A fetal wound healing program after intrauterine bile duct injury may contribute to biliary atresia.

BACKGROUND & AIMS: Biliary atresia (BA) is an obstructive cholangiopathy that initially affects the extrahepatic bile ducts (EHBDs) of neonates. The etiology is uncertain, but evidence points to a prenatal cause. Fetal tissues have increased levels of hyaluronic acid (HA), which plays an integral role in fetal wound healing. The objective of this study was to determine whether a program of fetal wound healing is part of the response to fetal EHBD injury. METHODS: Mouse, rat, sheep, and human EHBD samples were studied at different developmental time points. Models included a fetal sheep model of prenatal hypoxia, human BA EHBD remnants and liver samples taken at the time of the Kasai procedure, EHBDs isolated from neonatal rats and mice, and spheroids and other models generated from primary neonatal mouse cholangiocytes. RESULTS: A wide layer of high molecular weight HA encircling the lumen was characteristic of the normal perinatal but not adult EHBD. This layer, which was surrounded by collagen, expanded in injured ducts in parallel with extensive peribiliary gland hyperplasia, increased mucus production and elevated serum bilirubin levels. BA EHBD remnants similarly showed increased HA centered around ductular structures compared with age-appropriate controls. High molecular weight HA typical of the fetal/neonatal ducts caused increased cholangiocyte spheroid growth, whereas low molecular weight HA induced abnormal epithelial morphology; low molecular weight HA caused matrix swelling in a bile duct-on-a-chip device. CONCLUSION: The fetal/neonatal EHBD, including in human EHBD remnants from Kasai surgeries, demonstrated an injury response with prolonged high levels of HA typical of fetal wound healing. The expanded peri-luminal HA layer may swell and lead to elevated bilirubin levels and obstruction of the EHBD. IMPACT AND IMPLICATIONS: Biliary atresia is a pediatric cholangiopathy associated with high morbidity and mortality rates; although multiple etiologies have been proposed, the fetal response to bile duct damage is largely unknown. This study explores the fetal pathogenesis after extrahepatic bile duct damage, thereby opening a completely new avenue to study therapeutic targets in the context of biliary atresia.

Prominent Pseudoacini in Focal Nodular Hyperplasia: A Potential Diagnostic Pitfall.

Pseudoacini are generally a morphologic feature of hepatocellular carcinoma (HCC), being absent or rare in benign hepatocytic tumors, such as hepatocellular adenoma. However, rarely these can be seen in focal nodular hyperplasia (FNH) and may pose diagnostic challenges, especially when prominent. The study was aimed to evaluate the occurrence of pseudoacini in FNH and their clinicopathologic correlations. A total of 95 FNH cases diagnosed from 2005 to 2020 were included in the study. A pseudoacinus was defined as a circular arrangement of hepatocytes around a central dilated lumen present within the lobular parenchyma of the lesion with or without inspissated bile. Among the 95 FNH cases, 28 (29.5%) showed pseudoacini, which were prominent in 12 (12.6%) cases. Of these 3 occurred in patients above 50 years old. The pseudoacini were numerous in 3 cases, leading to an initial consideration of HCC in the differential diagnosis, and 1 case was diagnosed as well-differentiated hepatocellular neoplasm on initial biopsy. All 12 cases showed map-like staining pattern for glutamine synthetase. The hepatocytes forming the pseudoacini were positive for CK7 and HepPar1, while the inner lumina were highlighted by CD10 and bile salt export pump immunostains similar to adjacent canaliculi. The presence of prominent pseudoacini was not significantly associated with any clinical or pathologic features. The findings suggest that pseudoacini are likely manifestation of hepatocyte biliary transdifferentiation associated with chronic cholestasis in the lesion. This feature may pose a potential diagnostic pitfall especially on needle biopsies and awareness is needed to avoid misdiagnosing this as HCC.

Spindle epithelial tumor with thymus-like differentiation in fine needle aspiration of thyroid gland: Report of two cases.

Spindle epithelial tumor with thymus-like differentiation (SETTLE) is a rare, malignant tumor of the thyroid gland that typically affects young males and has a propensity for late metastasis. With fine needle aspiration (FNA) being a primary tool for diagnosis of thyroid lesions, there are rare reports of cytological features of SETTLE on FNA since its initial characterization 30 years ago . Here we report two cases of SETTLE, involving 9-year-old and 15-year-old male patients. Both patients underwent US-guided FNA with a subsequent resection confirming the diagnosis of SETTLE. In the first patient the thymic origin of the tumor was suspected on FNA, but the diagnosis of SETTLE was established only after resection. Five years later, this patient presented with an enlarged ipsilateral cervical lymph node. Needle biopsy confirmed it to be a metastatic tumor compatible with SETTLE. In the second patient the diagnosis of SETTLE was suggested on FNA. Cytology of the thyroid gland nodule on FNA from both patients showed loosely cohesive and single spindle-shaped epithelial cells associated with metachromatic stroma. The differential diagnosis of spindle cell lesions of the thyroid should include SETTLE based on characteristic morphological features, after more common entities of thyroid gland such as medullary carcinoma are excluded.

NTRK Fusions Identified in Pediatric Tumors: The Frequency, Fusion Partners, and Clinical Outcome.

PURPOSE: Neurotrophic tyrosine receptor kinase (NTRK) fusions have been described as oncogenic drivers in a variety of tumors. However, little is known about the overall frequency of NTRK fusion in unselected pediatric tumors. Here, we assessed the frequency, fusion partners, and clinical course in pediatric patients with NTRK fusion-positive tumors. PATIENTS AND METHODS: We studied 1,347 consecutive pediatric tumors from 1,217 patients who underwent tumor genomic profiling using custom-designed DNA and RNA next-generation sequencing panels. NTRK fusions identified were orthogonally confirmed. RESULTS AND DISCUSSION: NTRK fusions were identified in 29 tumors from 27 patients with a positive yield of 2.22% for all patients and 3.08% for solid tumors. Although NTRK2 fusions were found exclusively in CNS tumors and NTRK1 fusions were highly enriched in papillary thyroid carcinomas, NTRK3 fusions were identified in all tumor categories. The most canonical fusion was ETV6-NTRK3 observed in 10 patients with diverse types of tumors. Several novel NTRK fusions were observed in rare tumor types, including KCTD16-NTRK1 in ganglioglioma and IRF2BP2-NTRK3 in papillary thyroid carcinomas. The detection of an NTRK fusion confirmed the morphologic diagnosis including five cases where the final tumor diagnosis was largely based on the discovery of an NTRK fusion. In one patient, the diagnosis was changed because of the identification of an ETV6-NTRK3 fusion. One patient with infantile fibrosarcoma was treated with larotrectinib and achieved complete pathologic remission. CONCLUSION: NTRK fusions are more frequently seen in pediatric tumors than in adult tumors and involve a broader panel of fusion partners and a wider range of tumors than previously recognized. These results highlight the importance of screening for NTRK fusions as part of the tumor genomic profiling for patients with pediatric cancer.

Immune Dysregulation in Human ITCH Deficiency Successfully Treated with Hematopoietic Cell Transplantation.

BACKGROUND: Mutations in ITCH, which encodes an E3 ubiquitin-protein ligase, can result in systemic autoimmunity and immunodeficiency. The clinical phenotype and mechanism of disease have not been fully characterized, resulting in a paucity of therapeutic options for this potentially fatal disease. OBJECTIVE: We aimed to (1) expand the understanding about the phenotype of human ITCH deficiency (2) further characterize the associated immune dysregulation, and (3) report the first successful hematopoietic cell transplant (HCT) in a patient with ITCH deficiency. METHODS: Disease profiling was performed in a patient with multisystem immune dysregulation. Whole exome sequencing with trio analysis and functional validation of candidate disease variants were performed, including mRNA and protein expression. Analyses to further delineate the immunophenotype included quantitative evaluation of lymphoid and myeloid subsets with flow cytometry and mass cytometry. RESULTS: A patient with multisystem immune dysregulation presenting with growth failure, very-early-onset inflammatory bowel disease, arthritis, uveitis, psoriasis, and type 1 diabetes mellitus underwent whole exome sequencing, which identified novel compound heterozygous mutations in ITCH. Reduced expression of ITCH mRNA and absent ITCH protein were found. Abnormalities in both lymphoid and myeloid lineages were identified. The patient underwent HCT. He demonstrated excellent immune reconstitution and resolution of many manifestations of his systemic disease. CONCLUSIONS: Here we report ITCH deficiency with unique clinical features of colonic very-early-onset inflammatory bowel disease, arthritis, and uveitis in the setting of immune dysregulation and further characterize the underlying immune dysregulation. We demonstrate that HCT can be an effective, and potentially curative, therapy for ITCH deficiency.

Colonoids From Patients With Pediatric Inflammatory Bowel Disease Exhibit Decreased Growth Associated With Inflammation Severity and Durable Upregulation of Antigen Presentation Genes.

BACKGROUND: Defining epithelial cell contributions to inflammatory bowel disease (IBD) is essential for the development of much needed therapies for barrier repair. Children with very early onset (VEO)-IBD have more extensive, severe, and refractory disease than older children and adults with IBD and, in some cases, have defective barrier function. We therefore evaluated functional and transcriptomic differences between pediatric IBD (VEO and older onset) and non-IBD epithelium using 3-dimensional, biopsy-derived organoids. METHODS: We measured growth efficiency relative to histopathological and clinical parameters in patient enteroid (ileum) and colonoid (colon) lines. We performed RNA-sequencing on patient colonoids and subsequent flow cytometry after multiple passages to evaluate changes that persisted in culture. RESULTS: Enteroids and colonoids from pediatric patients with IBD exhibited decreased growth associated with histological inflammation compared with non-IBD controls. We observed increased LYZ expression in colonoids from pediatric IBD patients, which has been reported previously in adult patients with IBD. We also observed upregulation of antigen presentation genes HLA-DRB1 and HLA-DRA, which persisted after prolonged passaging in patients with pediatric IBD. CONCLUSIONS: We present the first functional evaluation of enteroids and colonoids from patients with VEO-IBD and older onset pediatric IBD, a subset of which exhibits poor growth. Enhanced, persistent epithelial antigen presentation gene expression in patient colonoids supports the notion that epithelial cell-intrinsic differences may contribute to IBD pathogenesis.

Coordinated development of the mouse extrahepatic bile duct: Implications for neonatal susceptibility to biliary injury.

BACKGROUND & AIMS: The extrahepatic bile duct is the primary tissue initially affected by biliary atresia. Biliary atresia is a cholangiopathy which exclusively affects neonates. Current animal models suggest that the developing bile duct is uniquely susceptible to damage. In this study, we aimed to define the anatomical and functional differences between the neonatal and adult mouse extrahepatic bile ducts. METHODS: We studied mouse passaged cholangiocytes, mouse BALB/c neonatal and adult primary cholangiocytes, as well as isolated extrahepatic bile ducts, and a collagen reporter mouse. The methods used included transmission electron microscopy, lectin staining, immunostaining, rhodamine uptake assays, bile acid toxicity assays, and in vitro modeling of the matrix. RESULTS: The cholangiocyte monolayer of the neonatal extrahepatic bile duct was immature, lacking the uniform apical glycocalyx and mature cell-cell junctions typical of adult cholangiocytes. Functional studies showed that the glycocalyx protected against bile acid injury and that neonatal cholangiocyte monolayers were more permeable than adult monolayers. In adult ducts, the submucosal space was filled with collagen I, elastin, hyaluronic acid, and proteoglycans. In contrast, the neonatal submucosa had little collagen I and elastin, although both increased rapidly after birth. In vitro modeling of the matrix suggested that the composition of the neonatal submucosa relative to the adult submucosa led to increased diffusion of bile. A Col-GFP reporter mouse showed that cells in the neonatal but not adult submucosa were actively producing collagen. CONCLUSION: We identified 4 key differences between the neonatal and adult extrahepatic bile duct. We showed that these features may have functional implications, suggesting the neonatal extrahepatic bile ducts are particularly susceptible to injury and fibrosis. LAY SUMMARY: Biliary atresia is a disease that affects newborns and is characterized by extrahepatic bile duct injury and obstruction, resulting in liver injury. We identify 4 key differences between the epithelial and submucosal layers of the neonatal and adult extrahepatic bile duct and show that these may render the neonatal duct particularly susceptible to injury.

Cardiac Magnetic Resonance-Derived Metrics Are Predictive of Liver Fibrosis in Fontan Patients.

BACKGROUND: Liver fibrosis is a serious complication of single ventricle Fontan survivors. Its causes are of great interest, and potential solutions to halt or delay progression are needed. The purpose of this study is to investigate if prior hemodynamics and anatomy can predict liver fibrosis severity in these patients. METHODS: Twenty-one Fontan patients with cardiac magnetic resonance (CMR) data obtained greater than 1 year before liver biopsy data were included. Computational fluid dynamic simulations were performed to quantify total cavopulmonary connection (TCPC) flow dynamics using patient-specific anatomies and blood flow waveforms reconstructed from CMR data. Collagen deposition (a measure of liver fibrosis) was quantified by digital image analysis of Sirius red-stained slides. Statistical analyses were performed to investigate potential relationships between Fontan hemodynamics and liver fibrosis. RESULTS: With an average time of 6.7 ± 2.9 years (range, 2-11 years) between CMR and biopsy, TCPC resistance and left pulmonary artery stenosis showed significant, positive correlations with magnitude of liver fibrosis (r = 0.54, P = .026; and r = 0.55, P = .028, respectively). The change in inferior vena cava flow rate over time also showed a significant positive correlation with magnitude of liver fibrosis (r = 0.91, P = .001). CONCLUSIONS: TCPC resistance, left pulmonary artery stenosis, and increased inferior vena cava flow are positively associated with liver fibrosis after Fontan operation and hold promise as important predictors of hepatic decline. These findings encourage preprocedural planning and interventional strategies to improve TCPC performance and reduce vessel stenosis. Further investigation is warranted to design the ideal Fontan circulation and optimize flow dynamics to reduce the risk of liver fibrosis.

Percutaneous liver biopsy in Fontan patients.

BACKGROUND: Patients who have undergone the Fontan operation for palliation of congenital heart disease with single-ventricle pathophysiology are at high risk for developing progressive liver fibrosis. Pathological assessment from percutaneous liver biopsy is central to the management of Fontan-associated liver disease, but liver biopsy in this vulnerable population poses unique challenges and potential risks. OBJECTIVE: This retrospective study describes our experience with percutaneous liver biopsy performed to assess changes of Fontan-associated liver disease, with particular regard to procedural outcomes. MATERIALS AND METHODS: Data from liver biopsy procedure reports, pathology reports, cardiac angiography pressure measurements and laboratory values of patients with single ventricle heart disease after the Fontan operation who underwent ultrasound-guided percutaneous liver biopsy performed in interventional radiology at a pediatric tertiary care center during a 3-year period were retrospectively analyzed. RESULTS: Sixty-eight liver biopsies were performed in 67 patients (mean age: 20.2 years, range: 7.2-39 years). The technical success rate was 100%, and tissue was adequate for assessing liver disease in 100% of the procedures, including biopsies performed with a single pass. Anticoagulation was routinely suspended before biopsy, and no cardiac complications were encountered due to this suspension. A coaxial biopsy system using an 18-gauge (G) full-core instrument through a 17-G introducer trocar was most commonly used, in 41/68 cases (60%). The most common trough length was 2.3 cm, used in 37 cases (54%). One pass was made in 27 procedures (40%) and two passes in 30 (44%); tract embolization with gelatin sponge was performed in 52 (76%). The only complication was hemorrhage, which occurred in 5/68 (7.4%) of the biopsies, minor in four (5.9%) and major in one (1.5%) -- similar to rates reported for liver biopsy in non-Fontan patients. Hemorrhage had a delayed presentation in three of these five cases. Immediate post-biopsy hemoglobin decrease of ≥2 mg/dL showed a low sensitivity for hemorrhage. The mean Fontan pressure measured during cardiac angiography was 13.8 mmHg, and shunt pressures were not associated with an increased risk of hemorrhage. CONCLUSION: Percutaneous liver biopsy in Fontan patients can be performed safely with high technical success rates and without increased complication rates. Meticulous technique and close observation are recommended to reduce post-biopsy complications. The degree of right heart pressure elevation was not associated with hemorrhage.

Impact of hemodynamics and fluid energetics on liver fibrosis after Fontan operation.

OBJECTIVE: The staged Fontan procedure has shown promising short-term outcomes in patients with single ventricles. However, Fontan-associated liver disease is a marked problem as patients age. The purpose of this study is to investigate the relationship between hemodynamics and liver fibrosis in patients undergoing the Fontan. METHODS: A total of 33 patients undergoing the Fontan with liver fibrosis were included in this study. Cardiac magnetic resonance and phase-contrast cardiac magnetic resonance data, as well as catheterization measurements and liver biopsies, were obtained for each patient. Computational fluid dynamic simulations were performed to quantify total cavopulmonary connection hemodynamics using patient-specific anatomies and blood flow waveforms reconstructed from cardiac magnetic resonance data. Collagen deposition (as a measure of liver fibrosis) was quantified by digital image analysis of Sirius Red stained slides. Statistical analyses were performed to investigate potential relationships between liver fibrosis and Fontan hemodynamics. RESULTS: Liver fibrosis was found to be related to global metrics (inferior vena cava flow, ventricle power output) rather than to local total cavopulmonary connection hemodynamics and efficiency. Indexed inferior vena cava flow showed a significant, positive correlation with liver fibrosis (rho = 0.624, P < .001). Upper and lower Sirius Red tertile comparisons showed a significant difference in indexed inferior vena cava flow (P = .008). CONCLUSIONS: Significant increases in inferior vena cava flow consistent with fibrosis induced arterialization and ventricular power output suggest a burden being placed on the single ventricle from liver fibrosis. Local total cavopulmonary connection flow dynamics do not seem to influence the degree of fibrosis. Favorable total cavopulmonary connection hemodynamics may not be enough to overcome the power shortage and elevated venous pressures inherent to a Fontan circulation.

Features of Adult Autoimmune Enteropathy Compared With Refractory Celiac Disease.

BACKGROUND & AIMS: Little is known about the features of immune-mediated non-celiac villous atrophies, such as autoimmune enteropathy (AIE). We investigated the demographic, clinical, and histologic features of adults with AIE compared to adults with refractory celiac disease type 1. We also report outcomes of treatment with open-label budesonide. METHODS: We performed a retrospective case-control of patients with AIE (n = 30) seen at the Mayo Clinic (in Rochester, Minnesota) from 2000 through 2015. Patients with refractory celiac disease type 1 who were treated with open-label budesonide served as controls (n = 42). Biopsy specimens were reviewed for all patients. We collected demographic, clinical, biochemical and histologic data from patients. We also collected data on responses to open-capsule budesonide from patients with AIE (available from 22 patients) and controls (available from 42 patients); the median duration of follow up was 28 months (range, 0-1421 months). RESULTS: Patients with AIE had a higher proportion of men (60%) and were younger (mean, 44 ± 18 years) than patients with refractory celiac disease type 1 (29% men; P = .002 and mean age, 57 ± 16 years; P = .007). A higher proportion of patients with AIE presented with chronic diarrhea (100%) and weight loss (90%) than patients with refractory celiac disease type 1 (71%; P < .001 and 71%; P = .05, respectively). Based on histologic analysis, there was no significant difference in degree of villous atrophy in intestinal tissues from patients with AIE vs controls (P = .68). However, a greater proportion of patients with RCD had increased intraepithelial lymphocytes (>40 per 100 epithelial cells in 100%) compared with patients with AIE (in 50%) (P = .003). Conventional therapy (systemic steroids) had failed in most patients with AIE (a complete clinical response was reported in only 7 patients) before treatment with open-capsule budesonide was initiated. A clinical response to open-capsule budesonide was reported for 85% of patients with AIE (50% complete response, 35% partial response) compared to 92% of controls (68% complete response, 24% partial response). CONCLUSIONS: In a retrospective study of 30 patients with AIE, followed for a median 28 months, we found this disease to have has distinct demographic, clinical, and histologic characteristics compared to refractory celiac disease type 1. Most patients with AIE (85%) have a clinical response to budesonide, all of whom were unsuccessfully treated with conventional therapies.

An Atypical Presentation of Alagille Syndrome.

Alagille syndrome is a multisystem disorder classically involving the liver, heart, vertebrae, facial features, and the eyes. In this case report, we document a case of Alagille syndrome with an atypical clinical and histopathologic presentation and subsequent identification of a novel JAG1 missense mutation. This case highlights that there may be both atypical clinical and pathologic findings in mutation-proven Alagille syndrome and that the diagnosis of Alagille syndrome should be considered in cases of ongoing bile duct damage in the setting of early-onset jaundice, cholestasis, hepatosplenomegaly, posterior embryotoxon in the eyes, and butterfly vertebrae.

A Rare BSEP Mutation Associated with a Mild Form of Progressive Familial Intrahepatic Cholestasis Type 2

ABSTRACT Progressive Familial Intrahepatic Cholestasis type 2 (PFIC2) is a rare cholestatic disorder diagnosed in infancy or childhood that can lead to severe hepatic fibrosis and liver failure. Mutations in the ABCB11 gene result in a deficiency of the bile salt export protein (BSEP) and accumulation of bile inside the hepatocytes. Hepatocellular carcinoma is another condition associated with severe forms of deletion mutations in the ABCB11 gene. Treatment options including ursodeoxycholic acid biliary diversion have mixed outcomes and some patients require liver transplantation. Here, we describe two siblings with an extremely mild form of PFIC2 inherited from heterozygous parents. The elder sibling had acute liver failure at the age of six months and both siblings had pruritus, cholestasis, coagulopathy and fat-soluble-vitamin deficiencies in infancy but have been asymptomatic past infancy. Genetic testing of the siblings revealed that each were compound heterozygotes for two missense mutations of the ABCB11 gene: p.C68Y and p.R832H. Medical treatment typical for PFIC2 has not been necessary for either patient. This is the first report of these variants following a mild course in two affected patients.(AU)

A Rare BSEP Mutation Associated with a Mild Form of Progressive Familial Intrahepatic Cholestasis Type 2.

Progressive Familial Intrahepatic Cholestasis type 2 (PFIC2) is a rare cholestatic disorder diagnosed in infancy or childhood that can lead to severe hepatic fibrosis and liver failure. Mutations in the ABCB11 gene result in a deficiency of the bile salt export protein (BSEP) and accumulation of bile inside the hepatocytes. Hepatocellular carcinoma is another condition associated with severe forms of deletion mutations in the ABCB11 gene. Treatment options including ursodeoxycholic acid biliary diversion have mixed outcomes and some patients require liver transplantation. Here, we describe two siblings with an extremely mild form of PFIC2 inherited from heterozygous parents. The elder sibling had acute liver failure at the age of six months and both siblings had pruritus, cholestasis, coagulopathy and fat-soluble-vitamin deficiencies in infancy but have been asymptomatic past infancy. Genetic testing of the siblings revealed that each were compound heterozygotes for two missense mutations of the ABCB11 gene: p.C68Y and p.R832H. Medical treatment typical for PFIC2 has not been necessary for either patient. This is the first report of these variants following a mild course in two affected patients.

The Role of MicroRNAs and Human Epidermal Growth Factor Receptor 2 in Proliferative Lupus Nephritis.

OBJECTIVE: To understand the roles of microRNAs (miRNAs) in proliferative lupus nephritis (LN). METHODS: A high-throughput analysis of the miRNA pattern of the kidneys of LN patients and controls was performed by molecular digital detection. Urinary miRNAs were measured by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Target gene expression in human mesangial cells was evaluated by arrays and qRT-PCR. Human epidermal growth factor receptor 2 (HER-2) was analyzed by immunohistochemistry in kidney samples from LN patients and in a murine model of lupus. Urinary levels of HER-2, monocyte chemotactic protein 1 (MCP-1), and vascular cell adhesion molecule 1 (VCAM-1) were measured by enzyme-linked immunosorbent assay. RESULTS: Levels of the miRNAs miR-26a and miR-30b were decreased in the kidneys and urine of LN patients. In vitro these miRNAs controlled mesangial cell proliferation, and their expression was regulated by HER-2. HER-2 was overexpressed in lupus-prone NZM2410 mice and in the kidneys of patients with LN, but not in other mesangioproliferative glomerulonephritides. HER-2 was found to be up-regulated by interferon-α and interferon regulatory factor 1. Urinary HER-2 was increased in LN and reflected disease activity, and its levels correlated with those of 2 other recognized LN biomarkers, MCP-1 and VCAM-1. CONCLUSION: The kidney miRNA pattern is broadly altered in LN, which contributes to uncontrolled cell proliferation. Levels of the miRNAs miR-26a and miR-30b are decreased in the kidneys and urine of LN patients, and they directly regulate the cell cycle in mesangial cells. The levels of these miRNAs are controlled by HER-2, which is overexpressed in NZM2410 mice and in the kidneys and urine of LN patients. HER-2, miR-26a, and miR-30b are thus potential LN biomarkers, and blocking HER-2 may be a promising new strategy to decrease cell proliferation and damage in this disease.

The inflammatory phenotype of the fibrous plate is distinct from the liver and correlates with clinical outcome in biliary atresia.

Biliary atresia is an inflammatory cholangiopathy of still undetermined etiology. Correlations between histologic findings and clinical outcome in this disease have largely been based on evaluation of liver parenchyma. This study aimed to characterize the pattern of inflammation within the biliary remnant and identify associations between the type and degree of inflammation and clinical outcome as reflected by the transplant-free interval. The inflammation within the fibrous plates and livers of 41 patients with biliary atresia was characterized using immunohistochemical markers and the cell populations were digitally quantified. The type and quantity of cells within the infiltrate were then correlated with length of time from Kasai portoenterostomy until transplant. Histologic and immunohistochemical features of the biliary remnant allowed stratification of patients into "inflammatory plate" and "fibrotic plate" groups. Overall there was no significant difference in transplant-free interval between the two cohorts; however, there was a trend towards a longer time to transplant among patients in the "fibrotic plate" group. In addition, the composition of the inflammatory infiltrate in the fibrous plate was distinctly different from that present in the liver and only the characteristics of the inflammation in the fibrous plate, in particular the number of Foxp3+ T regulatory lymphocytes correlated with clinical outcome. The results of this study support the view of the extra-hepatic biliary tree as the primary site of injury in BA with the changes seen in the liver as secondary manifestations of outflow obstruction. The association between specific inflammatory cell subtypes within the fibrous plate and the length of transplant-free interval also supports the role of the immune system in the initial process of bile duct damage in biliary atresia.

Replication of a GWAS signal in a Caucasian population implicates ADD3 in susceptibility to biliary atresia.

In the United States, biliary atresia (BA) is the most frequent indication for liver transplantation in pediatric patients. BA is a complex disease, with suspected environmental and genetic risk factors. A genome-wide association study in Chinese patients identified association to the 10q24.2 (hg18) genomic region. This signal was upstream of two genes, XPNPEP1 and ADD3, both expressed in intrahepatic bile ducts. We tested association to this region in 171 BA patients and 1,630 controls of European descent and found the strongest signal to be at rs7099604 (p = 2.5 × 10(-3)) in intron 1 of the ADD3 gene. Moreover, expression data suggest that ADD3, but not XPNPEP1, is differentially expressed in BA patients. The role of ADD3 in biliary development is unclear, but our findings suggest that this gene may be functionally relevant for the development of BA.

Extrahepatic cholangiocyte cilia are abnormal in biliary atresia.

OBJECTIVES: Biliary atresia (BA) is a rapidly progressive form of biliary fibrosis affecting neonates. We previously reported that primary cilia on the intrahepatic cholangiocytes of patients with both syndromic and nonsyndromic BA were structurally abnormal. Our objective was to determine whether extrahepatic cholangiocytes in human biliary atresia, intrahepatic and extrahepatic cholangiocytes of rhesus rotavirus (RRV)-infected neonatal mice, and RRV-infected primary neonatal extrahepatic cholangiocytes also demonstrate ciliary abnormalities. METHODS: The livers of neonatal BALB/c mice injected with RRV that developed jaundice, human extrahepatic bile duct samples obtained at time of hepatoportoenterostomy, and RRV-infected primary neonatal cholangiocytes were stained with antibodies against acetylated α tubulin to identify primary cilia. RESULTS: Extrahepatic cholangiocytes from RRV-treated mice demonstrated minimal loss of primary cilia at day 3 but almost complete loss at day 8 and partial loss at day 12. No changes were seen in mouse intrahepatic bile ducts at any of the time points. In the human BA samples, primary cilia were almost completely absent from extrahepatic duct cholangiocytes. There were, however, abundant cilia in the peribiliary glands adjacent to extrahepatic ducts in the BA sample. Cilia in RRV-infected primary neonatal cholangiocytes were significantly decreased compared with controls. CONCLUSIONS: Primary cilia are selectively lost from neonatal extrahepatic but not intrahepatic cholangiocytes after RRV infection in BALB/c mice. The cilia are also decreased in RRV-infected primary cholangiocytes and the extrahepatic ducts from human patients with BA. This suggests that ciliary abnormalities are part of the pathophysiology of BA.

Three linked vasculopathic processes characterize Kawasaki disease: a light and transmission electron microscopic study.

BACKGROUND: Kawasaki disease is recognized as the most common cause of acquired heart disease in children in the developed world. Clinical, epidemiologic, and pathologic evidence supports an infectious agent, likely entering through the lung. Pathologic studies proposing an acute coronary arteritis followed by healing fail to account for the complex vasculopathy and clinical course. METHODOLOGY/PRINCIPAL FINDINGS: Specimens from 32 autopsies, 8 cardiac transplants, and an excised coronary aneurysm were studied by light (n=41) and transmission electron microscopy (n=7). Three characteristic vasculopathic processes were identified in coronary (CA) and non-coronary arteries: acute self-limited necrotizing arteritis (NA), subacute/chronic (SA/C) vasculitis, and luminal myofibroblastic proliferation (LMP). NA is a synchronous neutrophilic process of the endothelium, beginning and ending within the first two weeks of fever onset, and progressively destroying the wall into the adventitia causing saccular aneurysms, which can thrombose or rupture. SA/C vasculitis is an asynchronous process that can commence within the first two weeks onward, starting in the adventitia/perivascular tissue and variably inflaming/damaging the wall during progression to the lumen. Besides fusiform and saccular aneurysms that can thrombose, SA/C vasculitis likely causes the transition of medial and adventitial smooth muscle cells (SMC) into classic myofibroblasts, which combined with their matrix products and inflammation create progressive stenosing luminal lesions (SA/C-LMP). Remote LMP apparently results from circulating factors. Veins, pulmonary arteries, and aorta can develop subclinical SA/C vasculitis and SA/C-LMP, but not NA. The earliest death (day 10) had both CA SA/C vasculitis and SA/C-LMP, and an "eosinophilic-type" myocarditis. CONCLUSIONS/SIGNIFICANCE: NA is the only self-limiting process of the three, is responsible for the earliest morbidity/mortality, and is consistent with acute viral infection. SA/C vasculitis can begin as early as NA, but can occur/persist for months to years; LMP causes progressive arterial stenosis and thrombosis and is composed of unique SMC-derived pathologic myofibroblasts.

Cholangiocyte cilia are abnormal in syndromic and non-syndromic biliary atresia.

Biliary atresia (BA) is a neonatal disorder characterized by aggressive fibroinflammatory obliteration of the biliary tract. Approximately 20 percent of BA patients demonstrate left-right laterality defects (syndromic BA). Cilia participate in important physiological functions in cholangiocytes, and as some ciliopathies have been associated with both laterality defects and hepatic fibrosis, we hypothesized that patients with syndromic BA exhibit abnormalities of cholangiocyte cilia that disrupt cholangiocyte homeostasis. Nine BA specimens were studied, including pre-Kasai diagnostic biopsies (n=7) and liver explants (n=2). Five specimens were from patients with laterality defects. These were compared with normal pediatric livers, as well as livers affected by primary sclerosing cholangitis, Wilson's disease, and cardiac cirrhosis. Biopsy sections were stained with antibodies against keratin 19 (a cholangiocyte marker) and acetylated α-tubulin (a cilia marker) and were visualized by confocal microscopy. Computer-assisted relative quantification was used to compare staining of cilia within bile ducts among samples. Surprisingly, cilia in BA specimens were significantly shorter, abnormal in their orientation, and less abundant compared with normal liver and disease controls regardless of the presence of a laterality defect. There are significant abnormalities of cholangiocyte cilia in both syndromic and non-syndromic BA livers compared with normal livers and livers affected by other cholestatic diseases. Although this may result from severe cholestasis or inflammation, it may also reflect common mechanistic pathways in different forms of BA and may have important implications for understanding the progression of the disease.