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.

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.

Microvillus inclusion disease: loss of Myosin vb disrupts intracellular traffic and cell polarity.

Microvillus inclusion disease (MVID) is a congenital enteropathy characterized by loss of apical microvilli and formation of cytoplasmic inclusions lined by microvilli in enterocytes. MVID is caused by mutations in the MYO5B gene, coding for the myosin Vb motor protein. Although myosin Vb is implicated in the organization of intracellular transport and cell surface polarity in epithelial cells, its precise role in the pathogenesis of MVID is unknown. We performed correlative immunohistochemistry analyses of sections from duodenal biopsies of a MVID patient, compound heterozygous for two novel MYO5B mutations, predicting loss of function of myosin Vb in duodenal enterocytes together with a stable MYO5B CaCo2 RNAi cell system. Our findings show that myosin Vb-deficient enterocytes display disruption of cell polarity as reflected by mislocalized apical and basolateral transporter proteins, altered distribution of certain endosomal/lysosomal constituents including Rab GTPases. Together, this severe disturbance of epithelial cell function could shed light on the pathology and symptoms of MVID.

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.

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.

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.

Carbonic anhydrase II mediates malignant behavior of pulmonary neuroendocrine tumors.

In normal lung, the predominant cytoplasmic carbonic anhydrase (CA) isozyme (CAII) is highly expressed in amine- and peptide-producing pulmonary neuroendocrine cells where its role involves CO2 sensing. Here, we report robust cytoplasmic expression of CAII by immunohistochemistry in the tumor cells of different native neuroendocrine tumor (NET) types, including typical and atypical carcinoids and small-cell lung carcinomas, and in NET and non-NET tumor cell lines. Because, in both pulmonary neuroendocrine cell and related NETs, the hypercapnia-induced secretion of bioactive serotonin (5-hydroxytryptamine) is mediated by CAII, we investigated the role of CAII in the biological behavior of carcinoid cell line H727 and the type II cell-derived A549 using both in vitro clonogenicity and in vivo xenograft model. We show that short hairpin RNA-mediated down-regulation of CAII resulted in significant reduction in clonogenicity of H727 and A549 cells in vitro, and marked suppression of tumor growth in vivo. CAII-short hairpin RNA cell-derived xenografts showed significantly reduced mitosis (phosphohistone H3 marker) and proliferation associated antigen Ki-67 (Ki67 marker), and significantly increased apoptosis by terminal deoxynucleotidyl transferase dUTP nick end labeling assay. Using an apoptosis gene array, we found no association with caspases 3 and 8, but with a novel association of CAII-mediated apoptosis with specific mitochondrial apoptosis-associated proteins. Furthermore, these xenografts showed a significantly reduced vascularization (CD31 marker). Thus, CAII may play a critical role in NET lung tumor growth, angiogenesis, and survival, possibly via 5-hydroxytryptamine, known to drive autocrine tumor growth. As such, CAII is a potential therapeutic target for the difficult-to-treat lung NETs.

Recent advances and contraversies on the role of pulmonary neuroepithelial bodies as airway sensors.

Pulmonary neuroepithelial bodies are polymodal sensors widely distributed within the airway mucosa of mammals and other species. Neuroepithelial body cells store and most likely release serotonin and peptides as transmitters. Neuroepithelial bodies have a complex innervation that includes vagal sensory afferent fibers and dorsal root ganglion fibers. Neuroepithelial body cells respond to a number of intraluminal airway stimuli, including hypoxia, hypercarbia, and mechanical stretch. This article reviews recent findings in the cellular and molecular biology of neuroepithelial body cells and their potential role as airway sensors involved in the control of respiration, particularly during the perinatal period. Alternate hypotheses and areas of controversy regarding potential function as mechanosensory receptors involved in pulmonary reflexes are discussed.

Loss of syntaxin 3 causes variant microvillus inclusion disease.

Microvillus inclusion disease (MVID) is a disorder of intestinal epithelial differentiation characterized by life-threatening intractable diarrhea. MVID can be diagnosed based on loss of microvilli, microvillus inclusions, and accumulation of subapical vesicles. Most patients with MVID have mutations in myosin Vb that cause defects in recycling of apical vesicles. Whole-exome sequencing of DNA from patients with variant MVID showed homozygous truncating mutations in syntaxin 3 (STX3). STX3 is an apical receptor involved in membrane fusion of apical vesicles in enterocytes. Patient-derived organoid cultures and overexpression of truncated STX3 in Caco-2 cells recapitulated most characteristics of variant MVID. We conclude that loss of STX3 function causes variant MVID.

Mutations in tetratricopeptide repeat domain 7A result in a severe form of very early onset inflammatory bowel disease.

BACKGROUND & AIMS: Very early onset inflammatory bowel diseases (VEOIBD), including infant disorders, are a diverse group of diseases found in children younger than 6 years of age. They have been associated with several gene variants. Our aim was to identify the genes that cause VEOIBD. METHODS: We performed whole exome sequencing of DNA from 1 infant with severe enterocolitis and her parents. Candidate gene mutations were validated in 40 pediatric patients and functional studies were carried out using intestinal samples and human intestinal cell lines. RESULTS: We identified compound heterozygote mutations in the Tetratricopeptide repeat domain 7 (TTC7A) gene in an infant from non-consanguineous parents with severe exfoliative apoptotic enterocolitis; we also detected TTC7A mutations in 2 unrelated families, each with 2 affected siblings. TTC7A interacts with EFR3 homolog B to regulate phosphatidylinositol 4-kinase at the plasma membrane. Functional studies demonstrated that TTC7A is expressed in human enterocytes. The mutations we identified in TTC7A result in either mislocalization or reduced expression of TTC7A. Phosphatidylinositol 4-kinase was found to co-immunoprecipitate with TTC7A; the identified TTC7A mutations reduced this binding. Knockdown of TTC7A in human intestinal-like cell lines reduced their adhesion, increased apoptosis, and decreased production of phosphatidylinositol 4-phosphate. CONCLUSIONS: In a genetic analysis, we identified loss of function mutations in TTC7A in 5 infants with VEOIBD. Functional studies demonstrated that the mutations cause defects in enterocytes and T cells that lead to severe apoptotic enterocolitis. Defects in the phosphatidylinositol 4-kinase-TTC7A-EFR3 homolog B pathway are involved in the pathogenesis of VEOIBD.

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.

A novel nonsense mutation in the EpCAM gene in a patient with congenital tufting enteropathy.

OBJECTIVES: Tufting enteropathy (TE) is a classical congenital disorder of the intestinal mucosa causing protracted diarrhea in infancy as a result of a dysfunctional epithelial cell barrier, which is mainly caused by mutations in the EpCAM gene and expression of a nonfunctional epithelial cell adhesion molecule in the intestine. We report here a novel nonsense mutation in a patient suspected of having TE, resulting in a complete absence of EpCAM in duodenal enterocytes. METHODS: A patient presenting with congenital diarrhea and suspected of having TE was screened for EpCAM mutations, and duodenal biopsies were stained for EpCAM using immunohistochemistry analysis. RESULTS: We identified a novel homozygous nonsense mutation in the EpCAM gene in a patient suspected of having TE, causing a complete loss of EpCAM expression in duodenal enterocytes. CONCLUSIONS: With screening analysis for EpCAM mutations and immunohistochemistry for EpCAM expression in duodenal enterocytes, we found a novel homozygous mutation in a patient with classical protracted diarrhea in infancy finally diagnosed as TE, which results in a complete absence of EpCAM and in dysfunctional barrier formation in duodenal enterocytes.

The distribution and frequency of pulmonary neuroendocrine cells in Down syndrome fetal lungs.

The pulmonary neuroendocrine cells (PNEC) are located in the epithelial lining of the airways and consist of solitary neuroendocrine cells (NEC) and NEC clusters, the neuroepithelial bodies (NEB). During fetal life, PNEC are the first to differentiate within the primitive airway epithelium, and bombesin expression favors branching of the respiratory tree. We investigated PNEC in Down syndrome (DS), where the lungs often show enlarged and reduced number of alveoli. Immunohistochemistry for bombesin and synaptophysin, PNEC markers, was evaluated in fetal lungs from 15 cases of DS and 11 age-matched controls from the 17th to 23rd week of gestation. Morphometric analysis assessed PNEC in the mucosal lining of each lung, expressed as number/mm. Nonparametric Mann-Whitney U test showed no statistical difference in frequency of PNEC in DS and controls. Our findings suggest that, at least in late second trimester, the distribution and frequency of PNEC in DS fetuses is not altered.

Pulmonary hypertensive vasculopathy following tandem autologous transplantation in pediatric patients with central nervous system tumors.

Tandem cycles of high-dose chemotherapy are an increasingly being used as alternative to radiation treatment in the management of infants and young children with central nervous system (CNS) tumors. Most of these protocols have a carboplatinum and thiotepa backbone. The toxicities of these regimens have been reported extensively; however, pulmonary arterial vasculopathy (PAV) with pulmonary arterial hypertension (PAH) has not been previously documented in patients treated with this approach. PAH is a disorder of the pulmonary vasculature characterized by a progressive increase in pulmonary vascular resistance, leading to right heart failure and potentially death. We evaluated PAH as a complication after high-dose chemotherapy and autologous stem cell transplantation (SCT). We performed a retrospective evaluation of all pediatric patients diagnosed with a CNS tumor between 2001 and 2010 scheduled to receive 3 cycles of high-dose chemotherapy with carboplatinum (17 mg/kd/day for 2 days) and thiotepa (10 mg/kg/day for 2 days), followed by autologous SCT. Our primary objective was to evaluate the incidence of PAV and PAH in this population, as well as patient outcomes after the development of PAH. Our cohort comprised 20 patients with a median age at diagnosis of 28 months (range, 3-131 months). Three patients developed biopsy-confirmed PAV with PAH, the 2 patients who developed PAV with PAH at the end of the third cycle succumbed to PAH. Death due to PAV and PAH was the sole toxic mortality observed during the study period. PAV with PAH is a major and possibly fatal complication after high-dose chemotherapy and sequential autologous SCT using carboplatinum and thiotepa in a tandem fashion. There is an urgent need to evaluate PAH as a potential complication after each cycle of high-dose chemotherapy when using such regimens in pediatric patients with CNS tumors.

Combination of carbonic anhydrase inhibitor, acetazolamide, and sulforaphane, reduces the viability and growth of bronchial carcinoid cell lines.

BACKGROUND: Bronchial carcinoids are pulmonary neuroendocrine cell-derived tumors comprising typical (TC) and atypical (AC) malignant phenotypes. The 5-year survival rate in metastatic carcinoid, despite multiple current therapies, is 14-25%. Hence, we are testing novel therapies that can affect the proliferation and survival of bronchial carcinoids. METHODS: In vitro studies were used for the dose-response (AlamarBlue) effects of acetazolamide (AZ) and sulforaphane (SFN) on clonogenicity, serotonin-induced growth effect and serotonin content (LC-MS) on H-727 (TC) and H-720 (AC) bronchial carcinoid cell lines and their derived NOD/SCID mice subcutaneous xenografts. Tumor ultra structure was studied by electron microscopy. Invasive fraction of the tumors was determined by matrigel invasion assay. Immunohistochemistry was conducted to study the effect of treatment(s) on proliferation (Ki67, phospho histone-H3) and neuroendocrine phenotype (chromogranin-A, tryptophan hydroxylase). RESULTS: Both compounds significantly reduced cell viability and colony formation in a dose-dependent manner (0-80 µM, 48 hours and 7 days) in H-727 and H-720 cell lines. Treatment of H-727 and H-720 subcutaneous xenografts in NOD/SCID mice with the combination of AZ + SFN for two weeks demonstrated highly significant growth inhibition and reduction of 5-HT content and reduced the invasive capacity of H-727 tumor cells. In terms of the tumor ultra structure, a marked reduction in secretory vesicles correlated with the decrease in 5-HT content. CONCLUSIONS: The combination of AZ and SFN was more effective than either single agent. Since the effective doses are well within clinical range and bioavailability, our results suggest a potential new therapeutic strategy for the treatment of bronchial carcinoids.

From diarrhea to obesity in prohormone convertase 1/3 deficiency: age-dependent clinical, pathologic, and enteroendocrine characteristics.

GOALS: The aim of this report is to delineate the clinical, pathologic, and enteroendocrine (EE) features of prohormone convertase 1/3 (PC1/3) deficiency in children. BACKGROUND: Prohormone convertases play a pivotal role in the activation of biologically inactive hormones. Congenital defects in the EE axis, such as PC1/3 deficiency, have been rarely reported and their pathophysiological mechanisms are largely unknown. STUDY: EE function and pathology was evaluated in 4 males (1, 2, 7, and 10 y old) from 2 families with PC1/3 deficiency at a university children's hospital. Clinical course, pathology analysis including immunohistochemistry for PC1/3, PC2, and glucagon-like peptide 1 (GLP-1) and electron microscopy, as well as EE function tests (GLP-1, GLP-2, oral glucose tolerance test) were performed. RESULTS: All (n=4) suffered from congenital severe diarrhea associated with malabsorption. The diarrhea improved during the first year of life and hyperphagia with excessive weight gain (BMI>97th percentile) became the predominant phenotype at an older age. Analysis of the enteroendocrine axis revealed high proinsulin levels (57 to 1116 pmol/L) in all patients, low serum GLP-2 levels, and impaired insulin and GLP-1 secretion after an oral glucose tolerance test at a young age, with improvement in 1 older child tested. Electron microscopy showed normal ultrastructure of enterocytes and EE cells. Immunohistochemistry revealed normal expression of chromogranin A, a marker of EE cells but markedly reduced immunostaining for PC1/3 and PC2 in all patients. CONCLUSIONS: PC1/3 deficiency is associated with an age dependent, variable clinical phenotype caused by severe abnormalities in intestinal and EE functions. Serum level of proinsulin can be used as an effective screening tool.

Successful liver transplantation in an infant with stage 4S(M) neuroblastoma.

We report a 2.5-month-old infant with bilateral adrenal neuroblastoma, stage 4S(M), with liver metastases and chemotherapy-induced veno-occlusive disease leading to cirrhosis requiring liver transplantation. Despite unknown tumour histology and MYCN-amplification status, we proceeded with liver transplant. This decision was based on clinical suspicion that our patient was MYCN-negative due to significant tumour regression, and was supported by evidence indicating that MYCN-amplification is rare in infants with favourable-stage neuroblastoma. This is the second case report of neuroblastoma requiring liver transplantation; however, in the previously reported case, the diagnosis of neuroblastoma was not established until after transplantation. We discuss this unique case to justify the potential use of life-saving liver transplants in infants with neuroblastoma.

Pulmonary alveolar proteinosis in patients with adenosine deaminase deficiency.

BACKGROUND: Inherited defects in the function of adenosine deaminase (ADA) cause severe combined immunodeficiency (SCID) and affect many other cells and tissues. OBJECTIVES: We sought to characterize the frequency and features of pulmonary alveolar proteinosis (PAP) in patients with ADA deficiency. METHODS: Clinical and laboratory features of all patients with SCID caused by ADA deficiency in a single center were analyzed. Bronchoalveolar lavage (BAL) fluid and lung biopsy specimens were stained with hematoxylin and eosin and periodic acid-Schiff, visualized by means of electron microscopy, and studied for associated infections. As a control group, BAL fluid and biopsy specimens from 22 patients with SCID caused by other genetic abnormalities were similarly assessed. RESULTS: Among 16 consecutive patients with ADA deficiency, 7 had BAL fluid containing periodic acid-Schiff-positive surfactant-like material with macrophages engulfing degenerating lamellar bodies and/or lung biopsy specimens with alveolar spaces filled with homogeneous granular eosinophilic material and large macrophages. The lung pathology was typical of PAP. Identification of various pathogens coincided with PAP in 3 of these patients. We have diagnosed PAP among patients with ADA deficiency more commonly in the last 10 years than previously (P= .05), likely reflecting increased awareness of this condition. There were no significant differences in the clinical or immunologic characteristics between patients with ADA deficiency with or without PAP. Similar findings of PAP were not found among patients with SCID caused by other genetic abnormalities (P= .001). ADA coupled to polyethylene glycol or allogeneic hematopoietic stem cell transplantation rapidly corrected this pulmonary complication. PAP seems to have contributed to the death of only 1 patient with ADA deficiency. CONCLUSIONS: ADA deficiency predisposes to the development of PAP, which could be reversed after enzyme replacement or transplantation.

NOX2 (gp91phox) is a predominant O2 sensor in a human airway chemoreceptor cell line: biochemical, molecular, and electrophysiological evidence.

Pulmonary neuroepithelial bodies (NEBs), composed of clusters of amine [serotonin (5-HT)] and peptide-producing cells, are widely distributed within the airway mucosa of human and animal lungs. NEBs are thought to function as airway O(2)-sensors, since they are extensively innervated and release 5-HT upon hypoxia exposure. The small cell lung carcinoma cell line (H146) provides a useful model for native NEBs, since they contain (and secrete) 5-HT and share the expression of a membrane-delimited O(2) sensor [classical NADPH oxidase (NOX2) coupled to an O(2)-sensitive K(+) channel]. In addition, both native NEBs and H146 cells express different NADPH oxidase homologs (NOX1, NOX4) and its subunits together with a variety of O(2)-sensitive voltage-dependent K(+) channel proteins (K(v)) and tandem pore acid-sensing K(+) channels (TASK). Here we used H146 cells to investigate the role and interactions of various NADPH oxidase components in O(2)-sensing using a combination of coimmunoprecipitation, Western blot analysis (quantum dot labeling), and electrophysiology (patchclamp, amperometry) methods. Coimmunoprecipitation studies demonstrated formation of molecular complexes between NOX2 and K(v)3.3 and K(v)4.3 ion channels but not with TASK1 ion channels, while NOX4 associated with TASK1 but not with K(v) channel proteins. Downregulation of mRNA for NOX2, but not for NOX4, suppressed hypoxia-sensitive outward current and significantly reduced hypoxia -induced 5-HT release. Collectively, our studies suggest that NOX2/K(v) complexes are the predominant O(2) sensor in H146 cells and, by inference, in native NEBs. Present findings favor a NEB cell-specific plasma membrane model of O(2)-sensing and suggest that unique NOX/K(+) channel combinations may serve diverse physiological functions.