Expression Analysis of ATP-Binding Cassette Transporters ABCB11 and ABCB4 in Primary Sclerosing Cholangitis and Variety of Pediatric and Adult Cholestatic and Noncholestatic Liver Diseases.

ATP-binding cassette (ABC) transporters are the members of the efflux pumps that are responsible for the removal of cytotoxic substances by active transport. ABCB11, the bile salt efflux pump of hepatocytes, coordinates cellular excretion of numerous conjugated bile salts into the bile canaliculi, whereas ABCB4 acts as an ATP-dependent floppase translocating phosphatidylcholine from the inner to the outer leaflet of the bile canalicular membrane. Loss of functional ABCB11 and ABCB4 proteins causes early-onset refractory cholestasis or cholangiopathy. In this study, we investigated the expression and localization pattern of ABCB11 and ABCB4 using immunohistochemistry and RNA profiling in liver samples from patients with different types and stages of chronic cholestatic liver disease, with emphasis on primary sclerosing cholangitis (PSC), compared to a variety of cholestatic and noncholestatic hepatopathies. Therefore, ABCB11 and ABCB4 expressions were investigated on formalin-fixed and paraffin-embedded (FFPE) material in a patient cohort of total 43 patients with or without cholestatic liver diseases, on protein level using immunohistochemistry and on RNA level using nanoString technology. Intriguingly, our results demonstrated increased expression of ABCB11 and ABCB4 on protein as well as RNA level in PSC, and the expression pattern correlated with disease progression. We concluded from our study that patients with PSC demonstrate altered expression levels and pattern of ABCB11 and ABCB4 which correlated with disease progression; thereby, ABCB11 and ABCB4 analysis may be a useful tool for assessment of disease stages in PSC.

Prolidase deficiency diagnosed by whole exome sequencing in a child with pulmonary capillaritis.

The case of a young boy with pulmonary haemorrhage who was ultimately diagnosed on whole exome sequencing with a rare condition called prolidase deficiency. This case demonstrates the utility of modern genomic testing in paediatric rare lung disease. http://ow.ly/rDGz30o8pcd.

Alveolar capillary dysplasia with misalignment of the pulmonary veins: clinical, histological, and genetic aspects.

Alveolar capillary dysplasia with misalignment of the pulmonary veins (ACD/MPV) is a rare and lethal disorder mainly involving the vascular development of the lungs. Since its first description, significant achievements in research have led to a better understanding of the underlying molecular mechanism of ACD/MPV and genetic studies have identified associations with genomic alterations in the locus of the transcription factor FOXF1. This in turn has increased the awareness among clinicians resulting in over 200 cases reported so far, including genotyping of patients in most recent reports. Collectively, this promoted a better stratification of the patient group, leading to new perspectives in research on the pathogenesis. Here, we provide an overview of the clinical aspects of ACD/MPV, including guidance for clinicians, and review the ongoing research into the complex molecular mechanism causing this severe lung disorder.

Pulmonary interstitial glycogenosis associated with a spectrum of neonatal pulmonary disorders.

Primary or isolated pulmonary interstitial glycogenosis (PIG) is a rare disease presenting as tachypnea and hypoxemia during the perinatal period. A diffuse interstitial infiltrate with focal hyperinflation is visible on chest imaging. The biopsy findings include diffuse expansion of the interstitium by spindle-shaped cells with pale cytoplasm that, on electron microscopy (EM), are poorly differentiated mesenchymal cells containing abundant monoparticulate glycogen. This glycogenosis appears to be a transient abnormality, usually with a favorable prognosis. Recently, cases of PIG, some associated with other pulmonary or systemic abnormalities, have been described. The clinical significance and potential role of PIG changes remain unknown. We report 28 cases of PIG associated with a spectrum of pediatric pulmonary and cardiovascular disorders, including arterial hypertensive changes with and without abnormal alveolar development (n=9), congenital heart disease (CHD; n=4), hyperplasia of pulmonary neuroendocrine cells resembling neuroendocrine hyperplasia of infancy (NEHI, n=5), congenital pulmonary airway malformation (n=5), congenital lobar emphysema (n=4), and Noonan syndrome (n=1). In all cases, PIG was confirmed by positive periodic acid-Schiff (PAS) staining, immunopositivity for vimentin, and EM. Although some patients improved with age, 7 died of respiratory failure or complications of CHD, suggesting that PIG may be clinically significant when associated with other severe disorders. The association of PIG with a spectrum of mostly congenital lung disorders supports its origin as a developmental abnormality of interstitial fibroblast differentiation rather than a nonspecific reactive process.

Disrupted apical exocytosis of cargo vesicles causes enteropathy in FHL5 patients with Munc18-2 mutations.

Familial hemophagocytic lymphohistiocytosis 5 (FHL5) is an autosomal recessive disease caused by mutations in STXBP2, coding for Munc18-2, which is required for SNARE-mediated membrane fusion. FHL5 causes hematologic and gastrointestinal symptoms characterized by chronic enteropathy that is reminiscent of microvillus inclusion disease (MVID). However, the molecular pathophysiology of FHL5-associated diarrhea is poorly understood. Five FHL5 patients, including four previously unreported patients, were studied. Morphology of duodenal sections was analyzed by electron and fluorescence microscopy. Small intestinal enterocytes and organoid-derived monolayers displayed the subcellular characteristics of MVID. For the analyses of Munc18-2-dependent SNARE-protein interactions, a Munc18-2 CaCo2-KO model cell line was generated by applying CRISPR/Cas9 technology. Munc18-2 is required for Slp4a/Stx3 interaction in fusion of cargo vesicles with the apical plasma membrane. Cargo trafficking was investigated in patient biopsies, patient-derived organoids, and the genome-edited model cell line. Loss of Munc18-2 selectively disrupts trafficking of certain apical brush-border proteins (NHE3 and GLUT5), while transport of DPPIV remained unaffected. Here, we describe the molecular mechanism how the loss of function of Munc18-2 leads to cargo-selective mislocalization of brush-border components and a subapical accumulation of cargo vesicles, as it is known from the loss of polarity phenotype in MVID.

Loss of the Arp2/3 complex component ARPC1B causes platelet abnormalities and predisposes to inflammatory disease.

Human actin-related protein 2/3 complex (Arp2/3), required for actin filament branching, has two ARPC1 component isoforms, with ARPC1B prominently expressed in blood cells. Here we show in a child with microthrombocytopenia, eosinophilia and inflammatory disease, a homozygous frameshift mutation in ARPC1B (p.Val91Trpfs*30). Platelet lysates reveal no ARPC1B protein and greatly reduced Arp2/3 complex. Missense ARPC1B mutations are identified in an unrelated patient with similar symptoms and ARPC1B deficiency. ARPC1B-deficient platelets are microthrombocytes similar to those seen in Wiskott-Aldrich syndrome that show aberrant spreading consistent with loss of Arp2/3 function. Knockout of ARPC1B in megakaryocytic cells results in decreased proplatelet formation, and as observed in platelets from patients, increased ARPC1A expression. Thus loss of ARPC1B produces a unique set of platelet abnormalities, and is associated with haematopoietic/immune symptoms affecting cell lineages where this isoform predominates. In agreement with recent experimental studies, our findings suggest that ARPC1 isoforms are not functionally interchangeable.

Abnormal Rab11-Rab8-vesicles cluster in enterocytes of patients with microvillus inclusion disease.

Microvillus inclusion disease (MVID) is a congenital enteropathy characterized by accumulation of vesiculo-tubular endomembranes in the subapical cytoplasm of enterocytes, historically termed "secretory granules." However, neither their identity nor pathophysiological significance is well defined. Using immunoelectron microscopy and tomography, we studied biopsies from MVID patients (3× Myosin 5b mutations and 1× Syntaxin3 mutation) and compared them to controls and genome-edited CaCo2 cell models, harboring relevant mutations. Duodenal biopsies from 2 patients with novel Myosin 5b mutations and typical clinical symptoms showed unusual ultrastructural phenotypes: aberrant subapical vesicles and tubules were prominent in the enterocytes, though other histological hallmarks of MVID were almost absent (ectopic intra-/intercellular microvilli, brush border atrophy). We identified these enigmatic vesiculo-tubular organelles as Rab11-Rab8-positive recycling compartments of altered size, shape and location harboring the apical SNARE Syntaxin3, apical transporters sodium-hydrogen exchanger 3 (NHE3) and cystic fibrosis transmembrane conductance regulator. Our data strongly indicate that in MVID disrupted trafficking between cargo vesicles and the apical plasma membrane is the primary cause of a defect of epithelial polarity and subsequent facultative loss of brush border integrity, leading to malabsorption. Furthermore, they support the notion that mislocalization of transporters, such as NHE3 substantially contributes to the reported sodium loss diarrhea.

PEPCK-C reexpression in the liver counters neonatal hypoglycemia in Pck1del/del mice, unmasking role in non-gluconeogenic tissues

Whole body cytosolic phosphoenolpyruvate carboxykinase knockout (PEPCK-C KO) mice die early after birth with profound hypoglycemia therefore masking the role of PEPCK-C in adult, non-gluconeogenic tissues where it is expressed. To investigate whether PEPCK-C deletion in the liver was critically responsible for the hypoglycemic phenotype, we reexpress this enzyme in the liver of PEPCK-C KO pups by early postnatal administration of PEPCK-C-expressing adenovirus. This maneuver was sufficient to partially rescue hypoglycemia and allow the pups to survive and identifies the liver as a critical organ, and hypoglycemia as the critical pathomechanism, leading to early postnatal death in the whole-body PEPCK-C knockout mice. Pathology assessment of survivors also suggest a possible role for PEPCK-C in lung maturation and muscle metabolism (AU)

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PEPCK-C reexpression in the liver counters neonatal hypoglycemia in Pck1 del/del mice, unmasking role in non-gluconeogenic tissues.

Whole body cytosolic phosphoenolpyruvate carboxykinase knockout (PEPCK-C KO) mice die early after birth with profound hypoglycemia therefore masking the role of PEPCK-C in adult, non-gluconeogenic tissues where it is expressed. To investigate whether PEPCK-C deletion in the liver was critically responsible for the hypoglycemic phenotype, we reexpress this enzyme in the liver of PEPCK-C KO pups by early postnatal administration of PEPCK-C-expressing adenovirus. This maneuver was sufficient to partially rescue hypoglycemia and allow the pups to survive and identifies the liver as a critical organ, and hypoglycemia as the critical pathomechanism, leading to early postnatal death in the whole-body PEPCK-C knockout mice. Pathology assessment of survivors also suggest a possible role for PEPCK-C in lung maturation and muscle metabolism.

Hyperplasia and hypertrophy of pulmonary neuroepithelial bodies, presumed airway hypoxia sensors, in hypoxia-inducible factor prolyl hydroxylase-deficient mice.

Pulmonary neuroepithelial bodies (NEBs), presumed polymodal airway sensors, consist of innervated clusters of amine (serotonin) and peptide-producing cells. While NEB responses to acute hypoxia are mediated by a membrane-bound O2 sensor complex, responses to sustained and/or chronic hypoxia involve a prolyl hydroxylase (PHD)-hypoxia-inducible factor-dependent mechanism. We have previously reported hyperplasia of NEBs in the lungs of Phd1-/- mice associated with enhanced serotonin secretion. Here we use a novel multilabel immunofluorescence method to assess NEB distribution, frequency, and size, together with the number and size of NEB cell nuclei, and to colocalize multiple cytoplasmic and nuclear epitopes in the lungs of Phd1-/-, Phd2+/-, and Phd3-/- mice and compare them with wild-type controls. To define the mechanisms of NEB cell hyperplasia, we used antibodies against Mash1 and Prox1 (neurogenic genes involved in NEB cell differentiation/maturation), hypoxia-inducible factor-1alpha, and the cell proliferation marker Ki67. Morphometric analysis of (% total lung area) immunostaining for synaptophysin (% synaptophysin), a cytoplasmic marker of NEB cells, was significantly increased in Phd1-/- and Phd3-/- mice compared to wild-type mice. In addition, NEB size and the number and size of NEB nuclei were also significantly increased, indicating that deficiency of Phds is associated with striking hyperplasia and hypertrophy of NEBs. In Phd2+/- mice, while mean % synaptophysin was comparable to wild-type controls, the NEB size was moderately increased, suggesting an effect even in heterozygotes. NEBs in all Phd-deficient mice showed increased expression of Mash1, Prox1, Ki67, and hypoxia-inducible factor-1alpha, in keeping with enhanced differentiation from precursor cells and a minor component of cell proliferation. Since the loss of PHD activity mimics chronic hypoxia, our data provide critical information on the potential role of PHDs in the pathobiology and mechanisms of NEB cell hyperplasia that is relevant to a number of pediatric lung disorders.

Hepatoblastoma in Explanted Livers of Patients With Biliary Atresia.

OBJECTIVES: Surveillance of hepatic nodules for malignant transformation to hepatocellular carcinoma is important in the monitoring of patients with biliary atresia (BA). To date, only 2 published case reports describe the finding of hepatoblastoma (HB) in this setting. The present study aimed to investigate this association of HB and BA, and to assess the utility of alpha-fetoprotein (aFP) as a marker in the diagnosis. METHODS: A retrospective study of all patients who underwent isolated liver transplantation (LTx) for the primary diagnosis of BA at a single center, between January 1999 and June 2014, was conducted. Patient demographics, pre-LTx aFP levels, and histologic examination of native liver explants were reviewed. RESULTS: One hundred two (44% men, median age 11 months) patients underwent LTx for BA. Two (2%) explants examinations were confirmatory for concomitant HB; both patients had abnormally elevated aFP. Overall, 56 (55%) patients had available pre-LTx aFP levels. Recipients with persistently abnormal aFP levels (n = 20, 36%) were older at hepatoportoenterostomy (107 vs 68 days, P = 0.02) and younger at LTx surgery (359 vs 1713 days, P < 0.01), compared to patients with constantly normal levels (n = 24, 43%). CONCLUSIONS: In our cohort, HB was found to coexist in approximately 2% of patients with BA undergoing LTx, far exceeding the hypothetical anticipated incidence of 1:10 billion for the concomitant diagnoses. Elevated serum aFP levels may be sensitive but not specific for HB in this context. Further research is required to identify specific mechanisms and risk factors.

Liver transplantation for children with acute liver failure associated with secondary hemophagocytic lymphohistiocytosis.

Hemophagocytic lymphohistiocytosis (HLH) is a rare life-threatening systemic disease, characterized by overwhelming stimulation of the immune system and categorized as primary or secondary types. Occasionally, acute liver failure (ALF) may dominate the clinical presentation. Given the systemic nature of HLH and risk of recurrence, HLH is considered by many a contraindication to liver transplantation (LT). The aim of this study is to review our single-center experience with LT in children with secondary HLH and ALF (HLH-ALF). This is a cross-sectional, retrospective study of children with secondary HLH-ALF that underwent LT in 2005-2014. Of 246 LTs, 9 patients (3 males; median age, 5 years; range, 0.7-15.4 years) underwent LT for secondary HLH-ALF. Disease progression was rapid with median 14 days (range, 6-27 days) between first symptoms and LT. Low fibrinogen/high triglycerides, elevated ferritin, hemophagocytosis on liver biopsy, and soluble interleukin 2 receptor levels were the most commonly fulfilled diagnostic criteria; HLH genetic studies were negative in all patients. Immunosuppressive therapy after LT included corticosteroids adjusted to HLH treatment protocol and tacrolimus. Thymoglobulin (n = 5), etoposide (n = 4), and alemtuzumab (n = 2) were used in cases of recurrence. Five (56%) patients experienced HLH recurrence, 1 requiring repeat LT, and 3 died. Overall graft and patient survival were 60% and 67%, respectively. Six patients are alive and well at a median of 24 months (range, 15-72 months) after transplantation. In conclusion, LT can be beneficial in selected patients with secondary HLH-ALF and can restore good health in an otherwise lethal condition. Liver Transplantation 22 1245-1253 2016 AASLD.

Multilabel immunofluorescence and antigen reprobing on formalin-fixed paraffin-embedded sections: novel applications for precision pathology diagnosis.

We report new methods for multilabel immunofluorescence (MIF) and reprobing of antigen epitopes on the same formalin-fixed paraffin-embedded (FFPE) sections. The MIF method includes an antigen-retrieval step followed by multilabel immunostaining and examination by confocal microscopy. As examples, we illustrate epitopes localized to the apical and basolateral membranes, and the cytoplasm of enterocytes of normal small intestine and in cases of congenital enteropathies (microvillous inclusion disease and congenital tufting enteropathy). We also demonstrate localization of the bile salt excretion pump protein (BSEP) in bile canalicular membrane of normal hepatocytes and in cases of primary sclerosing cholangitis. To demonstrate colocalization of cytoplasmic and nuclear epitopes we analyzed normal control and hyperplastic pulmonary neuroendocrine cells (PNEC) and neuroepithelial bodies (NEBs), presumed airway sensors in the lungs of infants with bronchopulmonary dysplasia (BPD). As cytoplasmic markers we used anti-bombesin or anti-synaptic vesicle protein 2 (SV2) antibody, respectively, and for nuclear localization, antibodies against neurogenic genes mammalian achaete-scute homolog (Mash1) and prospero homeobox 1 (Prox1), essential for NEB cells differentiation and maturation, hypoxia-inducible factor 1α (HIF1α) a downstream modulator of hypoxia response and a proliferation marker Ki67. The reprobing method consisted of removal of the previously immunolabeled target and immunostaining with different antibodies, facilitating colocalization of enterocyte brush border epitopes as well as HIF1α, Mash1 and Prox1 in PNEC/NEB PNEC and NEBs. As these methods are suitable for routine FFPE pathology samples from various tissues, allowing visualization of multiple epitopes in the same cells/sections with superior contrast and resolution, they are suitable for a wide range of applications in diagnostic pathology and may be particularly well suited for precision medicine diagnostics.

Variants in TRIM22 That Affect NOD2 Signaling Are Associated With Very-Early-Onset Inflammatory Bowel Disease.

BACKGROUND & AIMS: Severe forms of inflammatory bowel disease (IBD) that develop in very young children can be caused by variants in a single gene. We performed whole-exome sequence (WES) analysis to identify genetic factors that might cause granulomatous colitis and severe perianal disease, with recurrent bacterial and viral infections, in an infant of consanguineous parents. METHODS: We performed targeted WES analysis of DNA collected from the patient and her parents. We validated our findings by a similar analysis of DNA from 150 patients with very-early-onset IBD not associated with known genetic factors analyzed in Toronto, Oxford, and Munich. We compared gene expression signatures in inflamed vs noninflamed intestinal and rectal tissues collected from patients with treatment-resistant Crohn's disease who participated in a trial of ustekinumab. We performed functional studies of identified variants in primary cells from patients and cell culture. RESULTS: We identified a homozygous variant in the tripartite motif containing 22 gene (TRIM22) of the patient, as well as in 2 patients with a disease similar phenotype. Functional studies showed that the variant disrupted the ability of TRIM22 to regulate nucleotide binding oligomerization domain containing 2 (NOD2)-dependent activation of interferon-beta signaling and nuclear factor-κB. Computational studies demonstrated a correlation between the TRIM22-NOD2 network and signaling pathways and genetic factors associated very early onset and adult-onset IBD. TRIM22 is also associated with antiviral and mycobacterial effectors and markers of inflammation, such as fecal calprotectin, C-reactive protein, and Crohn's disease activity index scores. CONCLUSIONS: In WES and targeted exome sequence analyses of an infant with severe IBD characterized by granulomatous colitis and severe perianal disease, we identified a homozygous variant of TRIM22 that affects the ability of its product to regulate NOD2. Combined computational and functional studies showed that the TRIM22-NOD2 network regulates antiviral and antibacterial signaling pathways that contribute to inflammation. Further study of this network could lead to new disease markers and therapeutic targets for patients with very early and adult-onset IBD.

Hyperplasia of pulmonary neuroendocrine cells in infancy and childhood.

Pulmonary neuroendocrine cells (PNEC) are widely distributed throughout the airway mucosa of mammalian lung as solitary cells and as distinctive innervated clusters, neuroepithelial bodies (NEB). These cells differentiate early during lung development and are more prominent in fetal/neonatal lungs compared to adults. PNEC/NEB cells produce biogenic amine (serotonin) and a variety of peptides (i.e., bombesin) involved in regulation of lung function. During the perinatal period, NEB are thought to function as airway O(2)/CO(2) sensors. Increased numbers of PNEC/NEBs have been observed in a variety of perinatal and postnatal lung disorders. Recent advances in cellular and molecular biology of these cells, as they relate to perinatal and postnatal lung disorders associated with PNEC/NEB cell hyperplasia are reviewed and their possible role in pulmonary pathobiology discussed (WC 125).

Augmented 5-HT Secretion in Pulmonary Neuroepithelial Bodies from PHD1 Null Mice.

Sustained exposure to low oxygen concentration leads to profound changes in gene expression to restore oxygen homeostasis. Hypoxia-inducible factors (HIFs) comprise a group of transcription factors which accumulate under hypoxia and contribute to the complex changes in gene expression. Under normoxic conditions HIFs are degraded by prolyl-hydroxylases (PHD), however during hypoxia this degradation is inhibited causing HIF accumulation and subsequent changes in gene expression. Pulmonary neuroepithelial bodies (NEB) are innervated serotonin (5-HT)-producing cells distributed throughout the airway epithelium. These putative O(2) sensors are hypothesized to contribute to the ventilatory response to hypoxia. NEB dysfunction has been implicated in several paediatric lung diseases including neuroendocrine cell hyperplasia of infancy and sudden infant death syndrome, both characterized by a marked NEB hyperplasia with unknown functional significance. We have previously reported striking NEB hyperplasia in PHD1(-/-) mice making these mice a potential model to study the role of NEBs in paediatric lung diseases. Here we report in vitro studies on 5-HT release from NEB using this model.

Mutations in Plasmalemma Vesicle Associated Protein Result in Sieving Protein-Losing Enteropathy Characterized by Hypoproteinemia, Hypoalbuminemia, and Hypertriglyceridemia.

BACKGROUND & AIMS METHODS: Severe intestinal diseases observed in very young children are often the result of monogenic defects. We used whole exome sequencing (WES) to examine the genetic cause in a patient with a distinct severe form of protein losing enteropathy (PLE) characterized by hypoproteinemia, hypoalbuminemia, and hypertriglyceridemia. METHODS: WES was performed at the Centre for Applied Genomics, Hospital for Sick Children, Toronto, Canada. Exome library preparation was performed using the Ion Torrent AmpliSeq RDY Exome Kit. Functional studies were carried out based on the identified mutation. RESULTS: Using whole exome sequencing we identified a homozygous nonsense mutation (1072C>T; p.Arg358*) in the PLVAP (plasmalemma vesicle associated protein) gene in an infant from consanguineous parents who died at five months of age of severe protein losing enteropathy. Functional studies determined that the mutated PLVAP mRNA and protein were not expressed in the patient biopsy tissues, presumably secondary to nonsense-mediated mRNA decay. Pathological analysis showed that the loss of PLVAP resulted in disruption of endothelial fenestrated diaphragms. CONCLUSIONS: PLVAP p.Arg358* mutation resulted in loss of PLVAP expression with subsequent deletion of the diaphragms of endothelial fenestrae leading to plasma protein extravasation, protein-losing enteropathy and ultimately death.