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Trapianto di cellule staminali in un modello animale di danno osseo da glucocorticoidi
Full text linkBone, a specialized connective tissue, consists of cells and mineralized extracellular matrix. The main cell types of bone tissue are: the osteoblasts, the osteocytes and the osteoclasts. Osteoblasts produce extracellular matrix, osteoclasts are responsible of its resorption, hence bone physiology is a delicate balance between synthesis of new bone and resorption of the old one. Osteoporosis is a disease in which catabolic activity of osteoclasts overtakes anabolic activity of osteoblasts leading to increased bone resorption and progressive bone fragility.
Primary osteoporosis is a common disease among post-menopausal female population. Pathologies as diabetes mellitus, hyperparathyroidism and long-term treatment with glucocorticoids cause secondary osteoporosis. Glucocorticoid-induced osteoporosis is the most common type of secondary osteoporosis.
Glucocorticoid treatment is a well known method to induce osteoporosis in animal models, hence it could be an example of “translational model” in which injured bone could be repopulated by stem cells or progenitors in clinical trials.
The aim of this thesis is to investigate whether preosteoblasts could repopulate injured bone in an animal model treated with glucocorticoids. Preosteoblasts have been isolated from newborn calvariae of GFP mice.
In these cells, expression of the osteogenic marker Runx2 has been assessed by Real time PCR, while osteogenic potential has been analysed by cytochemistry assays to detect alkaline phosphatase and mineralized bone nodules (Alizarin Red and Von Kossa staining).
To realize the in vivo model, C57BL/6 three months aged mice have been divided into three groups [group I (n=4): mice not treated with drug and not infused with cells, group II (n=4): mice treated with drug and not infused with cells, group III (n=4): mice treated with drug and infused with cells]. Drug (methylprednisolone) has been administered for one month with a dose of 75 mg/Kg/week. In mice of group III, 5 x 105 GFP preosteoblasts, previously expanded in vitro, have been infused with injection into the tail vein. Mice have been sacrificed, tibial and femoral bones have been harvested, processed and analysed by istomorphometry and immunoistochemistry. Expression of Runx2, osteonectin (SPARC) and alkaline phosphatase (ALP) in these tissues has been detected with Real time PCR.
In vitro preosteoblasts produce alkaline phosphatase during early time in culture with normal medium, while the level decreases in differentiating conditions with medium containing ascorbic acid and β-glycerophosphate. Preosteoblasts maintained in differentiation medium for 30 days are positive to Alizarin and Von Kossa staining, hence they are able to produce mineralized extracellular matrix that is a feature of functional mature osteoblasts. Runx2 expression increases during differentiating conditions; in cells maintained in differentiation medium for 30 days there is an increase of 50% compared to cells maintained in normal medium (p<0.05).
In mice of group III an increased level of parameters concerning osteoid has been detected (O.Th, OS/BS, OV/BV) and an increased number of active osteoblasts (during synthetic activity) has been observed compared to group II. Between these two groups, significant variations of bone volume (BV/TV), trabecular thickness (Tb.Th), trabecular number (Tb.N) and trabecular separation (Tb.Sp) have not been detected.
Microarchitecture parameters (Nd.N/TV, Nd/Tm) have not been affected. Similar results have been obtained from inderect microarchitecture parameters as Marrow Star Volume and Fractal Dimension.
Real time PCR analysis revealed a reduction in osteogenic gene expression in group II compared to group I (ALP: -50%, p<0.01; Runx2: -56.75%, p<0.01; SPARC: -44.5%, p<0.05).
In group III there is a recovery of expression of osteogenic markers (ALP: +40%, p<0.05; Runx2: +66.28%, p<0.001; SPARC: +55%; p<0.01) compared to group II.
Immunoistochemistry is under investigation.
In our glucocorticoid-induced osteoporosis model we sacrificed mice only one week after infusion of cells, this preparatory investigation shows that our model induces the engraftment of preosteoblasts in injured bone. However, a longer time, at least of 1-2 months, is needed to investigate if preosteoblasts are able not only to graft onto host tissue, but also to proliferate in vivo and to differentiate in full mature and functional osteoblasts.L’osso è un tessuto connettivo specializzato costituito da cellule e matrice extracellulare mineralizzata. Le cellule principali sono gli osteoblasti, gli osteociti e gli osteoclasti. Gli osteoblasti depongono la matrice ossea, mentre gli osteoclasti sono i responsabili del suo riassorbimento.
La fisiologia dell’osso è quindi il risultato di un delicato equilibrio tra deposizione di matrice ossea e suo riassorbimento.
Quando l’azione catabolica degli osteoclasti è maggiore rispetto a quella anabolica degli osteoblasti si produce una progressiva fragilità ossea che porta ad un quadro clinico osteoporotico.
L’osteoporosi primaria colpisce soprattutto la popolazione femminile dopo la menopausa. Molte patologie come il diabete mellito, l’iperparatiroidismo ed il trattamento a lungo termine con glucocorticoidi causano l’osteoporosi secondaria. L’osteoporosi indotta da glucocorticoidi è la più comune causa di osteoporosi secondaria.
Il trattamento con glucocorticoidi è un noto procedimento di induzione dell’osteoporosi in modelli animali e può dunque rappresentare un primo esempio di modello “traslazionale” potenzialmente applicabile in clinica per indurre un ripopolamento dell’osso con cellule staminali mesenchimali o precursori osteogenici.
Lo scopo di questo lavoro è stato pertanto valutare nel modello animale se sia possibile ripopolare l’osso danneggiato con preosteoblasti.
I crani di topi neonati transgenici (GFP) sono stati prelevati e messi in coltura per ottenere preosteoblasti. Nelle colture in vitro è stata valutata l’espressione del gene Runx2 con la tecnica di Real time PCR, mentre la capacità osteogenica è stata analizzata con colorazioni citochimiche per la fosfatasi alcalina e per la deposizione di matrice ossea mineralizzata (Alizarin Red e Von Kossa). Per la realizzazione del modello in vivo topi C57BL/6 maschi di 3 mesi sono stati divisi in 3 gruppi [gruppo I (n=4): topi non trattati con farmaco e non infusi con cellule; gruppo II (n=4): topi trattati con farmaco non infusi con cellule; gruppo III (n=4): topi trattati con farmaco ed infusi con cellule]. Il farmaco (metilprednisolone) è stato somministrato per un mese alla dose di 75 mg/Kg/settimana.
Negli animali appartenenti al gruppo III sono state infuse, attraverso iniezione nella vena della coda, 5 x 105 preosteoblasti GFP precedentemente espansi in vitro. I topi sono stati sacrificati, le tibie ed i femori sono stati prelevati e processati per l’analisi istomorfometrica e della microarchitettura ossea e per l’ immunoistochimica. In questi tessuti, l’espressione genica di Runx2, osteonectina (SPARC) e fosfatasi alcalina (ALP) è stata valutata tramite Real time PCR.
In vitro i preosteoblasti producono fosfatasi alcalina durate i primi giorni di coltura in medium non differenziante, mentre il livello decresce in condizioni differenzianti, cioè in medium contenente acido ascorbico e β-glicerofosfato. I preosteoblasti mantenuti in medium di differenziamento per 30 giorni sono positivi alle colorazioni Alizarin Red e Von Kossa, quindi sono in grado di produrre matrice ossea mineralizzata, caratteristica degli osteoblasti funzionali e maturi. L’espressione del gene Runx2 aumenta durante il differenziamento, si ha un aumento del 50% nelle cellule differenziate per 30 giorni rispetto alle cellule non differenziate (p<0.05).
L’inoculazione dei preosteoblasti nei topi del gruppo III ha evidenziato un aumento dei parametri statici di neoformazione ossea relativi all’osteoide (O.Th, OS/BS, OV/BV) ed un aumento del numero di osteoblasti attivi, cioè in corso di deposizione di osteoide, rispetto al gruppo II. Tra questi due gruppi non si sono osservate, invece, variazioni significative in termini di volume osseo (BV/TV), spessore trabecolare (Tb.Th) numero delle trabecole (Tb.N) e separazione fra esse (Tb.Sp).
Non sono state rilevate, inoltre, differenze dei parametri di microarchitettura (Nd.N/TV, Nd/Tm). Risultati simili sono emersi dalla valutazione dei parametri indiretti di microarchitettura (Marrow Star Volume e Fractal Dimension).
L’espressione genica ha dimostrato che nel gruppo II si ha una riduzione dell’espressione dei geni osteogenici rispetto al gruppo I (ALP: -50%, p<0.01; Runx2: -56.75%, p<0.01; SPARC: -44.5%, p<0.05).
Nel gruppo III si è avuto un recupero dell’espressione dei geni osteogenici (ALP: +40%, p<0.05; Runx2: +66.28%, p<0.001; SPARC: +55%, p<0.01) rispetto al gruppo II. I campioni di tessuto per l’ immunoistochimica devono essere processati.
Nel nostro modello sperimentale di osteoporosi indotta da glucocorticoidi nel topo, abbiamo sacrificato gli animali solo una settimana dopo l’infusione delle cellule; questi dati preliminari dimostrano che il nostro modello induce l’engraftment dei preosteoblasti nell’osso danneggiato.
Tuttavia è richiesto un tempo di osservazione più lungo, di almeno 1-2 mesi per valutare se le cellule trapiantate siano in grado, non solo di integrarsi nel tessuto dell’ospite, ma anche di proliferare in vivo e di differenziare in osteoblasti maturi e funzionali
Predictive role of host constitutive variants in neoadjuvant therapy of esophageal cancer
No full textChemoradiotherapy followed by surgery is at present the standard therapeutic approach for esophageal cancer (EC) in patients with resectable tumor. However, response to neoadjuvant therapy is characterized by a strong interpatient variability, and the identification of markers predictive of outcome is mandatory. In this review, taking into account the currently available literature, we report the impact that host genetic variables can have on EC neoadjuvant therapy. We mainly focused on the gene variants involved in the pharmacokinetics and pharmacodynamics of the common chemotherapeutic drugs used to treat EC patients, commented on the weakness of the present knowledge, and discussed the future strategies to achieve a more personalized and effective EC 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
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
Liquid biopsy as a novel tool to monitor the carcinogenesis of Barrett's esophagus
No full textBarrett's esophagus (BE) is associated with an increased risk of developing esophageal adenocarcinoma. For this reason, endoscopic-based surveillance protocols have been developed. This prevention program is, however, burdensome for the patients and expensive for the national health systems. Thus, diagnostic strategies with a low invasiveness and a reduced economic impact are required. This study investigated the power of plasma circulating free DNA (cfDNA) in predicting neoplastic transformation in the natural history of two BE patients who progressed to esophageal adenocarcinoma. Longitudinally collected DNAs from plasma and paired formalin fixed paraffin embedded samples were examined for both loss of heterozygosity (LOH) in areas proximal to TP53, FHIT and BRCA2 genes, and mutations in TP53 gene. Results showed that: (i) early BE molecular alterations are mainly localized proximal to, or within, TP53 gene; (ii) LOH events present in cfDNA not only retrace the time-matched biopsy profile but better represent the total alterations of the BE epithelium. In conclusion, our findings suggested that LOH analysis in plasma cfDNA could represent an additional, less invasive, diagnostic tool to monitor neoplastic progression of BE epitheliu
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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