Anopheles mosquitoes, including the local An. coluzzii mosquito population. Cows were only used to attract mosquitoes into the huts and no tests were carried out directly on the cows. The author checklist is intended for use with studies where experiments are carried out on animals, which is why we have had such difficulty in completing this for the hut study, as many of the questions do not relate to how the cows were used
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