Characterization of the Gut Microbiota and Mycobiota in Italian Pediatric Patients With Primary Sclerosing Cholangitis and Ulcerative Colitis.

BACKGROUND: Primary sclerosing cholangitis (PSC) is a chronic, fibroinflammatory, cholestatic liver disease of unknown etiopathogenesis, often associated with inflammatory bowel diseases. Recent evidence ascribes, together with immunologic and environmental components, a significant role to the intestinal microbiota or its molecules in the PSC pathogenesis. METHODS: By metagenomic sequencing of 16S rRNA and ITS2 loci, we describe the fecal microbiota and mycobiota of 26 pediatric patients affected by PSC and concomitant ulcerative colitis (PSC-UC), 27 patients without PSC but with UC (UC), and 26 healthy subjects (CTRLs). RESULTS: Compared with CTRL, the bacterial and fungal gut dysbiosis was evident for both PSC-UC and UC groups; in particular, Streptococcus, Saccharomyces, Sporobolomyces, Tilletiopsis, and Debaryomyces appeared increased in PSC-UC, whereas Klebsiella, Haemophilus, Enterococcus Collinsella, Piptoporus, Candida, and Hyphodontia in UC. In both patient groups, Akkermansia, Bacteroides, Parabacteroides, Oscillospira, Meyerozyma and Malassezia were decreased. Co-occurrence analysis evidenced the lowest number of nodes and edges for fungi networks compared with bacteria. Finally, we identified a specific patient profile, based on liver function tests, bacterial and fungal signatures, that is able to distinguish PSC-UC from UC patients. CONCLUSIONS: We describe the gut microbiota and mycobiota dysbiosis associated to PSC-UC disease. Our results evidenced a gut imbalance, with the reduction of gut commensal microorganisms with stated anti-inflammatory properties (ie, Akkermansia, Bacteroides, Parabacteroides, Oscillospira, Meyerozyma, and Malassezia) and the increase of pathobionts (ie, Streptococcus, Saccharomyces, and Debaryomyces) that could be involved in PSC progression. Altogether, these events may concur in the pathophysiology of PSC in the framework of UC.

In this study, we report the gut microbiota and mycobiota dysbiosis in pediatric patients affected by primary sclerosing cholangitis (PSC) associated with ulcerative colitis (UC), with an increase in pro-inflammatory pathobionts and a reduction in anti-inflammatory commensals.

Impact of Two Antibiotic Therapies on Clinical Outcome and Gut Microbiota Profile in Liver Transplant Paediatric Candidates Colonized by Carbapenem-Resistant Klebsiella pneumoniae CR-KP.

Colonization by multidrug-resistant (MDR) organisms in liver transplant (LT) candidates significantly affects the LT outcome. To date, consensus about patient management is lacking, including microbiological screening indications. This pilot study aimed to evaluate the impact of carbapenem-resistant Klebsiella pneumoniae (CR-KP) colonization in LT paediatric candidates to enable optimal prevention and therapeutic strategies that exploit both clinical and microbiological approaches. Seven paediatric patients colonized by CR-KP were evaluated before and until one-year post LT. At the time of the transplant, patients were stratified based on antibiotic (ATB) prophylaxis into two groups: 'standard ATB' (standard ATB prophylaxis), and 'targeted ATB' (MDR antibiogram-based ATB prophylaxis). Twenty-eight faecal samples were collected during follow-up and used for MDR screening and gut microbiota 16S rRNA-based profiling. Post-transplant hospitalization duration was comparable for both groups. With the exception of one patient, no serious infections and/or complications, nor deaths were recorded. A progressive MDR decontamination was registered. In the 'standard ATB' group, overall bacterial richness increased. Moreover, 6 months after LT, Lactobacillus and Bulleidia were increased and Enterobacteriaceae and Klebsiella spp. were reduced. In the 'targeted ATB' group Klebsiella spp., Ruminococcus gnavus, Erysipelotrichaceae, and Bifidobacterium spp. were increased 12 months after LT. In conclusion, both antibiotics prophylaxis do not affect nor LT outcomes or the risk of intestinal bacterial translocation. However, in the 'standard ATB' group, gut microbiota richness after LT was increased, with an increase of beneficial lactic acid- and short-chain fatty acids (SCFA)-producing bacteria and the reduction of harmful Enterobacteriaceae and Klebsiella spp. It could therefore be appropriate to administer standard prophylaxis, reserving the use of ATB-based molecules only in case of complications.

Proteomics and Metabolomics Approaches towards a Functional Insight onto AUTISM Spectrum Disorders: Phenotype Stratification and Biomarker Discovery.

Autism spectrum disorders (ASDs) are neurodevelopmental disorders characterized by behavioral alterations and currently affect about 1% of children. Significant genetic factors and mechanisms underline the causation of ASD. Indeed, many affected individuals are diagnosed with chromosomal abnormalities, submicroscopic deletions or duplications, single-gene disorders or variants. However, a range of metabolic abnormalities has been highlighted in many patients, by identifying biofluid metabolome and proteome profiles potentially usable as ASD biomarkers. Indeed, next-generation sequencing and other omics platforms, including proteomics and metabolomics, have uncovered early age disease biomarkers which may lead to novel diagnostic tools and treatment targets that may vary from patient to patient depending on the specific genomic and other omics findings. The progressive identification of new proteins and metabolites acting as biomarker candidates, combined with patient genetic and clinical data and environmental factors, including microbiota, would bring us towards advanced clinical decision support systems (CDSSs) assisted by machine learning models for advanced ASD-personalized medicine. Herein, we will discuss novel computational solutions to evaluate new proteome and metabolome ASD biomarker candidates, in terms of their recurrence in the reviewed literature and laboratory medicine feasibility. Moreover, the way to exploit CDSS, performed by artificial intelligence, is presented as an effective tool to integrate omics data to electronic health/medical records (EHR/EMR), hopefully acting as added value in the near future for the clinical management of ASD.

Infections and malignancies risks related to TNF-α-blocking agents in pediatric inflammatory bowel diseases.

Introduction: Tumor necrosis factor-α (TNF-α)-blocking agents are drugs approved for the treatment of inflammatory bowel diseases (IBDs). Infliximab and adalimumab are approved for the treatment of IBD in the pediatric setting with the improvement of therapeutic management. Biological agents, also in the pediatric population, can be administered either alone or in combination with immunomodulators. Their use has raised safety concerns regarding the risk of infections and malignancies.Areas covered: A broad review of the safety concerns for the use of anti-TNF-α drugs in children with IBD was performed, and information regarding the risk of infections and malignancies were updated, also in comparison with the safety of traditional drugs such as steroids and/or immunosuppressants.Expert commentary: Anti-TNF-α drugs have shown favorable safety profiles, and adalimumab treatment is associated with lower immunogenicity compared with infliximab. Heightened awareness and vigilant surveillance leading to prompt diagnosis and treatment are important for optimal management.

The activating p.Ser466Arg change in STAT1 causes a peculiar phenotype with features of interferonopathies.

Signal Transducer and Activator of Transcription 1 (STAT1) is a DNA-binding signal transducer that regulates transcription of specific genes in response to IFNγ and IFNα/ß stimulation. Loss-of-function mutations impairing STAT1 activity are known to confer susceptibility to intracellular bacterial and viral diseases. Conversely, the few known activating mutations of STAT1 allow predisposition to chronic mucocutaneous candidiasis disease, and occur in patients with combined immunodeficiency and defective Th1 and Th17 responses. Here, we report on a de novo gain-of-function (GoF) STAT1 mutation (c.1398C>G, p.Ser466Arg) identified by exome sequencing in an individual with brain calcification, arthritis, recurrent pericarditis, leukopenia, thrombocytopenia and low C3 levels, a phenotype resembling an interferonopathy. The Ser466Arg change affects a highly conserved residue located in the DNA binding domain of the protein and the amino acid substitution was documented to have an activating role both in vitro and in vivo. Altogether, clinical features and functional studies are compatible with hyperactivation of the Interferon pathways, highlighting a role of STAT1 GoF mutation in clinical phenotypes fitting interferonopathies.

Phase I/II Trial of Liver-derived Mesenchymal Stem Cells in Pediatric Liver-based Metabolic Disorders: A Prospective, Open Label, Multicenter, Partially Randomized, Safety Study of One Cycle of Heterologous Human Adult Liver-derived Progenitor Cells (HepaStem) in Urea Cycle Disorders and Crigler-Najjar Syndrome Patients.

BACKGROUND: Regenerative medicine using stem cell technology is an emerging field that is currently tested for inborn and acquired liver diseases. OBJECTIVE: This phase I/II prospective, open label, multicenter, randomized trial aimed primarily at evaluating the safety of Heterologous Human Adult Liver-derived Progenitor Cells (HepaStem) in pediatric patients with urea cycle disorders (UCDs) or Crigler-Najjar (CN) syndrome 6 months posttransplantation. The secondary objective included the assessment of safety up to 12 months postinfusion and of preliminary efficacy. METHODS: Fourteen patients with UCDs and 6 with CN syndrome were divided into 3 cohorts by body weight and intraportally infused with 3 doses of HepaStem. Clinical status, portal vein hemodynamics, morphology of the liver, de novo detection of circulating anti-human leukocyte antigen antibodies, and clinically significant adverse events (AEs) and serious adverse events to infusion were evaluated by using an intent-to-treat analysis. RESULTS: The overall safety of HepaStem was confirmed. For the entire study period, patient-month incidence rate was 1.76 for the AEs and 0.21 for the serious adverse events, of which 38% occurred within 1 month postinfusion. There was a trend of higher events in UCD as compared with CN patients. Segmental left portal vein thrombosis occurred in 1 patient and intraluminal local transient thrombus in a second patient. The other AEs were in line with expectations for catheter placement, cell infusion, concomitant medications, age, and underlying diseases. CONCLUSIONS: This study led to European clinical trial authorization for a phase II study in a homogeneous patient cohort, with repeated infusions and intermediate doses.

Liver Transplantation and Gut Microbiota Profiling in a Child Colonized by a Multi-Drug Resistant Klebsiella pneumoniae: A New Approach to Move from Antibiotic to "Eubiotic" Control of Microbial Resistance.

The increase of microorganisms multi-drug resistant (MDR) to antibiotics (ATBs) is becoming a global emergency, especially in frail subjects. In chronic liver disease (LD) with indications for liver transplantation (LT), MDR colonization can significantly affect the LT outcome. However, no clear guidelines for microbial management are available. A novel approach toward MDR-colonized patients undergoing LT was developed at our Center refraining from ATBs use during the transplant waiting list, and use of an intensive perioperative prophylaxis cycle. This study aimed to couple clinical evaluation with monitoring of gut microbiota in a pediatric LD patient colonized with MDR Klebsiella pneumoniae (KP) who underwent LT. No peri-transplant complications were reported, and a decontamination from the MDR bacteria occurred during follow-up. Significant changes in gut microbiota, especially during ATB treatment, were reported by microbiota profiling. Patterns of Klebsiella predominance and microbiota diversity revealed opposite temporal trends, with Klebsiella ecological microbiota niches linked to ATB-driven selection. Our infection control program appeared to control complications following LT in an MDR-KP-colonized patient. The perioperative ATB regimen, acting as LT prophylaxis, triggered MDR-KP overgrowth and gut dysbiosis, but buffered infectious processes. Mechanisms modulating the gut ecosystem should be taken into account in MDR colonization clinical management.

Autoimmune liver diseases and inflammatory bowel diseases in children: current issues and future perspectives.

Inflammatory bowel diseases (IBDs) represent a group of intestinal disorders with a chronic and relapsing inflammation of the gut, and with a potential risk of systemic involvement of other organs and systems. Over the pediatric age, an incidence higher than 20% of developing extraintestinal manifestation during follow-up has been reported. The liver and the biliary system are frequently involved, and primary sclerosing cholangitis (PSC) is the most predominant entity with an incidence rate of 6.4-7.8% in children. PSC recognizes a multifactorial pathogenesis, and so far a not fully known mechanism for this association. The peculiar phenotype and the distinct clinical course of patients with IBD and PSC-associated make this 'linkage' an attractive study model to better understand mechanisms underlying these diseases. Approaching to these patients is complex and multidisciplinary, and a unique therapeutic strategy has not been standardized yet. New medications are being studied; however, further studies are needed to fully understand the pathogenesis and to improve the care of these patients. The aim of this paper is to review the recent literature regarding hepatobiliary involvement in IBD patients, with particular attention to PSC, and to provide the latest information for a correct diagnosis and appropriate management.

Liver transplant in ethylmalonic encephalopathy: a new treatment for an otherwise fatal disease.

Ethylmalonic encephalopathy is a fatal, rapidly progressive mitochondrial disorder caused by ETHE1 mutations, whose peculiar clinical and biochemical features are due to the toxic accumulation of hydrogen sulphide and of its metabolites, including thiosulphate. In mice with ethylmalonic encephalopathy, liver-targeted adeno-associated virus-mediated ETHE1 gene transfer dramatically improved both clinical course and metabolic abnormalities. Reasoning that the same achievement could be accomplished by liver transplantation, we performed living donor-liver transplantation in an infant with ethylmalonic encephalopathy. Unlike the invariably progressive deterioration of the disease, 8 months after liver transplantation, we observed striking neurological improvement with remarkable achievements in psychomotor development, along with dramatic reversion of biochemical abnormalities. These results clearly indicate that liver transplantation is a viable therapeutic option for ETHE1 disease.

Clinical Features and Risk Factors of Autoimmune Liver Involvement in Pediatric Inflammatory Bowel Disease.

OBJECTIVES: Autoimmune liver disease is reported in up to 7.8% of children with inflammatory bowel disease. A distinct inflammatory bowel disease phenotype has been suggested in adults and in small pediatric cohorts. The aim of the study was to evaluate the features of inflammatory bowel disease associated with autoimmune liver diseases and to analyze the characteristics of the liver disease. METHODS: Information on patients was obtained from the Italian Pediatric Inflammatory Bowel Disease Registry. Data of patients with and without autoimmune liver disease were compared. RESULTS: Autoimmune liver disease was detected in 6.8% of the 677 patients enrolled and was significantly associated with the diagnosis of ulcerative colitis (83%), with pancolonic involvement (84%), and with perinuclear antineutrophil cytoplasmic antibody positivity (41%) (all Ps < 0.05). Autoimmune liver disease was defined as sclerosing cholangitis in 61% of the patients and as an overlap syndrome in 33%. Concomitant intra- and extrahepatic biliary involvement was reported in 61% of cases, whereas exclusive extrahepatic lesions were reported in 21%. Hepatobiliary complications were observed in 9% of the patients during follow-up. CONCLUSIONS: Autoimmune liver disease, especially sclerosing cholangitis, was significantly more common in patients with extensive ulcerative colitis. Although complications are relatively rare in the pediatric age, monitoring is recommended.

Macrophage recruitment by fibrocystin-defective biliary epithelial cells promotes portal fibrosis in congenital hepatic fibrosis.

UNLABELLED: Congenital hepatic fibrosis (CHF) is a disease of the biliary epithelium characterized by bile duct changes resembling ductal plate malformations and by progressive peribiliary fibrosis, in the absence of overt necroinflammation. Progressive liver fibrosis leads to portal hypertension and liver failure; however, the mechanisms leading to fibrosis in CHF remain elusive. CHF is caused by mutations in PKHD1, a gene encoding for fibrocystin, a ciliary protein expressed in cholangiocytes. Using a fibrocystin-defective (Pkhd1(del4/del4)) mouse, which is orthologous of CHF, we show that Pkhd1(del4/del4) cholangiocytes are characterized by a ß-catenin-dependent secretion of a range of chemokines, including chemokine (C-X-C motif) ligands 1, 10, and 12, which stimulate bone marrow-derived macrophage recruitment. We also show that Pkhd1(del4/del4) cholangiocytes, in turn, respond to proinflammatory cytokines released by macrophages by up-regulating αvß6 integrin, an activator of latent local transforming growth factor-ß1. While the macrophage infiltrate is initially dominated by the M1 phenotype, the profibrogenic M2 phenotype increases with disease progression, along with the number of portal myofibroblasts. Consistent with these findings, clodronate-induced macrophage depletion results in a significant reduction of portal fibrosis and portal hypertension as well as of liver cysts. CONCLUSION: Fibrosis can be initiated by an epithelial cell dysfunction, leading to low-grade inflammation, macrophage recruitment, and collagen deposition; these findings establish a new paradigm for biliary fibrosis and represent a model to understand the relationship between cell dysfunction, parainflammation, liver fibrosis, and macrophage polarization over time.

Genetics and Molecular Modeling of New Mutations of Familial Intrahepatic Cholestasis in a Single Italian Center.

Familial intrahepatic cholestases (FICs) are a heterogeneous group of autosomal recessive disorders of childhood that disrupt bile formation and present with cholestasis of hepatocellular origin. Three distinct forms are described: FIC1 and FIC2, associated with low/normal GGT level in serum, which are caused by impaired bile salt secretion due to defects in ATP8B1 encoding the FIC1 protein and defects in ABCB11 encoding bile salt export pump protein, respectively; FIC3, linked to high GGT level, involves impaired biliary phospholipid secretion due to defects in ABCB4, encoding multidrug resistance 3 protein. Different mutations in these genes may cause either a progressive familial intrahepatic cholestasis (PFIC) or a benign recurrent intrahepatic cholestasis (BRIC). For the purposes of the present study we genotyped 27 children with intrahepatic cholestasis, diagnosed on either a clinical or histological basis. Two BRIC, 23 PFIC and 2 BRIC/PFIC were identified. Thirty-four different mutations were found of which 11 were novel. One was a 2Mb deletion (5'UTR- exon 18) in ATP8B1. In another case microsatellite analysis of chromosome 2, including ABCB11, showed uniparental disomy. Two cases were compound heterozygous for BRIC/PFIC2 mutations. Our results highlight the importance of the pathogenic role of novel mutations in the three genes and unusual modes of their transmission.

The clinical implications of thalidomide in inflammatory bowel diseases.

Thalidomide has anti-inflammatory and anti-angiogenetic activity that makes it suitable for treating inflammatory bowel diseases (IBD). The recent guidelines from the European Crohn's and Colitis Organization/European Society for Pediatric Gastroenterology Hepatology and Nutrition conclude that thalidomide cannot be recommended in refractory pediatric Crohn's disease but that it may be considered in selected cohorts of patients who are not anti-TNFα agent responders. The main adverse effect is the potential teratogenicity that renders the long-term use of thalidomide problematic in young adults due to the strict need for contraceptive use. In short-term use it is relatively safe; the most likely adverse effect is the neuropathy, which is highly reversible in children. So far the use of thalidomide is reported in 223 adult and pediatric IBD patients (206 with Crohn's disease). In the following sections, the authors will discuss efficacy and safety of thalidomide, in the short-term treatment of IBD.

Long-term outcome of home parenteral nutrition in patients with ultra-short bowel syndrome.

OBJECTIVE: The patients with ultra-short bowel syndrome (U-SBS) have been considered potential candidates for a preemptive/rehabilitative intestinal transplantation owing to the high risk of death from the underlying disease. We hypothesized that children with U-SBS, in the absence of intestinal failure-associated liver disease (IFALD), could also have a good rate of survival on home parenteral nutrition (HPN). METHODS: A prospective database from the "Bambino Gesù" Artificial Nutrition and Intestinal Failure Program was used to evaluate outcomes and morbidities of consecutive patients with ≤ 10 cm of small bowel enrolled since 2000. RESULTS: Eleven patients were identified with a median bowel length of 7.5 (3-9) cm. Eight patients developed IFALD, which reversed in 7 of them; the IFALD progressively worsened in 1 patient until death. One patient underwent isolated intestinal transplantation and 1 patient is no longer receiving parenteral nutrition (PN) and both are fully enterally fed. The other patients remained at least partially dependent on HPN. The number of days of inpatient care decreased in all of the patients except for the 1 who had repeated episodes of central line infections. CONCLUSIONS: The survival of patients with U-SBS receiving HPN was good. Although IFALD was frequent, it had been manageable in most of the patients, but in a single complex case, it led to death. The multidisciplinary management warranted to these patients to approach the school age, to grow, and to maintain the oral intake. Patients with U-SBS are rare, and to better understand their long-term survival, further studies, including more large patient populations, are required.

The expression of CD90/Thy-1 in hepatocellular carcinoma: an in vivo and in vitro study.

Although the CD90 (Thy-1) was proposed as biomarker of several tumors and cancer stem cells, the involvement of this molecule in the progression of hepatocellular carcinoma (HCC) and other less frequent hepatic neoplasms is still undefined. The distribution of CD90 was investigated both in in vivo (human tissues samples) and in vitro (human HCC cell line JHH-6). A total of 67 liver tumors were analyzed: 51 HCC, 6 cholangiocarcinoma and 10 hepatoblastoma. In all cases, paired tissue sample of both the tumor and cirrhotic liver was available. Hepatic tissue obtained in 12 healthy livers was used as control. CD90 gene expression was studied by RT-qPCR, protein expression was assessed by quantitative Western Blot, immunofluorescence and flow cytometry. The CD90 expression analysis showed a significant increment in tumor compared to both its paired cirrhotic tissue and normal liver (p<0.05 and p<0.001, respectively). This increase was accompanied by the up-regulation of stromal component in the cancer, as demonstrated by alpha smooth muscle actin staining. In vitro analysis of JHH-6 cell line showed a higher proliferation capacity of CD90(+) compared to CD90(-) cells (p<0.001), also noticed in 3D clonogenic assay (p<0.05), associated by a significant higher expression of the promoting factors (hepatocyte growth factor, fibroblast associated protein and alpha smooth muscle actin 2). A higher expression of the breast cancer resistance protein was found in CD90(+) subpopulation while the multidrug resistance protein 1 showed an opposite behavior. Collectively, these results point to the importance of CD90 in the HCC.

Chronic pancreatitis as presentation of Crohn's disease in a child.

It is reported that a pancreatic disease may precede the diagnosis of inflammatory bowel disease (IBD) both in children and in adults. Idiopathic chronic pancreatitis, however, occasionally co-exists with the IBD, mainly at pediatric age. We report a case of a patient who progressively developed the features of a chronic pancreatitis, before the diagnosis of Crohn's Disease (CD). Ten months after the onset of the first episode of pancreatitis the patient developed bloody diarrhea, mucus stools and biochemical findings of inflammation. The colonoscopy revealed a diffuse colitis without involvement of the last loop and the gastroscopy showed inflammation of the iuxta-papillary area. The histological findings confirmed the diagnosis of CD that involved the colon and the duodenum. In conclusion, in children the idiopathic chronic pancreatitis may be an unusual presentation of CD. Thus, if other known cause of chronic pancreatitis are not found, a not invasive work up to exclude the IBD should be warranted. An early coincidental diagnosis of the IBD may delay the progression of the pancreatic disease.

MEDNIK syndrome: a novel defect of copper metabolism treatable by zinc acetate therapy.

MEDNIK syndrome-acronym for mental retardation, enteropathy, deafness, neuropathy, ichthyosis, keratodermia-is caused by AP1S1 gene mutations, encoding σ1A, the small subunit of the adaptor protein 1 complex, which plays a crucial role in clathrin coat assembly and mediates trafficking between trans-Golgi network, endosomes and the plasma membrane. MEDNIK syndrome was first reported in a few French-Canadian families sharing common ancestors, presenting a complex neurocutaneous phenotype, but its pathogenesis is not completely understood. A Sephardic-Jewish patient, carrying a new AP1S1 homozygous mutation, showed severe perturbations of copper metabolism with hypocupremia, hypoceruloplasminemia and liver copper accumulation, along with intrahepatic cholestasis. Zinc acetate treatment strikingly improved clinical conditions, as well as liver copper and bile-acid overload. We evaluated copper-related metabolites and liver function retrospectively in the original French-Canadian patient series. Intracellular copper metabolism and subcellular localization and function of copper pump ATP7A were investigated in patient fibroblasts. Copper metabolism perturbation and hepatopathy were confirmed in all patients. Studies in mutant fibroblasts showed abnormal copper incorporation and retention, reduced expression of copper-dependent enzymes cytochrome-c-oxidase and Cu/Zn superoxide dismutase, and aberrant intracellular trafficking of Menkes protein ATP7A, which normalized after rescue experiments expressing wild-type AP1S1 gene. We solved the pathogenetic mechanism of MEDNIK syndrome, demonstrating that AP1S1 regulates intracellular copper machinery mediated by copper-pump proteins. This multisystem disease is characterized by a unique picture, combining clinical and biochemical signs of both Menkes and Wilson's diseases, in which liver copper overload is treatable by zinc acetate therapy, and can now be listed as a copper metabolism defect in humans. Our results may also contribute to understand the mechanism(s) of intracellular trafficking of copper pumps.