Inhibition of heparanase protects against chronic kidney dysfunction following ischemia/reperfusion injury

Renal ischemia/reperfusion (I/R) injury occurs in patients undergoing renal transplantation and with acute kidney injury and is responsible for the development of chronic allograft dysfunction as characterized by parenchymal alteration and fibrosis. Heparanase (HPSE), an endoglycosidase that regulates EMT and macrophage polarization, is an active player in the biological response triggered by ischemia/reperfusion (I/R) injury. I/R was induced in vivo by clamping left renal artery for 30 min in wt C57BL/6J mice. Animals were daily treated and untreated with Roneparstat (an inhibitor of HPSE) and sacrificed after 8 weeks. HPSE, fibrosis, EMT-markers, inflammation and oxidative stress were evaluated by biomolecular and histological methodologies together with the evaluation of renal histology and measurement of renal function parameters. 8 weeks after I/R HPSE was upregulated both in renal parenchyma and plasma and tissue specimens showed clear evidence of renal injury and fibrosis. The inhibition of HPSE with Roneparstat-restored histology and fibrosis level comparable with that of control. I/R-injured mice showed a significant increase of EMT, inflammation and oxidative stress markers but they were significantly reduced by treatment with Roneparstat. Finally, the inhibition of HPSE in vivo almost restored renal function as measured by BUN, plasma creatinine and albuminuria. The present study points out that HPSE is actively involved in the mechanisms that regulate the development of renal fibrosis arising in the transplanted organ as a consequence of ischemia/reperfusion damage. HPSE inhibition would therefore constitute a new pharmacological strategy to reduce acute kidney injury and to prevent the chronic pro-fibrotic damage induced by I/R

CDX2 hox gene product in a rat model of esophageal cancer

<p>Abstract</p> <p>Background</p> <p>Barrett's mucosa is the precursor of esophageal adenocarcinoma. The molecular mechanisms behind Barrett's carcinogenesis are largely unknown. Experimental models of longstanding esophageal reflux of duodenal-gastric contents may provide important information on the biological sequence of the Barrett's oncogenesis.</p> <p>Methods</p> <p>The expression of <it>CDX2 </it>hox-gene product was assessed in a rat model of Barrett's carcinogenesis. Seventy-four rats underwent esophago-jejunostomy with gastric preservation. Excluding perisurgical deaths, the animals were sacrificed at various times after the surgical treatment (Group A: <10 weeks; Group B: 10–30 weeks; Group C: >30 weeks).</p> <p>Results</p> <p>No Cdx2 expression was detected in either squamous epithelia of the proximal esophagus or squamous cell carcinomas. <it>De novo </it>Cdx2 expression was consistently documented in the proliferative zone of the squamous epithelium close to reflux ulcers (Group A: 68%; Group B: 64%; Group C: 80%), multilayered epithelium and intestinal metaplasia (Group A: 9%; Group B: 41%; Group C: 60%), and esophageal adenocarcinomas (Group B: 36%; Group C: 35%). A trend for increasing overall Cdx2 expression was documented during the course of the experiment (<it>p </it>= 0.001).</p> <p>Conclusion</p> <p><it>De novo </it>expression of Cdx2 is an early event in the spectrum of the lesions induced by experimental gastro-esophageal reflux and should be considered as a key step in the morphogenesis of esophageal adenocarcinoma.</p

New Insights In Experimental Esophageal Carcinogenesis

Gastro-esophageal reflux disease (GERD) is a relevant health problem worldwide. It impairs patients' quality of life and predisposes to intestinal-like esophageal metaplasia (i.e. Barrett's esophagus, BE), that is recognized as the major risk factor for the development of esophageal adenocarcinoma (Eac). The incidence of Eac has dramatically increased since the mid of the 1970s in the USA and Western countries for unknown reasons, while the prognosis of Eac has only slightly been improved. During the same time potent acid suppressors have been introduced for the treatment of GERD. Nowadays these drugs lead the list of the world best seller drugs with a US$ 8 billion market per year. Some authors have linked the increase of the incidence of Eac with the wide diffusion of acid suppression in the general population and among patients with GERD(1). The risk of Eac in patients with GERD is too low to justify endoscopic surveillance. On the other hand endoscopic surveillance for patients with BE is generally accepted. Clinical evidence is still lacking on the best treatment for BE, in order to minimize the risk for neoplastic progression(2). Pharmacological, surgical and endoscopic therapies have been used, without a clear evidence about the benefit of a treatment on the others (chapter 1). The experimental surgical models of reflux-induced esophageal carcinogenesis can reproduce in laboratory animals the stepwise progression from inflammation to Eac, through BE(3,4). In Chapter 2 we provide a detailed description of the microsurgical technique we used for the reflux induced esophageal carcinogenesis model, in order to increase its reproducibility and minimize the number of animals needed to set up the experiments. Chapter 3 shows the results of a study about the effect of chronic GERD on animal welfare. The main short and long-term clinical complications are analyzed, as well as the significance and prognostic value of two different scoring systems based on clinical parameters. Using these methods, humane endpoints can be defined. A time-course experiment of long-lasting GERD in the rat is presented in chapter 4 with both the histological findings and Cdx2 immunostaining(5). Two types of metaplastic lesions are described: intestinal metaplasia (BE) and multi-layered epithelium (MLE). MLE consists of four to seven layers of cells that appear as basaloid squamous cells in the basal part and columnar cells in the superficial layer. Therefore MLE is a hybrid epithelium in which both squamous and columnar epithelia coexist and is considered a "protometaplasia" (i.e. a precursor of BE). Consistently with its phenotype, MLE expresses markers of both squamous and columnar differentiation6. The presence of MLE has been associated with reflux(1). Cdx2 is a transcription factor that regulates the expression of differentiation-related molecules and it is specifically involved in intestinal cells commitment. The prevalence of Cdx2 expression (i.e. the prevalence of BE and MLE) increases significantly with time in the study, suggesting a time-dependent relationship between the "chemical" injury and the severity of the lesions. De novo Cdx2 expression is shown to be an early event among the morphological changes caused by the refluxate, consistently with the results by Pera and collaborators(7), who described Cdx2 immunostaining in the basal cell layer close to esophageal ulcers 16 weeks after surgery. Chapter 5 provides evidence of both esophagitis cystica profunda, metaplasia and Eac in the model in use. Esophagitis cystica profunda has been defined as a highly differentiated mucinous lesion commonly found in the submucosa at the site of surgical anastomosis(8). This entity has to be considered an inflammatory reaction, caused by the surgical insult. On the other side we present an external validation that both intestinal metaplasia (i.e. BE) and true Eac can be obtained using our surgically-induced reflux model. Chapter 6 is an experimental study on the effect of long-term proton pump inhibitor (PPI) treatment in the rat model of reflux-induced esophageal carcinogenesis. Consistently with the literature, describing PPIs as very effective drugs in ulcer healing, ulcers resulted more severe in the placebo group, compared with the PPI group, in our study. Surprisingly, esophagitis cystica profunda was more common among PPI-treated animals. This mucous-producing lesion has been generally described as well differentiated mucinous adenocarcinoma. However, we consider these tumours as inflammatory reactions, consistently with Ten Kate and collaborators(8). Of note, the misinterpretation of those lesions as adenocarcinomas could lead to the false belief that PPI treatment had increased the incidence of adenocarcinoma in the present study. On the contrary, an effect of the drug on the incidence of carcinomas was not demonstrated by our study. Surgical anti-reflux treatments and acid-suppressors in humans aim primarily to relieve symptoms of GERD. Anti-reflux surgery offers the advantage of reducing both acid and bile reflux, which has been shown to act synergistically in the pathogenesis of Barrett’s esophagus(9). On the other hand, PPIs are acid suppressors. The effect of PPIs in preventing or inducing Eac progression in patients with GERD or BE is controversial. In vivo experimental studies of reflux treated with proton pump inhibitor have not revealed a reduction in adenocarcinoma risk(10-12). However using the esophagoduodenostomy model for esophageal reflux in the rat a recent study comparing refluxates of different pH found that non-acidic refluxate increases the occurrence of intestinal metaplasia with dysplasia and EAC while the low-pH gastric juice exerts a protective effect in the presence of duodenal juice(13). Acid has been recently shown to have antiproliferative effects in nonneoplastic Barrett's epithelial cells cultured in vitro and it has been suggested that the prescription of acid suppressors in dosages beyond those required to control GERD symptoms could be detrimental(14). Gastric acid secretion is a complex, tightly regulated, physiological mechanism, with neural, hormonal, paracrine, and intracellular pathways. Gastrin, histamine, acetylcholine are the major stimuli for acid secretion, that is primarily inhibited by somatostatin, and to a lesser extent by cholecystokinin, atrial natriuretic peptide, and nitric oxide(15). PPIs act on the final common pathway of gastric acid secretion, permanently inactivating the H+/K+ ATPase (proton pump) in the parietal cell. The consequent increase in gastric pH removes the negative feedback for gastrin production by G cells. As a consequence, hypergastrinemia develops in patients with GERD treated with PPIs chronically or life-long. Concerns have been expressed about the potential role of gastrin on esophageal carcinogenesis. In vitro studies suggested that BE is sensitive to the proliferative effects of gastrin via its cholecystokinin-type 2/gastrin receptor (CCK-2R)(16). An antiapoptotic role for gastrin through up-regulation of PKB/Akt in BE samples has been recently suggested and the treatment with a CCK-2R antagonist has been shown to reduce the levels of activated PKB/Akt(17). A better understanding of the effect of pathways regulating gastric secretions could lead to new pharmacological strategies to treat gastroesophageal reflux disease.La malattia da reflusso gastroesofageo (MRGE) è un problema clinico di rilevanza mondiale. Influisce negativamente sulla qualità di vita dei pazienti e predispone alla metaplasia esofagea di tipo intestinale (Esofago di Barrett, EB), che è riconosciuta essere il principale fattore di rischio per lo sviluppo di adenocarcinoma esofageo (ACE). L'incidenza di ACE è aumentata drasticamente negli USA e paesi occidentali dalla metà degli anni Settanta per ragioni sconosciute, mentre la prognosi di ACE rimane infausta. Nello stesso periodo sono stati introdotti efficaci soppressori acidi per il trattamento della MRGE. Allo stato attuale queste terapie guidano la classifica dei farmaci più venduti al mondo con un mercato annuo di 8 miliardi di dollari. Alcuni Autori hanno collegato l'aumento nell'incidenza di ACE con l'ampia diffusione di soppressori acidi nella popolazione generale e tra i pazienti con MRGE(1). Il rischio di ACE nei pazienti con MRGE è troppo basso per giustificare una sorveglianza endoscopica. D'altro canto il follow up endoscopico per i pazienti con EB è generalmente accettato. Tuttora manca evidenza clinica sul miglior trattamento per BE al fine di rendere minimo il rischio di progressione neoplastica(2). Le varie terapie in uso, farmacologiche, chirurgiche ed endoscopiche, non hanno ancora dimostrato una chiara evidenza di beneficio di un trattamento sugli altri (capitolo 1). I modelli chirurgici sperimentali di carcinogenesi esofagea indotta da reflusso possono riprodurre negli animali da laboratorio la progressione a tappe dall'infiammazione all'ACE, attraverso il BE(3,4). Nel capitolo 2 viene fornita una descrizione dettagliata della tecnica microchirurgica in uso per il modello di carcinogenesi esofagea indotta da reflusso, al fine di aumentare la riproducibilità dei dati e minimizzare il numero di animali necessari per il set up sperimentale. Il capitolo 3 riporta i risultati di uno studio sugli effetti della MRGE cronica sul benessere animale. Le principali complicanze a breve e lungo termine vengono analizzate, così come l'importanza e il valore prognostico di due sistemi di valutazione del benessere basati su parametri clinici. Un esperimento time-course di MRGE cronica nel ratto viene presentato nel capitolo 4 con i risultati istologici e immunoistochimici per Cdx2(5)). Vengono descritti 2 tipi di lesioni metaplastiche: la metaplasia intestinale (EB) e il multi-layered epithelium (MLE). MLE consiste di diversi strati di cellule, da 4 a 7, che appaiono squamose basaloidi nella parte basale e colonnari nello strato superficiale. Per questo MLE è un epitelio ibrido nel quale sia l'epitelio squamoso che il colonnare coesistono e viene considerato un precursore di EB. Coerentemente con il proprio fenotipo, MLE esprime marcatori sia di differenziazione squamosa che colonnare(6). La presenza di MLE è stata associata a reflusso(1). Cdx2 è un fattore di trascrizione che regola l'espressione di molecole collegate alla differenziazione ed è coinvolto specificamente nel commitment delle cellule intestinali. La prevalenza dell'espressione di Cdx2 (vale a dire di EB e MLE) aumenta significativamente con il tempo, ad indicare una relazione tempo-dipendente tra l'insulto “chimico” e la gravità delle lesioni. L'espressione di Cdx2 de novo risulta essere un evento precoce nelle modifiche morfologiche secondarie a reflusso, in accordo con i risultati del gruppo di Pera(7), che descrive positività per Cdx2 nello strato di cellule basali in vicinanza di ulcere esofagee già dalla sedicesima settimana dopo l'intervento. Il capitolo 5 dimostra la presenza sia di esophagitis cystica profunda, che di metaplasia e ACE nel modello in uso. L'esophagitis cystica profunda è stata definita come una lesione mucinosa altamente differenziata di comune riscontro a livello dell'anastomosi chirurgica(8). Questa lesione deve essere considerata di natura infiammatoria, secondaria all'atto chirurgico. Il capitolo fornisce un autorevole parere esterno che sia la metaplasia intestinale (EB) che veri ACE possono essere ottenuti utilizzando il nostro modello di reflusso indotto chirurgicamente. Il capitolo 6 è uno studio sperimentale sugli effetti del trattamento con inibitore di pompa protonica (PPI) nel modello sperimentale di carcinogenesi esofagea. Coerentemente con i dati di letteratura, che riconoscono i PPI come farmaci molto efficaci nella guarigione delle ulcere, nel nostro studio la gravità delle ulcere è risultata inferiore nel gruppo trattato con il farmaco rispetto al placebo. Al contrario l'esophagitis cystica profunda è risultata più frequente tra gli animali trattati. L'interpretazione di queste lesioni come carcinomatose ci avrebbe portato a ritenere erroneamente che l'incidenza di cancro fosse più alta tra i trattati, mentre un effetto del farmaco sull'incidenza di carcinomi non è dimostrato nel nostro studio. I trattamenti chirurgici antireflusso e i farmaci soppressori acidi hanno l'indicazione clinica principale di controllare i sintomi nei pazienti con MRGE. La chirurgia antireflusso offre inoltre il vantaggio di ridurre sia il reflusso acido sia quello biliare, che hanno mostrato azione sinergistica nello sviluppo di EB(9). Al contrario i PPI sono soppressori acidi. L'effetto dei PPI nella prevenzione o nell'induzione di ACE nei pazienti con MRGE o EB è controverso. Esperimenti in vivo di reflusso trattato con PPI non hanno rilevato una riduzione nel rischio di adenocarcinoma(10-12). Tuttavia uno studio recente che utilizzava un modello di esofagoduodenostomia nel ratto e confrontava i pH del reflussato ha dimostrato che il reflusso alcalino aumenta il rischio di EB, displasia e EAC, mentre un pH basso esercita un effetto protettivo in presenza di succo duodenale(13). L'acido ha dimostrato avere effetti antiproliferativi in celllule epiteliali di Barrett non neoplastico coltivate in vitro ed è stato suggerito che la prescrizione di soppressori acidi non dovrebbe superare i dosaggi necessari per il controllo dei sintomi di MRGE(14). La secrezione acida gastrica è un meccanismo fisiologico complesso e finemente regolato da vie nervose, ormonali, paracrine e intracellulari. La gastrina, l'istamina e l'acetilcolina costituiscono i maggiori stimoli per la secrezione acida, che viene principalmente inibita dalla somatostatina e in misura minore dalla colecistochinina, dal peptide natriuretico atriale e dall'ossido nitrico(15). I PPI agiscono a livello della tappa finale della secrezione acida gastrica, inattivando la pompa protonica (H+/K+ ATPasi) nella cellula parietale. Di conseguenza, l'aumento del pH intragastrico rimuove il feedback negativo per la produzione di gastrina dalle cellule G. Nei pazienti con MRGE trattati cronicamente con PPI si sviluppa perciò un quadro di ipergastrinemia. Preoccupazione è stata espressa su un potenziale ruolo della gastrina nella carcinogenesi esofagea. Studi in vitro hanno suggerito che EB sia sensibile agli effetti proliferativi della gastrina attraverso il suo recettore CCK-2R(16). Recentemente è stato proposto un ruolo antiapoptotico per la gastrina nell'EB attraverso l'up-regulation di PKB/Akt in BE e il trattamento di campioni di EB con un antagonista per CCK-2R ha dimostrato di ridurre il livello di attivazione di PKB/Akt(17). Una più profonda comprensione degli effetti dei regolatori della secrezione acida potrebbe portare allo sviluppo di nuove strategie farmacologiche per trattare la malattia da reflusso

Neutralizing antibody titers six months after Comirnaty vaccination: kinetics and comparison with SARS-CoV-2 immunoassays

Objectives: mRNA vaccines, including Comirnaty (BNT162b2 mRNA, BioNTech-Pfizer), elicit high IgG and neutralizing antibody (NAb) responses after the second dose, but the progressive decrease in serum antibodies against SARS-CoV-2 following vaccination have raised questions concerning long-term immunity, decreased antibody levels being associated with breakthrough infections after vaccination, prompting the consideration of booster doses. Methods: A total number of 189 Padua University-Hospital healthcare workers (HCW) who had received a second vaccine dose were asked to collect serum samples for determining Ab at 12 (t12) and 28 (t28) days, and 6 months (t6m) after their first Comirnaty/BNT162b2 inoculation. Ab titers were measured with plaque reduction neutralization test (PRNT), and three chemiluminescent immunoassays, targeting the receptor binding domain (RBD), the trimeric Spike protein (trimeric-S), and surrogate viral neutralization tests (sVNT). Results: The median percentages (interquartile range) for decrease in antibodies values 6 months after the first dose were 86.8% (67.1-92.8%) for S-RBD IgG, 82% (58.6-89.3%) for trimeric-S, 70.4% (34.5-86.4%) for VNT-Nab, 75% (50-87.5%) for PRNT50 and 75% (50-93.7%) for PRNT90. At 6 months, neither PRNT titers nor VNT-Nab and S-RBD IgG bAb levels correlated with age (p=0.078) or gender (p=0.938), while they were correlated with previous infection (p<0.001). Conclusions: After 6 months, a method-independent reduction of around 90% in anti-SARS-CoV-2 antibodies was detected, while no significant differences were found between values of males and females aged between 24 and 65 years without compromised health status. Further efforts to improve analytical harmonization and standardization are needed

Host-derived circulating cells generate hybrid cardiomyocytes by cell fusion in heterotopic heart xenotransplantations

The possibility to regenerate dead myocardium by cell therapy using either extra-cardiac or cardiac stem cells is the object of an intense investigation. Whereas much work has been done to assess the potential of injected cells to form new myocardium, little is known about the spontaneous recruitment of stem cells through the circulation. Host-derived cardiomyocytes were found in human sex-mismatched heart transplantations, however the relative proportion of this phenomenon was estimated very high by some authors and negligible by others. In heterotopic heart allo-transplantation, we previously demonstrated that circulating cells engraft the heart transplants but do not significantly contribute to cardiac repair. Possible fusion events, as opposed to transdifferentiation, had been hypothesized but not proved. Here, we took advantage by the use of xeno-transplantations, in which markers of both donor and recipient cells are available, to address this issue. Xeno-transplantations were performed using GFP transgenic rats as hosts, and either Syrian hamsters (n8) or transgenic mice expressing the lacZ reporter gene under the control of the cardiac troponin I promoter (n6), as heart donors. All transplants, which were retrieved 15 days after surgery, contained a large quantity of GFP inflammatory cells. In 7 of 8 hamster-to-rat heart transplants GFP mature cardiomycytes were found, with percentages ranging from 0.0001% to 0.034%. No more than 15 GFP cardiomyocytes were detected in all mouse-to-rat transplants. We also found rare small GFP cells expressing markers of cardiac progenitor cells, such as GATA-4 and MEF2C, in all xenografts. All GFP cardiomyocytes identified in our study co-expressed GFP (the host\u2019s marker) and either hamster-specific antigens or the LacZ marker of mouse origin (the donor\u2019s markers). Thus, using both an immunological and a genetic approach, we conclusively demonstrate that in our experimental model circulating cells do not significantly contribute to form new myocardium, rather they generate hybrid cardiomyocytes by cell fusion

Heparanase regulates the M1 polarization of renal macrophages and their crosstalk with renal epithelial tubular cells after ischemia/reperfusion injury

Heparanase (HPSE) is part of the biologic network triggered by ischemia/reperfusion (I/R) injury, a complication of renal transplantation and acute kidney injury. During this period, the kidney or graft undergoes a process of macrophages recruitment and activation. HPSE may therefore control these biologic effects. We measured the ability of HPSE and its inhibitor, SST0001, to regulate macrophage polarization and the crosstalk between macrophages and HK-2 renal tubular cells during in vitro hypoxia/reoxygenation (H/R). Furthermore, we evaluated in vivo renal inflammation, macrophage polarization, and histologic changes in mice subjected to monolateral I/R and treated with SST0001 for 2 or 7 d. The in vitro experiments showed that HPSE sustained M1 macrophage polarization and modulated apoptosis, the release of damage associated molecular patterns in post-H/R tubular cells, the synthesis of proinflammatory cytokines, and the up-regulation of TLRs on both epithelial cells and macrophages. HPSE also regulated M1 polarization induced by H/R-injured tubular cells and the partial epithelial-mesenchymal transition of these epithelial cells by M1 macrophages. All these effects were prevented by inhibiting HPSE. Furthermore, the inhibition of HPSE in vivo reduced inflammation and M1 polarization in mice undergoing I/R injury, partially restored renal function and normal histology, and reduced apoptosis. These results show for the first time that HPSE regulates macrophage polarization as well as renal damage and repair after I/R. HPSE inhibitors could therefore provide a new pharmacologic approach to minimize acute kidney injury and to prevent the chronic profibrotic damages induced by I/R.-Masola, V., Zaza, G., Bellin, G., Dall'Olmo, L., Granata, S., Vischini, G., Secchi, M. F., Lupo, A., Gambaro, G., Onisto, M. Heparanase regulates the M1 polarization of renal macrophages and their crosstalk with renal epithelial tubular cells after ischemia/reperfusion injury

Stem cell engraftment in a heterotopic cardiac hamster-to-rat xenotransplantation model

Introduction: The hamster-to-rat species combination is considered a concordant difficult xenotransplantation model that resembles the immunological situation observed when transplanting hDAF pig organs into non-human primates. The aim of the present study was to investigate in a hamster-to-rat cardiac model the capacity of progenitor stem cells to engraft the transplanted heart and transdifferentiate into cardiomyocytes. Material and methods: A heterotopic cardiac transplantation procedure was performed between Golden Syrian hamsters (Harlan Italia) which served as organ donors and green fluorescent protein (GFP) transgenic Sprague\u2013 Dawley rat (Japan SLC) recipients. Recipients underwent splenectomy during the transplantation procedure and were treated by intramuscular cyclosporine A (Novartis) at a dose of 15 mg/kg/day throughout the postoperative period. As a control, a group of GFP transgenic recipients underwent allogeneic heterotopic heart transplantation and immunosuppression by the administration of cyclosporine A. The transplanted hearts were harvested on postoperative day 15 and analyzed histologically for the presence of GFP positive cells. The GFP positive cells were characterized for specific muscle differentiation markers and analyzed by immunofluoresence and confocal microscopy. Results: A high number of GFP positive cells were found in the xenotransplanted hamster hearts, however the majority were inflammatory in nature. In four out of six (67%) cases analyzed, a limited number of GFP positive cardiac cells with shape and morphology compatible with that of mature cardiomyocytes were found. It is noteworthy that these cells expressed cardiomyocyte-specific markers such as desmin and myosin heavy chain. In the allotransplanted group similar GFP positive cardiac cells were observed in 10 out of 12 (83%) cases. Conclusions: The heterotopic cardiac hamster-to-rat xenotransplantation model is an appropriate animal model to study stem cell engraftment in damaged hearts after a transplant procedure between different species. Our preliminary data also suggest that also a xenogeneic environment may provide adequate mediators and cell signals to allow donor stem cell engraftment and differentiation into cardiomyocytes. Fusion vs. transdifferentiation events are currently under examination

Hybrid cardiomyocytes derived by cell fusion in heterotopic cardiac xenografts

Cardiomyocytes expressing host markers, such as the Y chromosome in sex-mismatched transplants, have been described in human allografts, suggesting that circulating cells can contribute to cardiac regeneration. It has not been established, however, whether host-derived cardiomyocytes result from transdifferentiation of stem cells or cell fusion. To address this issue, we used heterotopic heart xenografts and looked for markers of donor and recipient cells. Golden Syrian hamsters or transgenic mice expressing nuclear beta-galactosidase under the control of the cardiac troponin I promoter served as organ donors, while GFP+ transgenic rats were used as recipients. GFP+ cells, including abundant CD-45+ inflammatory cells and rare undifferentiated cells expressing early cardiac markers (GATA-4 or MEF2C), were found in xenografts harvested two weeks after surgery. In addition, rare GFP+ mature cardiomyocytes were found in 7 of 8 hamster xenografts and 6 of 6 mouse xenografts. The proportion of these cells was very low (0.0001% to 0.0344% in hamster xenografts) but similar to the one observed in control rat heart allografts. Without exception, all GFP+ cardiomyocytes also expressed donor markers, i.e., hamster membrane antigens or lacZ, so they must derive from cell fusion, not transdifferentiation

Transcribed ultraconserved noncoding RNAs (T-UCR) are involved in Barrett's esophagus carcinogenesis.

Barrett's esophagus (BE) involves a metaplastic replacement of native esophageal squamous epithelium (Sq) by columnar-intestinalized mucosa, and it is the main risk factor for Barrett-related adenocarcinoma (BAc). Ultra-conserved regions (UCRs) are a class non-coding sequences that are conserved in humans, mice and rats. More than 90% of UCRs are transcribed (T-UCRs) in normal tissues, and are altered at transcriptional level in tumorigenesis. To identify the T-UCR profiles that are dysregulated in Barrett's mucosa transformation, microarray analysis was performed on a discovery set of 51 macro-dissected samples obtained from 14 long-segment BE patients. Results were validated in an independent series of esophageal biopsy/surgery specimens and in two murine models of Barrett's esophagus (i.e. esophagogastric-duodenal anastomosis). Progression from normal to BE to adenocarcinoma was each associated with specific and mutually exclusive T-UCR signatures that included up-regulation of uc.58-, uc.202-, uc.207-, and uc.223- and down-regulation of uc.214+. A 9 T-UCR signature characterized BE versus Sq (with the down-regulation of uc.161-, uc.165-, and uc.327-, and the up-regulation of uc.153-, uc.158-, uc.206-, uc.274-, uc.472-, and uc.473-). Analogous BE-specific T-UCR profiles were shared by human and murine lesions. This study is the first demonstration of a role for T-UCRs in the transformation of Barrett's muco

Heterotopic cardiac xenotransplantation in rodents: Report of a refined technique in a hamster-to-rat model.

Hamster-to-rat heterotopic cardiac xenotransplantation is widely used as an experimental model to study xenograft rejection, accommodation, and tolerance, as well as in studies aimed at developing immunosuppressive strategies in xenotransplantation. Despite its widespread application, no detailed description of a surgical technique for this model has been provided in the literature. Indeed, all publications so far on the use of this species combination refer to the rat allotransplantation technique. Hence the present paper provides a detailed, up-to-date description of the surgical method adopted at our center for the hamster-to-rat heterotopic cardiac xenotransplantation model. Considerable effort went into developing a reliable, reproducible experimental model in rodents, and the description given here is enriched with "tips" that we learned in the process. The discussion of the technique also addresses several significant related issues, e.g., the anesthesia and organ preservation solution used (aspects that, in our experience, are crucial to a good surgical outcome)