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Amniotic Fluid Stem Cells Improve Survival And Enhance Repair Of Damaged Intestine In Experimental Necrotizing Enterocolitis Via A Cox-2 Dependent Mechanism
Get PDFBackground. Necrotizing enterocolitis (NEC) is a major cause of morbidity and death in neonates. No specific therapy is available and the treatment is only supportive. Amniotic Fluid Stem (AFS) cells represent a novel class of pluripotent stem cells with intermediate characteristics between embryonic and adult stem cells, as they are able to differentiate into lineages representative of all embryonic germ layers and do not form tumors after implantation in vivo. These characteristics, together with the absence of ethical issues concerning their obtainment, make AFS cells good candidates for cell therapy of human diseases.
Aim. The aim of this study was to explore the therapeutic potential of Amniotic Fluid Stem (AFS) cells in a rat model of NEC. Methods. AFS cells were obtained from green fluorescent (GFP+) transgenic pregnant rats at 16 days p.c. by c-kit selection. NEC was induced in newborn rats by hyperosmolar milk formula, oral lipopolysaccharide and hypoxia. Rats were divided into 2 groups receiving at 24 and 48 hours of life an intraperitoneal injection of: (i) phosphate buffered saline (PBS; n=120) or (ii) 2x106 AFS cells (n= 121). Additional groups of animals, either injected with bone marrow-derived mesenchymal stem cells (i.e. rat BM-MSCs) or committed cells (i.e. rat myoblasts), or non subjected to NEC induction (i.e. healthy breast fed newborn rats), were used as additional controls. All groups were blindly compared regarding survival, clinical status, radiological features (abdominal MRI), gut motility (carmine red transit time) and intestinal permeability (plasma lactulose/mannitol ratio). Intestines were blindly analyzed for macro- and microscopic appearance, transcriptional profile (microarray-based expression analysis), neutrophil infiltration (myeloperoxidase activity), enterocyte proliferation (EdU assay) and apoptosis (cleaved caspase 3 immunohistochemistry). AFS cell integration in the gut was evaluated by GFP amplification and immunostaining. Cyclooxygenase 2 (COX2+) cells in the lamina propria were evaluated by immunofluorescence. COX2 activity was inhibited in vivo using selective (celecoxib) and non-selective (ibuprofen) inhibitors; the effects of COX2 pharmacological inhibition on rat survival and clinical status were evaluated.
Results. Compared to animals injected with PBS, rats receiving AFS cells survived longer (p<0.0001), and showed: improved clinical conditions (p<0.001), better abdominal appearance at MRI, restored intestinal transit (p<0.01), decreased intestinal permeability (p<0.05), reduced macroscopical (p<0.001) and histological gut damage (p<0.001). These beneficial effects were specific to AFS cells as neither BM-MSCs nor myoblasts were able to improve animal morbidity/mortality in comparison to PBS (p=n.s.). AFS cells integrated in the intestine with various degrees of spreading in all the animals. cDNA arrays comparing the intestinal transcriptional profile of PBS vs. AFS cell rats showed differences in the expression of genes involved in inflammation, apoptosis and cell proliferation which were respectively down-regulated (inflammation and apoptosis) and up-regulated (proliferation) in the AFS cell group. At a protein level, AFS cell rats had lower gut neutrophil infiltration (p<0.05), reduced enterocyte apoptosis (p<0.05) and increased enterocyte proliferation (p<0.0001) compared to PBS rats. In rats treated with AFS cells vs. rats injected with PBS, COX2+ cells in the lamina propria were increased (p<0.001) and repositioned under crypts (p<0.001). Moreover, both the total number of COX-2+ cells per villus unit and the number of cryptal COX-2+ cells inversely correlated with the degree of intestinal damage (p=0.014). The pharmacological inhibition of COX2 activity did not exert any effect in PBS rats, whereas it completely abolished AFS cell beneficial effects on animal survival and clinical behavior.
Conclusions. In experimental NEC, AFS cell administration via the intraperitoneal route is associated with reduced animal morbidity/mortality and decreased incidence of NEC. AFS cell beneficial effects seems to be related to decreased intestinal neutrophil infiltration, enhanced enterocyte proliferation and reduced epithelial apoptosis. We hypothesize that is achieved through activation of COX2+ cells in the lamina propria. Stem cell therapy may represent a new therapeutic option for infants with NEC.Premesse. L’enterocolite necrotizzante (NEC) rappresenta la causa più frequente di insufficienza intestinale in età pediatrica. Non esistono tuttora terapie specifiche per la NEC ed il suo trattamento si basa unicamente sulla terapia medica di supporto e sulla rimozione chirurgica delle porzioni di intestino affetto. Le cellule staminali derivanti da liquido amniotico (AFSC) sono una popolazione di cellule staminali di origine fetale descritta per la prima volta nel 2007. Esse possiedono delle caratteristiche intermedie fra le cellule staminali embrionali (i.e. pluripotenza) e le cellule staminali adulte (i.e. mancata tumorigenicità dopo iniezione in vivo) che le rendono candidati ideali per la terapia cellulare.
Scopo dello studio. Valutare il potenziale terapeutico delle cellule staminali derivanti da liquido amniotico (AFSC) in un modello animale di NEC. Materiali e metodi. AFSC sono state derivate da ratti Sprague-Dawley GFP+ (i.e. esprimenti in modo costitutivo la proteina reporter “Green Fluorescent Protein”) al 16^ giorno p.c. tramite immunoselezione per il loro caratterstico marcatore di superficie (i.e. c-kit/CD117). Le cellule ottenute sono state caratterizzate per morfologia e immunofenotipo. La NEC e’ stata indotta in ratti neonati tramite l’utilizzo di elementi simili ai fattori patogenetici implicati nell’insorgenza della NEC umana: alimentazione con latte formulato iperosmolare, eventi ipossici, somministrazione di lipopolisaccaride. I ratti, suddivisi in due gruppi principali, hanno ricevuto a 24 e 48 ore di vita, per via intraperitoneale: i. 50 ul di soluzione salina (PBS; n=120) o ii. 2x106 AFSC (n= 121). Altri gruppi di animali, trattati con cellule staminali mesenchimali di ratto derivanti da midollo osseo o con mioblasti, oppure non sottoposti all’induzione di NEC (i.e. neonati sani allattati al seno), sono stati utilizzati come gruppi aggiuntivi di controllo. I diversi gruppi di animali sono stati valutati in cieco per i seguenti parametri: sopravvivenza, stato clinico, aspetto radiologico intestinale (RM ad alta risoluzione), motilita’ intestinale (studio del tempo di transito con coloranti vitali), permeabilita’ intestinale (rapporto lattulosio/mannitolo plasmatici). L’intestino e’ stato valutato in cieco per: aspetto macroscopico ed istologico, profilo di espressione genica (tramite tecnologia cDNA-microarray), infiltrazione neutrofila (saggio di attivita’ della mieloperossidasi), proliferazione (EdU) e apoptosi degli enterociti (immunoistochimica per caspasi 3 attivata). L’integrazione di AFSC nell’intestino e’ stata analizzata sia tramite PCR (amplificazione del gene gfp) che tramite immunoistochimica (immunofluorescenza per GFP). Il numero e la localizzazione delle cellule stromali esprimenti COX2 nella mucosa sono stati valutati con immunofluorescenza. L’attivita’ di COX2, in vivo, e’ stata inibita farmacologicamente con inibitori selettivi (celecoxib) e non selettivi di COX2 (ibuprofene); gli effetti di tale inibizione sulla sopravvivenza e sulla morbidita’ degli animali trattai con AFSC o PBS sono stati analizzati in cieco.
Risultati. La somministrazione di AFSC, per via intraperitoneale a ratti neonati affetti da NEC: migliora significativamente la sopravvivenza degli animali sia rispetto alla somministrazione di PBS (p<0.0001) che di linee cellulari di controllo (i.e. cellule staminali mesenchimali di ratto derivanti da midollo osseo [p=0.024] e mioblasti di ratto [p<0.0001]). Rispetto alla somministrazione di PBS, inoltre, il trattamento con AFSC: i. riduce la morbidita’ degli animali migliorandone l’aspetto clinico (p<0.001); ii. riduce significativamente il danno intestinale sia alla valutazione dell’addome con RM ad alta risoluzione che all’esame macroscopico (p<0.001) ed istologico dell’intestino (p<0.001); iii. migliora significativamente la funzionalità dell’intestino sia per quanto concerne la motilità (p<0.01) che l’assorbimento di nutrienti (p<0.05). AFSC somministrate per via intraperitoneale migrano preferenzialmente verso l’intestino dove, seppur con un basso tasso di integrazione tissutale, sono in grado di localizzarsi in tutti gli strati della parete e talora di differenziarsi in cellule con fenotipo mesenchimale (i.e. cellule muscolari lisce). La somministrazione di AFSC in ratti neonati affetti da NEC è in grado di modificare il profilo di espressione genica dell’intestino incrementando l’espressione di geni coinvolti nella proliferazione e riducendo l’espressione di geni coinvolti in apoptosi e infiammazione. Tali dati di espressione sono stati confermati a livello proteico dimostrando che nell’intestino dei ratti affetti da NEC trattati con AFSC v.s. PBS è maggiore la proliferazione delle cellule epiteliali (p<0.0001), minore l’apoptosi degli enterociti (p<0.05) e ridotta l’infiltrazione neutrofila tissutale (p<0.05). La somministrazione di AFSC, inoltre, determina l’attivazione di una popolazione di cellule stromali esprimenti la ciclossigenasi 2 (COX2) nella lamina propria della mucosa intestinale. Più in dettaglio la somministrazione di AFSC v.s. PBS causa un significativo aumento del numero delle cellule COX2+ nella lamina propria (p<0.001) e un loro spostamento dall’asse del villo alla niche delle cripte intestinali (p<0.001). Tale effetto costituisce il meccanismo d’azione di AFSC poiché la somministrazione in vivo di inibitori selettivi e non selettivi di COX2 (ma non di COX1) a ratti affetti da NEC abolisce gli effetti positivi di AFSC su morbidità e mortalità degli animali ma non ha alcun effetto sugli animali trattati con PBS.
Conclusioni. In un modello animale di NEC, AFSC sono in grado di migliorare in modo significativo la mortalita’ e la morbidita’ degli animali e il danno intestinale. AFSC non determinano direttamente tali effetti rigenerando di per sé l’intestino ma indirettamente attivando le cellule stromali esprimenti COX2 presenti nella lamina propria le quali a loro volta stimolano la proliferazione e riducono l’apoptosi delle cellule epiteliali intestinali residenti. Sebbene ulteriori studi siano necessari (e.g. per identificare i fattori/meccanismi molecolari responsabili dell’attivazione delle cellule COX2+), riteniamo che la terapia con cellule staminali derivanti da liquido amniotico possa rappresentare una nuova prospettiva terapeutica per i pazienti affetti da NEC
NAFLD and Liver Transplantation in Children - Working Group Report from the ILTS Single Topic Conference on NAFLD
No full textBackground Nonalcoholic fatty liver disease (NAFLD) has now become a common cause of chronic liver disease in children; however, unlike adults liver transplantation is rarely required as treatment. It is important that in children presenting with NAFLD, secondary causes of fatty liver particularly inherited metabolic defects should be excluded. Methods A pediatric working group comprised of 3 hepatologists and a liver transplant surgeon was tasked with a set of questions to address the current state of evidence and knowledge about NAFLD in children with particular focus on liver transplantation. A systematic review of the English literature regarding pediatric NAFLD (from birth to 18 years of age) published in the last 2 decades (2000-2018) was carried out. The evidence was evaluated by the subgroup members and further discussed with the wider workshop faculty leading to the recommendations for best practice. Results Given the paucity of literature on the subject good quality of evidence was only available on risk factors for NAFLD and medical treatment where the group could make recommendation with high/moderate strength. The evidence on natural history and indications for liver transplantation was poor hence group could not make any recommendations. Conclusions Based on the existing literature and subgroups, collective experience NAFLD unlike in adults is a very rare indication for liver transplantation in children. No definitive recommendations could be made about the natural history, indications, and outcome of liver transplantation for NAFLD in children
Educazione alla salute in giovani pazienti sottoposti a trapianto di fegato. Progetto pilota di strutturazione e implementazione di strumenti di dialogo educativo condotto presso l’Azienda Ospedaliera di Padova
No full textIl presente progetto pilota è finalizzato alla creazione e attuazione di interventi di educazione alla salute (mediati dall’uso di strumenti dialogici originali) in un gruppo di 14 pazienti sottoposti a trapianto di fegato in età infantile. I pazienti, in una fascia di età compresa tra tarda adolescenza e giovane età adulta, sono parimenti coinvolti in un processo di transizione dal servizio pediatrico al servizio per adulti. Ciascuna persona è stata coinvolta nello studio per 10 mesi; ogni paziente è stato allocato mediante randomizzazione nel gruppo sperimentale (partecipa agli incontri) o di controllo (non partecipa). Sono stati utilizzati metodi di ricerca misti: i) analisi statistica dell’efficacia degli interventi (somministrazione pre e post di una batteria di 5 questionari); ii) analisi qualitativa di interviste semi-strutturate (condotte con l’intero gruppo) e dei contenuti emersi nel corso degli incontri (con gruppo sperimentale). Lo studio, iniziato a luglio 2015, si è concluso a ottobre 2016
Progetto pilota di strutturazione e implementazione di interventi di cura ed educazione sanitaria ad hoc per giovani ragazzi trapiantati di fegato
No full textAssessing pediatric ileocolonic Crohn's disease activity based on global MR enterography scores
Get PDFObjectives This study was aimed at correlating a magnetic
resonance index of activity (MaRIA) and a magnetic resonance
enterography global score (MEGS) with activity indexes
in a paediatric population with Crohn’s disease (CD).
Methods This retrospective study included 32 paediatric patients
(median age 14.5 years, 18 male) with proven CD who
underwent magnetic resonance enterography (MRE). A correlation
analysis was performed on the MRE-based scores, the
simplified endoscopic score for CD (SES-CD), the paediatric
Crohn’s disease activity index (PCDAI), and C-reactive protein
(CRP) levels. Based on PCDAI, comparison of both global
MaRIA and MEGS was made between patients with mild
and moderate/severe disease activity.
Results Global MaRIA correlated with SES-CD (r = 0.70,
p =0.001) and PCDAI (r=0.42, p =0.016). MEGS correlated
with PCDAI (r = 0.46, p = 0.007) and CRP levels (r = 0.35,
p =0.046). MEGS differed significantly (p=0.027) between
patients grouped by clinical disease severity.
Conclusions MRE-based global scores correlated with clinical
indexes of CD activity. Therefore, they represent a potential useful tool to predict CD activity and severity, as
well as a possible promising alternative to endoscopy, to monitor
paediatric patients with CD during their follow-up
"Rendezvous" Procedure in Children with Cholecysto-Choledocholithiasis
No full textIntroduction: Cholecysto-choledocolithiasis is a rare entity in children and its management is still challenging and controversial. The laparoendoscopic rendezvous (LERV) procedure, consisting of laparoscopic cholecystectomy and simultaneous endoscopic retrograde cholangiopancreatography for the management of symptomatic choledocholithiasis is well described in adult patients. However, in the literature, few reports about its application in the pediatric population have been recorded. Aim of the Study: The aim of the present study is to report our first successful cases of symptomatic cholecysto-choledocholithiasis LERV treatment. Methods: Two girls suffering of hemolytic disease presented to our third referral center with acute abdominal pain due to cholecysto-choledocholithiasis. Preoperative, perioperative, and postoperative data were retrospectively reviewed. Results: Surgery was performed without complications. The girls were dismissed once re-alimentation and re-canalization were achieved and had no other episodes of biliary cholic. Reported advantages of LERV include: a shorter in-hospital stay, a reduction in the number of procedures and anesthesia, and a reduced overall risk of complications. Conclusions: The promising result with our 2 cases suggest that, when performed in highly specialized centers, LERV is a safe procedure, which leads to considerable benefits, despite logistic and organizational difficulties
High level of oxysterols in neonatal cholestasis: A pitfall in analysis of biochemical markers for Niemann-Pick type C disease
No full textBackground: Niemann-Pick disease type C (NPC) is a rare lipid storage disorder characterized by progressive neurological deterioration. Diagnosing NPC is challenging as clinical signs and symptoms are variable and non-specific. Two oxysterols, cholestane-3 beta,5 alpha,6 beta-triol (triol) and 7-ketocholesterol (7KC), have been proposed as biomarkers for aiding diagnosis of NPC. This study evaluated the use of triol and 7KC as biomarkers in cholestatic neonates with suspected NPC.
Methods: Plasma triol and 7KC were analysed as - dimethylglycine esters using an liquid chromato-graphy tandem mass spectrometry (LC-MS/MS) assay in selected neonates with severe cholestasis and suspected NPC (n=7), adults with cholestasis (n=15), patients with confirmed NPC (positive controls; n=11 [one child and 10 adults]), healthy subjects (negative controls; n=40 [20 children and 20 adults]), and cholestatic adults (comparative reference; n=15). The LC-MS/MS method was subjected to a number of tests for accuracy and consistency.
Results: Triol and 7KC levels were substantially and significantly increased in NPC positive patients compared with healthy controls (p<0.001). However, positive results (markedly increased levels of both oxysterols) were identified in 6/7 (86%) neonates with cholestasis. Genetic testing confirmed NPC only in one neonate who had increased triol and 7KC, and increased oxysterol levels among neonates with no identified NPC gene mutations were considered likely due to biliary atresia (BA).
Conclusions: While the potential of oxysterols as NPC biomarkers has been well evaluated in older patient populations (without cholestasis), our data suggest that cholestasis might represent a pitfall in oxysterol measurements intended to aid diagnosis of NPC in affected patients
Disseminated Mycobacterial Infection With Reactive Polyarthritis (Poncet’s Disease) During Immune-suppressive Treatment Including Ustekinumab for Pediatric Crohn’s Disease
No full textBackground: The incidence of pediatric inflammatory bowel disease is increasing. tumor necrosis factor alpha inhibitors medicines improved the prognosis of affected subjects. Nonetheless, a proportion of patients do not respond or lose response to treatment. Newer biologics, like ustekinumab, have been approved for adults. The pediatric off-label use of these drugs is increasing, despite limited safety evidence. We report a case of disseminated mycobacterial infection (MI) presenting with reactive polyarthritis (Poncet's disease, PD) in a girl with Crohn's disease receiving various immunosuppressants, including ustekinumab. Case report: A 12-year-old girl with Crohn's disease was admitted for acute-onset migratory polyarthritis of large and small joints and opioid-resistant pain. She had recently received adalimumab and methotrexate and was currently under treatment with ustekinumab. She was vaccinated with Bacillus Calmette-Guérin and screened for tuberculosis before starting immunosuppressants. Interferon-gamma release assay, Mantoux test and chest computed tomography scan were negative. Disseminated MI with PD was diagnosed following positive cultures for Mycobacterium tuberculosis complex in blood and intestinal biopsies (with negative in synovial fluid and gastric aspirate). Whole-exome sequencing did not identify any genetic susceptibility to MI. Antituberculosis treatment eradicated MI. Conclusions: Children with inflammatory bowel disease receiving combination immunosuppressive treatments including tumor necrosis factor alpha inhibitors and anti-IL-12/23 agents are at higher risk for MI. Disseminated MI should be considered and ruled out in these patients when presenting with pulmonary, extrapulmonary or unusual clinical manifestations, like PD. The collection of multiple specimens (including intestinal biopsies) for mycobacterial culture is recommended when mycobacterial disease is suspected
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