Conservative treatment for cystic duct stenosis in a child.

Introduction. Few cases of common bile duct stenosis have been reported in the literature, and observations of strictures in the cystic duct are even more rare. Surgical cholecystectomy is the treatment needed in most cases of gallbladder hydrops. This paper describes the diagnosis and successful medical treatment of a rare pediatric case of cystic duct stenosis and gallbladder hydrops. Case Report. A formerly healthy one-year-old girl was admitted with colicky abdominal pain. Blood tests were normal, except for an increase in transaminases. Abdominal ultrasound excluded intestinal intussusception and identified a distended gallbladder with biliary sludge. MR cholangiography revealed a dilated gallbladder containing bile sediment and no detectable cystic duct, while the rest of the intra- and extrahepatic biliary tree and hepatic parenchyma were normal. This evidence was consistent with gallbladder hydrops associated with cystic duct stenosis. The baby was treated with i.v. hydration, corticosteroids, antibiotics, and ursodeoxycholic acid. Her general condition rapidly improved, with no further episodes of abdominal pain and normalization of liver enzymes. This allowed to avoid cholecystectomy, and the child is well 1.5 years after diagnosis. Conclusions. Although cholecystectomy is usually necessary in case of gallbladder hydrops, our experience suggests that surgical procedures can be avoided when the distension is caused by a cystic duct stenosis

Autologous transplantation of amniotic fluid-derived mesenchymal stem cells into sheep fetuses

Long-term engraftment and phenotype correction has been difficult to achieve in humans after in utero stem cell transplantation mainly because of allogeneic rejection. Autologous cells could be obtained during gestation from the amniotic fluid with minimal risk for the fetus and the mother. Using a sheep model, we explored the possibility of using amniotic fluid mesenchymal stem cells (AFMSCs) for autologous in utero stem cell/gene therapy. We collected amniotic fluid (AF) under ultrasound-guided amniocentesis in early gestation pregnant sheep (n = 9, 58 days of gestation, term = 145 days). AFMSCs were isolated and expanded in all sampled fetal sheep. Those cells were transduced using an HIV vector encoding enhanced green fluorescent protein (GFP) with 63.2% (range 38.3-96.2%) transduction efficiency rate. After expansion, transduced AFMSCs were injected into the peritoneal cavity of each donor fetal sheep at 76 days under ultrasound guidance. One ewe miscarried twin fetuses after amniocentesis. Intraperitoneal injection was successful in the remaining 7 fetal sheep giving a 78% survival for the full procedure. Tissues were sampled at postmortem examination 2 weeks later. PCR analysis detected GFP-positive cells in fetal tissues including liver, heart, placenta, membrane, umbilical cord, adrenal gland, and muscle. GFP protein was detected in these tissues by Western blotting and further confirmed by cytofluorimetric and immunofluorescence analyses. This is the first demonstration of autologous stem cell transplantation in the fetus using AFMSCs. Autologous cells derived from AF showed widespread organ migration and could offer an alternative way to ameliorate prenatal congenital diseas

Novel CARMIL2 loss-of-function variants are associated with pediatric inflammatory bowel disease

CARMIL2 is required for CD28-mediated co-stimulation of NF-kappa B signaling in T cells and its deficiency has been associated with primary immunodeficiency and, recently, very early onset inflammatory bowel disease (IBD). Here we describe the identification of novel biallelic CARMIL2 variants in three patients presenting with pediatric-onset IBD and in one with autoimmune polyendocrine syndrome (APS). None manifested overt clinical signs of immunodeficiency before their diagnosis. The first patient presented with very early onset IBD. His brother was found homozygous for the same CARMIL2 null variant and diagnosed with APS. Two other IBD patients were found homozygous for a nonsense and a missense CARMIL2 variant, respectively, and they both experienced a complicated postoperative course marked by severe infections. Immunostaining of bowel biopsies showed reduced CARMIL2 expression in all the three patients with IBD. Western blot and immunofluorescence of transfected cells revealed an altered expression pattern of the missense variant. Our work expands the genotypic and phenotypic spectrum of CARMIL2 deficiency, which can present with either IBD or APS, aside from classic immunodeficiency manifestations. CARMIL2 should be included in the diagnostic work-up of patients with suspected monogenic IBD

Enteric Neurospheres Are Not Specific to Neural Crest Cultures: Implications for Neural Stem Cell Therapies

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited

Incidence of Isolated Biliary Atresia during the COVID Lockdown in Europe: Results from a Collaborative Project by RARE-Liver

Background: Biliary atresia (BA) is a rare cholangiopathy where one of the proposed aetiological mechanisms is an infectious viral trigger. Coronavirus disease-19 (COVID) lockdown restrictions were implemented to reduce the transmission of infections. Strictness of lockdown varied across European countries. This study aimed to investigate if there was an association between strictness of lockdown and change in isolated BA (IBA) incidence in Europe. Methods: We approached European centres involved in the European Reference Network RARE-LIVER. We included IBA patients born between 2015 and June 2020. We calculated the number of IBA patients born per centre per month. The Stringency Index (SI) was used as lockdown strictness indicator. The association between percentage change of mean number of IBA patients born per month and the SI was assessed. Results: We included 412 IBA patients from thirteen different centres. The median number of patients per month did not change: 6 (1–15) pre-lockdown and 7 (6–9) during lockdown (p = 0.34). There was an inverse association between SI and percentage change in IBA (B = -0.73, p = 0.03). Median age at Kasai portoenterostomy (days) did not differ between time periods (51 (9–179) vs. 53 (19–126), p = 0.73). Conclusion: In this European study, a stricter COVID-lockdown was seemingly accompanied by a simultaneous larger decrease in the number of IBA patients born per month in the lockdown. Results should be interpreted with caution due to the assumptions and limitations of the analysis

Genotype-phenotype relationships of truncating mutations, p.E297G and p.D482G in bile salt export pump deficiency

Background &amp; Aims: Bile salt export pump (BSEP) deficiency frequently necessitates liver transplantation in childhood. In contrast to two predicted protein truncating mutations (PPTMs), homozygous p.D482G or p.E297G mutations are associated with relatively mild phenotypes, responsive to surgical interruption of the enterohepatic circulation (siEHC). The phenotype of patients with a compound heterozygous genotype of one p.D482G or p.E297G mutation and one PPTM has remained unclear. We aimed to assess their genotype-phenotype relationship. Methods: From the NAPPED database, we selected patients with homozygous p.D482G or p.E297G mutations (BSEP1/1; n = 31), with one p.D482G or p.E297G, and one PPTM (BSEP1/3; n = 30), and with two PPTMs (BSEP3/3; n = 77). We compared clinical presentation, native liver survival (NLS), and the effect of siEHC on NLS. Results: The groups had a similar median age at presentation (0.7-1.3 years). Overall NLS at age 10 years was 21% in BSEP1/3 vs. 75% in BSEP1/1 and 23% in BSEP3/3 (p &lt;0.001). Without siEHC, NLS in the BSEP1/3 group was similar to that in BSEP3/3, but considerably lower than in BSEP1/1 (at age 10 years: 38%, 30%, and 71%, respectively; p = 0.003). After siEHC, BSEP1/3 and BSEP3/3 were associated with similarly low NLS, while NLS was much higher in BSEP1/1 (10 years after siEHC, 27%, 14%, and 92%, respectively; p &lt;0.001). Conclusions: Individuals with BSEP deficiency with one p.E297G or p.D482G mutation and one PPTM have a similarly severe disease course and low responsiveness to siEHC as those with two PPTMs. This identifies a considerable subgroup of patients who are unlikely to benefit from interruption of the enterohepatic circulation by either surgical or ileal bile acid transporter inhibitor treatment. Impact and implications: This manuscript defines the clinical features and prognosis of individuals with BSEP deficiency involving the combination of one relatively mild and one very severe BSEP deficiency mutation. Until now, it had always been assumed that the mild mutation would be enough to ensure a relatively good prognosis. However, our manuscript shows that the prognosis of these patients is just as poor as that of patients with two severe mutations. They do not respond to biliary diversion surgery and will likely not respond to the new IBAT (ileal bile acid transporter) inhibitors, which have recently been approved for use in BSEP deficiency.</p

Clonal Characterization of Rat Muscle Satellite Cells: Proliferation, Metabolism and Differentiation Define an Intrinsic Heterogeneity

Satellite cells (SCs) represent a distinct lineage of myogenic progenitors responsible for the postnatal growth, repair and maintenance of skeletal muscle. Distinguished on the basis of their unique position in mature skeletal muscle, SCs were considered unipotent stem cells with the ability of generating a unique specialized phenotype. Subsequently, it was demonstrated in mice that opposite differentiation towards osteogenic and adipogenic pathways was also possible. Even though the pool of SCs is accepted as the major, and possibly the only, source of myonuclei in postnatal muscle, it is likely that SCs are not all multipotent stem cells and evidences for diversities within the myogenic compartment have been described both in vitro and in vivo. Here, by isolating single fibers from rat flexor digitorum brevis (FDB) muscle we were able to identify and clonally characterize two main subpopulations of SCs: the low proliferative clones (LPC) present in major proportion (∼75%) and the high proliferative clones (HPC), present instead in minor amount (∼25%). LPC spontaneously generate myotubes whilst HPC differentiate into adipocytes even though they may skip the adipogenic program if co-cultured with LPC. LPC and HPC differ also for mitochondrial membrane potential (ΔΨm), ATP balance and Reactive Oxygen Species (ROS) generation underlying diversities in metabolism that precede differentiation. Notably, SCs heterogeneity is retained in vivo. SCs may therefore be comprised of two distinct, though not irreversibly committed, populations of cells distinguishable for prominent differences in basal biological features such as proliferation, metabolism and differentiation. By these means, novel insights on SCs heterogeneity are provided and evidences for biological readouts potentially relevant for diagnostic purposes described

Genotype-phenotype relationships of truncating mutations, p.E297G and p.D482G in bile salt export pump deficiency

Background & Aims: Bile salt export pump (BSEP) deficiency frequently necessitates liver transplantation in childhood. Homozygous p.D482G or p.E297G mutations are associated with relatively mild phenotypes, responsive to surgical interruption of the enterohepatic circulation (siEHC), in contrast to patients with two predicted protein truncating mutations (PPTM). The phenotype of patients with a compound heterozygous genotype of one p.D482G or p.E297G mutation and one PPTM has remained unclear. We aimed to assess their genotype-phenotype relationship. Methods: From the NAPPED database, we selected patients with homozygous p.D482G or p.E297G mutations (BSEP1/1; n=31), with one p.D482G or p.E297G, and one PPTM (BSEP1/3; n=30), and with two PPTMs (BSEP3/3; n=77). We compared presentation, native liver survival (NLS), and effect of siEHC on NLS. Results: The groups had a similar median age at presentation (0.7-1.3 years). Overall NLS at age 10 years was 21% in BSEP1/3 vs. 75% in BSEP1/1 and 23% in BSEP3/3 (P<0.001). Without siEHC in their follow-up, NLS of BSEP1/3 was similar to BSEP3/3 patients, but considerably lower than BSEP1/1 patients (at age 10 years: 38%, 30%, and 71%, resp; P=0.003). After siEHC, BSEP1/3 and BSEP3/3 patients had similarly low NLS, while this was much higher in BSEP1/1 patients (10 years after siEHC, 27%, 14%, and 92%, resp.; P<0.001). Conclusions: BSEP deficiency patients with one p.E297G or p.D482G mutation and one PPTM have a similarly severe disease course and low responsiveness to siEHC as patients with two PPTMs. This identifies a considerable subgroup of patients who are unlikely to benefit from interruption of the enterohepatic circulation by either surgical or ileal bile acid transporter inhibitor treatment

Amniotic Fluid Stem Cells Improve Survival And Enhance Repair Of Damaged Intestine In Experimental Necrotizing Enterocolitis Via A Cox-2 Dependent Mechanism

Background. 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