Comprehensive analyses of neurodevelopmental outcomes and quality of life of children with biliary atresia.

OBJECTIVES: To holistically evaluate neurodevelopmental outcomes and quality of life (QOL) of Japanese patients with biliary atresia (BA) and to investigate the factors associated with the outcomes. METHODS: This study enrolled patients with BA aged 5-18 years who visited Osaka University Hospital in 2021. Neurodevelopmental assessments were performed to evaluate intellectual ability, cognitive functions and adaptive skill levels. Furthermore, emotional and behavioral issues, characteristics of attention deficit hyperactivity disorder, and QOL were concomitantly assessed in the same cohort. Biochemical and social factors associated with the results were examined. RESULTS: Fifty-three patients, with a median age of 11.2 years were included in the analyses. Patients with BA had a significantly lower Full-Scale Intelligence Quotient or developmental quotient (FSIQ/DQ) score and Vineland Adaptive Behavior Scale (VABS) composite score than the general Japanese population. Household education level and short stature were associated with low and borderline FSIQ/DQ and VABS composite scores, respectively. Among patients with low and borderline FSIQ/DQ scores, those with average or high VABS composite scores received significantly less neuroeducational care than those with low and borderline VABS composite scores. Despite the low FSIQ/DQ and VABS composite scores, the total QOL scores were higher than those of the general population. CONCLUSION: Patients with BA had intellectual and behavioral impairments. Notably, patients with intellectual impairments are overlooked and not followed up, especially if adaptive skills are maintained.

Balloon dilatation for congenital esophageal stenosis associated with esophageal atresia.

PURPOSE: Congenital esophageal stenosis (CES) associated with esophageal atresia (EA) is rare, and no standard treatment has been established. We reviewed cases of EA-associated CES to assess the clinical characteristics and treatment outcomes, especially the feasibility of endoscopic dilatation. METHODS: We retrospectively examined patients with EA-associated CES. We also compared treatment outcomes of EA-associated CES with those of EA patients without CES who developed postoperative anastomotic stricture. RESULTS: Among 44 patients with EA, ten had CES (23%). Postoperative complications were not significantly different between EA patients with CES and those without CES but with anastomotic stricture. All CES patients underwent balloon dilatation as initial treatment. Eight of nine patients (89%) were successfully treated by dilatation only, and one patient underwent surgical resection. The median number of balloon dilatations for CES was five (2-17), which was higher than that for anastomotic stricture in patients without CES (p = 0.012). Esophageal perforation occurred in five patients with CES (5/9, 56%) after dilatation, but all perforations were successfully managed conservatively with an uneventful post-dilatation course. CONCLUSIONS: Twenty-three percent of patients with EA had CES. Although balloon dilatation for EA-associated CES required multiple treatments and carried a risk of perforation, balloon dilatation showed an 89% success rate and all perforations could be managed conservatively.

Tension-free thoracoscopic repair of congenital diaphragmatic hernia combined with a percutaneous extracorporeal closure technique: how to do it.

PURPOSE: Thoracoscopic repair (TR) of congenital diaphragmatic hernia (CDH) is associated with a higher recurrence rate than the conventional open method. We evaluated the effectiveness of our strategy for quality improvement, named "tension-free TR of CDH". METHODS: The subjects of this retrospective analysis were 11 consecutive patients with CDH who underwent TR at our hospital between 2017 and 2021. Tension-free TR of CDH included the proactive use of an oversized patch for dome-shaped reconstruction and gapless suturing. We developed a percutaneous extracorporeal closure technique for secure suturing using a commercially available needle. RESULTS: Patch repair was performed in 8 (73%) patients and none required conversion to open surgery because of technical difficulties. Recurrence developed in one patient (9%), who underwent successful reoperation via TR. All patients had an uneventful postoperative course. CONCLUSION: Tension-free TR combined with extracorporeal closure could reduce the difficulty of suturing and the risk of recurrence of CDH.

Present state of intestinal transplantation in Japan.

INTRODUCTION: Intestinal transplantation (ITx) is the ultimate treatment for intestinal failure (IF). In Japan, most cases of IF are a result of pediatric disease, including secondary or congenital intestinal disease or allied disorders of Hirschsprung's disease. Here, we report the results of the Japanese ITx registry. METHODS: A web-based survey form was completed. We investigated the number, age, sex, indication, surgical procedure, immunosuppressants, postoperative course, and the effects of transplantation in patients who underwent cadaveric or living-donor ITx. RESULTS: By the end of 2022, 42 cases of ITx have been performed in 38 patients in Japan. The donor sources included cadavers (29 cases) and living donors (13 cases). The surgical method was isolated ITx (N = 40) and combined liver and ITx (n = 2). Survival rates were 92%, 73%, and 59% at 1 year, 5 years, and 10 years, respectively. Ninety percent of patients completely discontinued parenteral nutrition. Approximately 80% of the patients had a performance status of 1 or less, indicating that the QOL of patients after ITx was extremely good. CONCLUSION: The results of ITx are acceptable to treat IF patients and the QOL after transplantation is also good.

Treatment and follow-up of late onset intra hepatic bile duct stones in congenital biliary dilatation.

PURPOSE: The postoperative course after surgery for congenital biliary dilatation (CBD) has some complications. Intrahepatic bile duct (IHBD) stones were known as a late complication. We report on the treatment and long-term follow-up of postoperative IHBD stones in our department. METHODS: Patients who underwent CBD surgery at age 15 years or younger in our department were identified. Those followed up for 5 years or more were enrolled. Annual blood chemistry tests and abdominal ultrasonography were performed. Each patient's surgical procedure, IHBD stone diagnosis, treatments, and outcomes were retrospectively assessed. RESULTS: Fifty-one patients were analyzed. The median age at the last visit was 24 years (range 7-45 years), and the median age at CBD surgery was 3 years. Eight patients (16%) developed late-onset IHBD stones. The median age at onset was 25 years, and the median duration after surgery was 20 years. The initial treatment was double-balloon enteroscopy (DBE) in 4 cases, which resulted in stone removal in 3 of the 4 patients (75%). CONCLUSION: Since CBD may cause late-onset IHBD stones, continuous follow-up is required even in adulthood. In this study, DBE was effective and minimally invasive, and it is recommended as the initial treatment.

Elevation of microRNA-214 is associated with progression of liver fibrosis in patients with biliary atresia.

BACKGROUND: MicroRNAs (miRNAs) play an important role in regulating fibrogenesis in the liver. The current study examined the ability of microRNA-214 (miR-214) level in liver and serum samples obtained from patients with BA to predict progressive liver fibrosis in patients with biliary atresia (BA). METHODS: We examined miR-214 level in relation to conventional markers of liver fibrosis, with liver and serum samples from BA patients. Fifty-two patients with BA who underwent Kasai portoenterostomy and four control patients underwent liver biopsy. In 28 patients with BA, blood samples were collected to analyze circulating serum miR-214. RESULTS: MiR-214 levels in liver tissue were significantly upregulated in patients with BA who had severe liver fibrosis (F3-4) compared to those with none to mild fibrosis (F0-2), whereas suppressors-of-fused homolog (Sufu) mRNA levels were significantly suppressed in F3-4. Serum miR-214 levels were significantly higher in patients with F3-4 compared with F0-2. Area under the curve analysis showed that the serum miR-214 cut-off level for predicting F3-4 was 0.805 (p = 0.0046). CONCLUSION: Hepatic overexpression of miR-214 is associated with progression of liver fibrosis in patients with BA, and the circulating miR-214 level may serve as a non-invasive predictor of liver fibrosis.

Early detection of liver fibrosis with serum Mac-2 binding protein glycosylation-modified isomer (M2BPGi) during follow-up intestinal failure patients without intestinal failure-associated liver disease (IFALD).

PURPOSE: Mac-2 binding protein glycosylation-modified isomer (M2BPGi) is a new marker for hepatic fibrosis progression. We examined the relationship between serum M2BPGi levels and liver histological findings in intestinal failure (IF) patients without IF-associated liver disease (IFALD). METHODS: This study included IF patients without IFALD followed at our hospital. All patients underwent routine liver biopsies per protocol every 1-2 years. We examined M2BPGi levels and histological findings in relation to aspartate aminotransferase (AST) to platelet ratio index, fibrosis-4 index, and AST/ALT ratio. Liver fibrosis was evaluated based on the METAVIR score. RESULTS: Total 18 liver biopsies out of eight patients were included. The median age was 11.5 years. Mean M2BPGi was 0.44 cutoff index (COI) in patients with F0 fibrosis, 0.78 COI in patients with F1 fibrosis and 1.63 COI in patients with F2 fibrosis. Mean M2BPGi was significantly higher in patients with F2 versus F1 or F0 fibrosis (P < 0.016 and P < 0.028, respectively). M2BPGi levels were more strongly correlated with fibrosis stage than with other conventional fibrosis markers. CONCLUSION: Serum M2BPGi is a novel marker of liver fibrosis in patients with IF. It is useful for follow-up prior to IFALD. Serum M2BPGi levels can support the interpretation of liver status.

Effect of microscopy-assisted portoenterostomy (MAPE) for biliary atresia.

PURPOSE: Portoenterostomy (PE) is the standard treatment for biliary atresia (BA). However, micro-bile ducts are difficult to identify with surgical loupes and dissect systematically. We report the effects of our attempts to dissect hilar tissue using a surgical microscope. METHODS: Microscopy-assisted portoenterostomy (MAPE) was initiated in 2014. Patients born between 2000 and 2013 who underwent PE until day 70 without a surgical microscope for BA were gathered as historical control. MAPE in re-do PE cases (Re-MAPE) was evaluated in the same manner. RESULTS: Ten patients underwent MAPE for BA during the study period. 17 patients in the conventional PE group were gathered. In the MAPE group, the jaundice clearance rate was 80%, compared with 53% in the conventional PE group. Re-MAPE was performed in four patients, who had a jaundice clearance rate of 75%, essentially identical to the rate with initial MAPE. At age 4 years, the native liver survival rate was 58% in the MAPE group and 38% in the conventional PE group. The native liver survival rate in the Re-MAPE group was 75%. CONCLUSION: MAPE is useful for sharing the surgical field during open PE in patients with BA. It may improve the rate of jaundice clearance.

Thoracoscopic repair of esophageal atresia.

Thoracoscopic repair (TR) of esophageal atresia (EA) has been performed with increasing frequency over the last two decades, with the expectation of improved outcomes by avoiding thoracotomy. To understand the current practice and outcomes of TR of EA, we reviewed the relevant literature, including 15 case series, 7 comparative studies, and 3 meta-analysis comparing TR with conventional open repair (COR). Most of the studies had a retrospective design and small numbers of patients. Although the evidence level is low because of the lack of prospective studies, this review found that TR is as safe as COR, with comparative outcomes. Moreover, there were several advantages of TR over COR, such as less blood loss and a shorter hospital stay. The long-term outcomes of TR remain unclear because of limited data. Moreover, there is a significant learning curve over the first 10-20 TRs performed. We conclude that TR of EA, when conducted by experienced surgeons, is a safe and minimally invasive alternative to COR and may yield better results than COR in appropriately selected patients.

Can plain chest X-ray replace computed tomography for the follow-up of children who have undergone the Nuss procedure?

PURPOSE: To establish whether new indices on plain chest X-ray (CXR) can replace those on computed tomography (CT) for the follow-up of children who have undergone the Nuss procedure. METHODS: The subjects of this retrospective study were 45 children who underwent the Nuss procedure between 2000 and 2016. The Haller index (HI) was measured by preoperative CT. Preoperative and postoperative chest deformities were evaluated by two CXR measurements: the concave rate on the lateral view (CR; the depth of the concavity divided by the anterior-posterior diameter of the rib cage) and the tracheal bifurcation angle (TBA) on the anterior view. Data are expressed as the median with range. RESULTS: The median age and HI of the children, when they underwent the Nuss procedure, was 9.3 (3.8-17.3) years and 4.5 (3.2-10.1), respectively. The preoperative CR was correlated significantly with the HI. The postoperative CR was significantly lower than the preoperative CR [pre: 0.17 (0.08-0.37), post: 0.09 (0.01-0.18), p < 0.05], and the low value was sustained after bar removal. The TBA decreased significantly after the Nuss procedure from 74.2° (55-104) preoperatively to 65.0° (45-92) postoperatively (p < 0.05). CONCLUSIONS: These results suggest that CXR can replace CT for the follow-up of patients after the Nuss procedure, with lower radiation exposure.

Current status of intestinal transplantation in East Asia.

PURPOSE OF REVIEW: Intestinal transplantation (ITx) activity remains low in East Asia. We conducted a multinational, retrospective study on patients who underwent ITx in Korea, Taiwan, and Japan, to provide an overview and to foresee future developments and collaborations in this region. RECENT FINDINGS: Total 71 ITx were performed in 67 patients. Living donor ITx was most commonly conducted in Japan (n = 13). Despite the low caseload, all three countries demonstrated acceptable patient survival rates of approximately 70% at 5 years. Over 70% of recipients with a functioning graft were free from total parenteral nutrition. SUMMARY: There is an urgent need to establish a nationwide and multinational registry of ITx recipients and patients with intestinal failure in East Asia. An efficient referral system to specialized intestinal rehabilitation and ITx centers and a multidisciplinary team approach is also warranted to provide state-of-the-art treatment for patients desperately waiting for a chance to survive.

CYP7A1 expression in hepatocytes is retained with upregulated fibroblast growth factor 19 in pediatric biliary atresia.

AIM: Bile acid biosynthesis is strictly regulated under physiological conditions. The expression of fibroblast growth factor (FGF) 19 is induced when bile acids bind to the farnesoid X receptor in the intestinal epithelium. Fibroblast growth factor 19 is then transported by the portal flow, causing transcriptional inhibition of cytochrome P450, family 7, subfamily A, polypeptide 1 (CYP7A1), a key enzyme in bile acid biosynthesis, through the extracellular signal-regulated kinase (ERK) pathway. However, the regulatory mechanisms of these signaling pathways in hepatocytes under chronic cholestasis remain unclear. We investigated the regulation of these signaling pathways in patients with biliary atresia (BA). METHODS: We analyzed the regulation of molecules in these signaling pathways using liver and serum samples from eight BA children and four non-cholestatic disease controls. RESULTS: CYP7A1 mRNA expression was not inhibited in BA microdissected hepatocyte-enriched tissue (HET) despite high serum bile acid concentrations. The FGF19 protein was synthesized in BA HET, and its serum concentration was elevated. Fibroblast growth factor receptor 4 was phosphorylated in BA livers. However, ERK phosphorylation was significantly reduced. We examined SPRY2 expression to determine how the ERK pathway was inactivated downstream of the FGF receptor; the expression was significantly increased in BA HET. CONCLUSIONS: This is the first study to measure the CYP7A1 mRNA levels in human BA HET. Fibroblast growth factor 19 was increased in BA hepatocytes. By focusing on its regulation in hepatocytes, we showed that the FGF19 pathway did not suppress bile acid synthesis, probably due to an altered mechanism involving upregulated SPRY2 in BA patients.

Laparoscopic ligation of a congenital extrahepatic portosystemic shunt for children with hyperammonemia: a single-institution experience.

PURPOSE: A congenital extrahepatic portosystemic shunt (CEPS) associated with hyperammonemia requires occlusion of the shunt vessels. We evaluated the effectiveness and safety of laparoscopic ligation of a CEPS in children with hyperammonemia. METHODS: The subjects of this retrospective study were seven children with hyperammonemia who underwent laparoscopic ligation of a CEPS. Their median age was 5.2 years (range 1-16 years). Before the laparoscopic procedure, a catheter was inserted through the femoral vein and placed in the portal vein via the shunt vessel. The shunt vessel was dissected and taped laparoscopically. After measuring the portal vein pressure under temporal occlusion, the shunt vessels were ligated. RESULTS: The types of shunts according to location were patent ductus venosus (n = 2), splenorenal shunt (n = 2), gastrorenal shunt (n = 2), and superior mesenteric vein-inferior vena cava shunt (n = 1). Laparoscopic ligation of the shunt vessel was completed uneventfully in all patients. The median portal vein pressure was 19 mmHg after ligation. The median preoperative blood ammonia level was 94 µg/dL (range 71-259 µg/dL), which decreased after ligation in all patients. There was no incidence of postoperative liver failure. CONCLUSION: Laparoscopic ligation of a CEPS is safe and effective for children with hyperammonemia.

Recombinant human soluble thrombomodulin reduces the severity and incidence of necrotizing enterocolitis in a newborn rat model.

PURPOSE: Necrotizing enterocolitis (NEC) remains the leading cause of death in preterm infants. Recombinant human soluble thrombomodulin (rTM) has been reported to have anti-inflammatory effects as well as antithrombogenic effects. The aim of this study was to evaluate the effect of rTM in a rat NEC model. METHODS: NEC was induced by enteral feeding with hyperosmolar formula, gavage administration of lipopolysaccharide and asphyxia stress. Controls were fed by their mother ad libitum. In the treatment group, rTM was administered subcutaneously twice (once each on the first and second day). All animals surviving beyond 96 h or that developed signs of distress were euthanized. The ileum was harvested for a histological evaluation and the measurement of the mRNA and protein expression. RESULTS: The rate of NEC-like intestinal injury in the treatment group (9/25, 36%) was significantly lower than in the NEC group (25/34, 73.5%). Tissue levels of TNF-α, IL-6 and HMGB1 were significantly elevated in the NEC group, whereas those in the treatment group were decreased to similar values as in the control group. CONCLUSIONS: Our experimental study showed that rTM is able to reduce the severity and incidence of NEC. It may be an alternative option for the treatment of NEC.

Serum Mac-2-binding protein (M2BPGi) as a marker of chronological liver fibrosis in biliary atresia patients with cirrhosis.

PURPOSE: Biliary atresia (BA) is characterized by progressive liver fibrosis, but it is difficult to assess the progression after the patient develops cirrhosis. Mac-2-binding protein glycosylation isomer (M2BPGi) is a new marker for hepatic fibrosis. We examined the chronological changes in M2BPGi levels in BA patients with cirrhosis. METHODS: Patients with cirrhosis were selected from among pediatric BA patients who had their native livers. Serum M2BPGi levels and Child-Pugh classification were evaluated. A total of 11 pediatric BA patients with cirrhosis were recruited. RESULTS: Initial M2BPGi level after diagnosis of liver cirrhosis based on liver biopsy was on average 3.4, and the most recent M2BPGi level under observation was on average 4.3. The follow-up period from the initial M2BPGi measurement averaged 22.6 months. The ratio of the initial and most recent values (M2BPGi ratio) was on average 1.3 (0.5-2.4). Three cases with improved fibrosis (M2BPGi ratio < 1.0) remained in Child A, as did six cases (1.0 ≤ M2BPGi ratio < 2.0), but two cases with marked fibrosis progression (2.0 ≤ M2BPGi ratio) advanced to decompensated cirrhosis Child B. CONCLUSION: M2BPGi is useful as a prognostic factor for BA patients with liver cirrhosis. In addition, fibrosis improved even after the development of cirrhosis.

Postoperative decrease in plasma acyl ghrelin levels after pediatric living donor liver transplantation in association with hepatic damage due to ischemia and reperfusion injury.

PURPOSE: Ghrelin was recently reported to promote recovery from hepatic injury. We hypothesized that it could also be associated with clinical recovery of the transplanted liver from ischemia and reperfusion injury. Our aims were to investigate perioperative ghrelin changes following pediatric living donor liver transplantation (LDLT) and to analyze the association of these changes with postoperative hepatic function. METHODS: We measured plasma acyl ghrelin (AG) concentrations before surgery, at the end of surgery and on postoperative days (PODs) 1, 3 and 7 in 12 children who underwent LDLTs, and, as controls, pre- and post-operatively and on POD1 in 7 children who underwent benign abdominal mass resection. The correlations between the participants' ghrelin profiles and hepatic function-related data were evaluated. RESULTS: AG levels significantly declined to 15.6% of preoperative levels after LDLT and almost returned to baseline on POD3. Post-operative AG levels were significantly reduced to a greater extent following LDLT than benign abdominal mass resection. AG levels on POD1 inversely correlated with aspartate aminotransferase levels and cold/total ischemia time (P < 0.05). CONCLUSION: These results suggest that reduced AG levels on POD1 may reflect the degree of damage to the transplanted liver due to ischemia and reperfusion injury.

The effect of maternal grafts in early acute cellular rejection after pediatric living-donor liver transplantation.

PURPOSE: Living-donor liver transplantations (LDLTs) with maternal grafts can be more successful than those with paternal grafts because of their tolerance to non-inherited maternal antigens. We reviewed LDLT patients to investigate the relationship between acute rejection and donor sex. METHODS: LDLT patients between January 2010 and November 2015 were enrolled. ACR was defined by a rejection activity index of > 3. RESULTS: Forty-six patients (22 males and 24 females), of whom 28 had biliary atresia, were enrolled. The median age of the patients was 2.8 years and the donor types were maternal (n = 25) and paternal (n = 21). Acute cellular rejection (ACR) was observed in 22 patients. Twelve (48%) of the 25 patients in the maternal group had at least one episode of rejection compared with 10 (48%) of the 21 in the paternal group. Among the patients with ACR, the first rejection in the maternal group occurred significantly earlier than that in the paternal group (p < 0.01). In the multivariable analysis, the only variable significantly related to the first rejection day after LDLT was donor sex (male) (p < 0.005). CONCLUSION: Our results showed that maternal grafts had an effect on causing earlier ACR in LDLT.

Total Bile Acid Concentration in Duodenal Fluid Is a Useful Preoperative Screening Marker to Rule Out Biliary Atresia.

OBJECTIVES: Duodenal tube test (DTT) is used as a preoperative screening to rule out biliary atresia (BA). In previous reports, DTT was assessed by the color of the duodenal fluid, but there were no quantitative criteria. The aim of this study was to examine the efficacy of DTT based on the total bile acid (TBA) concentration in duodenal fluid. METHODS: This is a single-center retrospective study of infants with cholestasis who underwent DTT from 2008 to 2016 at the Osaka University Hospital. The cut-off values of maximum TBA in duodenal fluid (dTBA), dTBA/serum TBA ratio (sTBA), and dTBA/serum gamma-glutamyl transpeptidase (sGGT) ratio were assessed for the accuracy in excluding BA. RESULTS: A total of 37 infants were included in this study; 16 infants with BA and 21 infants with other causes of intrahepatic cholestasis. dTBA demonstrated sensitivity of 100% and specificity of 90.5% with the cut-off value of 16.8 µmol/L. Specificity was further improved to 95.2% with dTBA/sTBA ratio (cut-off value: 0.088) and 100% with dTBA/sGGT ratio (cut-off value: 0.076 µmol/U). DTT could be performed 0.8 ±â€Š1.4 days after admission. Hypoglycemia was developed in 1 infant. CONCLUSIONS: DTT evaluated by dTBA, dTBA/sTBA ratio, and dTBA/sGGT ratio had high accuracy to rule out BA and could avoid unnecessary surgery in some infants.

Clinical implications of serum Mac-2-binding protein (M2BPGi) during regular follow-up of patients with biliary atresia.

PURPOSE: The Mac-2-binding protein glycosylation-modified isomer (M2BPGi) is a new marker for progression of hepatic fibrosis. We examined the relationship between serum M2BPGi levels and liver histological findings in patients with biliary atresia (BA) who were not transplant candidates. METHODS: Patients with BA who were not transplant candidates with good liver function were included. We examined M2BPGi levels and histological findings in relation to other laboratory markers of liver fibrosis, including aspartate aminotransferase (AST) to platelet ratio index, fibrosis-4 index, and type IV collagen 7s domain. Liver fibrosis was evaluated based on the METVIR score. RESULTS: 37 patients were included. The median age was 18 years (range 3-38 years). M2BPGi values ranged from 0.3 to 6.9 cutoff index (COI) (median 1.6). The degree of liver fibrosis varied with M2BPGi level. For predicting cirrhosis (F4) and advanced liver fibrosis (≥ F3), M2BPGi had higher areas under the curve (AUCs; 0.93, respectively) with cutoff COIs of 1.84 and 1.67, respectively, than for the four conventional markers for fibrosis. CONCLUSION: M2BPGi is a novel marker for liver fibrosis in patients with BA. It is especially useful for following patients with BA with a native liver and supporting liver biopsy interpretation findings.

Bile salt export pump-reactive antibodies form a polyclonal, multi-inhibitory response in antibody-induced bile salt export pump deficiency.

UNLABELLED: Progressive familial intrahepatic cholestasis type 2 (PFIC-2) is caused by mutations in ABCB11, encoding the bile salt export pump (BSEP). In 2009, we described a child with PFIC-2 who developed PFIC-like symptoms after orthotopic liver transplantation (OLT). BSEP-reactive antibodies were demonstrated to account for disease recurrence. Here, we characterize the nature of this antibody response in 7 more patients with antibody-induced BSEP deficiency (AIBD). Gene sequencing and immunostaining of native liver biopsies indicated absent or strongly reduced BSEP expression in all 7 PFIC-2 patients who suffered from phenotypic disease recurrence post-OLT. Immunofluorescence, western blotting analysis, and transepithelial transport assays demonstrated immunoglobulin (Ig) G-class BSEP-reactive antibodies in these patients. In all cases, the N-terminal half of BSEP was recognized, with reaction against its first extracellular loop (ECL1) in six sera. In five, antibodies reactive against the C-terminal half also were found. Only the sera recognizing ECL1 showed inhibition of transepithelial taurocholate transport. In a vesicle-based functional assay, transport inhibition by anti-BSEP antibodies binding from the cytosolic side was functionally proven as well. Within 2 hours of perfusion with antibodies purified from 1 patient, rat liver showed canalicular IgG staining that was absent after perfusion with control IgG. CONCLUSIONS: PFIC-2 patients carrying severe BSEP mutations are at risk of developing BSEP antibodies post-OLT. The antibody response is polyclonal, targeting both extra- and intracellular BSEP domains. ECL1, a unique domain of BSEP, likely is a critical target involved in transport inhibition as demonstrated in several patients with AIBD manifest as cholestasis.