Inflammation patterns in early post-operative cholangitis predict long-term outcomes in biliary atresia: a potential role of non-suppurative cholangitis.

PURPOSE: Frequent post-operative cholangitis in biliary atresia (BA) affects the long-term native liver survival. This study assessed the characteristics of early cholangitis and their influence on the prognosis. METHODS: Forty-three patients with BA who underwent surgery between 2000 and 2020 were analyzed for routine inflammatory markers. Early cholangitis characteristics were compared between native liver survivor (NLS) and living donor liver transplant (LDLT) patients. RESULTS: Among the 43 patients, 30 (69.8%) experienced 130 episodes of cholangitis. In the area under the receiver operating characteristics curve (AUROC) analysis, the cutoff value of the total cholangitis episodes was 3, with an area under the AUROC curve of 0.695 (95% confidence interval 0.522-0.868). Before 3 years old, 113 episodes (86.9%) of cholangitis were observed. The white blood cell, C-reactive protein, and alanine aminotransferase values at cholangitis onset did not markedly differ between the LDLT and NLS groups. Conversely, the neutrophil-to-lymphocyte ratio in the NLS group was significantly lower than in the LDLT group (0.85 vs. 1.63, p < 0.001). CONCLUSIONS: Cholangitis in the NLS group was lymphocyte-dominant and atypical in its pathogenesis. Lymphocyte-dominant cholangitis is non-suppurative, and future research should clarify its pathogenesis to improve the treatment and prognosis of BA.

Clinical outcome and neurological development of patients with biliary atresia associated with a bleeding tendency: a single institution experience.

PURPOSE: We compared the clinical features of patients with biliary atresia (BA) associated with a bleeding tendency (BT) at the time of the diagnosis with those of patients without a bleeding tendency (NBT). METHODS: The patients' background characteristics, age in days at the first visit, Kasai portoenterostomy (KPE), and postoperative course were retrospectively analyzed. RESULTS: Nine of the 93 BA patients (9.7%) showed a BT, including 7 with intracranial hemorrhaging (ICH), 1 with gastrointestinal bleeding, and 1 with a prothrombin time (PT) of 0%. The age at the first visit was 62 ± 12 days old for BT patients and 53 ± 27 days old for NBT patients (p = 0.4); the age at KPE was 77 ± 9 days old for BT patients and 65 ± 24 days old for NBT patients (p = 0.2); the time from the first visit to surgery was 13 ± 7 days for BT patients and 11 ± 10 days for NBT patients (p = 0.5); and the native liver survival rate was 56% for BT patients and 58% for NBT patients (p = 1), with no significant difference in any of the parameters. The neurological outcomes of survivors of ICH were favorable. CONCLUSIONS: Appropriate BT correction allowed early KPE even after ICH, resulting in native liver survival rates comparable to those of NBT patients without significant neurological complications.

New insights in understanding biliary atresia from the perspectives on maternal microchimerism.

Biliary atresia (BA) is a fibroinflammatory cholangiopathy and portal venopathy. It is of unknown etiology and is associated with systemic immune dysregulation, in which the first insult begins before birth. Maternal microchimerism is a naturally occurring phenomenon during fetal life in which maternal alloantigens promote the development of tolerogenic fetal regulatory T-cells in utero. However, maternal cells may alter the fetus's response to self-antigens and trigger an autoimmune response under certain histocompatibility combinations between the mother and the fetus. A recent report on a set of dizygotic discordant twins with BA, one of whose placentae showed villitis of unknown etiology, implies a certain immune-mediated conflict between the fetus with BA and the mother. Maternal chimeric cells persist postnatally for various time spans and can cause cholangitis, which ultimately leads to liver failure. In contrast, patients who eliminate maternal chimeric cells may retain their liver function.

Circulating maternal chimeric cells have an impact on the outcome of biliary atresia.

Introduction: We aimed to quantify the DNA of maternal chimeric (MC) cells in the peripheral blood of the BA patients and investigated the impact on the outcome. Methods: Patients with progressive jaundice because of no bile flow, which necessitated liver transplantation, or who showed inadequate bile flow with or without episodes of cholangitis and progressive hepatic fibrosis and portal hypertension were classified into the poor group. Those with adequate bile flow with completely normal liver function tests beyond 2 years were classified into the good group. The qPCR were separately carried out in buffy coat samples and plasma samples, targeting the non-inherited maternal HLA alleles in the DNA samples. Results: MC-DNA was present in the buffy coat (10-328 gEq per 106 host cells) in seven patients. There was no MC-DNA in the remaining five patients. MC-DNA (214-15,331 gEq per 106 host cells) was observed in the plasma of five patients. The quantity of MC-DNA in the buffy coat showed a significant difference between the two prognostic groups (p = 0.018), whereas there was no significant difference in the quantity of MC-DNA in plasma (p = 0.205). MC-DNA in the buffy coat was significantly associated with the outcome (p = 0.028), whereas MC-DNA in the plasma did not influence the outcome (p = 0.56). Conclusions: Poor outcomes in BA were correlated with circulating maternal chimeric lymphocytes.

The preoperative lymphocyte ratio and postoperative C-reactive protein are related to the surgical outcome in biliary atresia: an analysis of serial ubiquitous markers of inflammation.

PURPOSE: Various prognostic predictors for biliary atresia (BA) have been identified. This study aimed to evaluate the serial changes in the preoperative and postoperative ubiquitous inflammatory biomarkers and their relationship with the outcomes in patients with BA. PATIENTS AND METHODS: Forty-three BA patients were retrospectively reviewed to investigate serial levels of ubiquitous inflammatory biomarkers, including C-reactive protein (CRP) and lymphocyte ratio, and outcomes. The patients with BA were divided based on their outcomes into two prognostic groups: the native liver survivor group (n = 30) and the survivors with living-donor liver transplant group (n = 13). RESULTS: The area under the receiver operating characteristic (ROC) curve analysis showed that a preoperative lymphocyte ratio of < 61% and CRP value > 0.1 mg/dl predicted a poor outcome. In the ROC curve analysis, the timing of reaching the cut-off value of CRP after Kasai portoenterostomy was postoperative day (POD) 57. The third postoperative week, which was the timing of the discontinuation of steroid therapy, was the branchpoint of inflammatory markers between the two prognostic groups. CONCLUSION: The POD 57 CRP level predicts the surgical outcome of Kasai portoenterostomy. The postoperative anti-inflammatory management of BA can be monitored by the ubiquitous inflammatory biomarkers CRP and the preoperative lymphocyte ratio.

Biliary atresia: graft-versus-host disease with maternal microchimerism as an etiopathogenesis.

Biliary atresia (BA) is an inflammatory disease of the biliary system in newborns and infants. The etiology is largely unknown. Approximately half of BA patients require liver transplantation by 20 years of age, even after surgical correction due to progressive fibrosis of the liver. Regarding the disease mechanism, there is circumstantial evidence to support the hypothesis of graft-versus-host disease because of the existence of maternal cells in the liver (maternal microchimerism, MMC), histopathological similarity of the liver and an intense maternal response to the BA patient with mixed lymphocyte culture. Immune dysregulation with decreased Treg and increased Th1 and Th17 cells are the pathogenic features of BA, which are homologous to the pathogenic features of GvHD. Further elucidation of the etiopathogenetic mechanism of BA is warranted for development of new therapeutic strategies for native liver survival.

Segmental Atrophy of Explanted Livers in Biliary Atresia: Pathological Data From 63 Cases of Failed Portoenterostomy.

OBJECTIVES: Atrophy of the left lateral segment (LLS) is often encountered in liver transplantation (LT) for biliary atresia (BA). To clarify the meaning of the heterogeneous atrophy, we compared the pathological characteristics of the LLS with the right posterior segment (RPS) of BA livers obtained during LT. METHODS: Among the 116 patients with BA who underwent LT at our hospital between 2014 and 2018, 63 patients with persistent cholestasis after the Kasai portoenterostomy (KP) were selected. Three pathologists evaluated tissues from the LLS and RPS for 5 pathological parameters. Positive areas in whole-slide image observed as portal inflammation, fibrosis, cholestasis, and ductular reaction, were analyzed with automated image quantitation. Moreover, we examined the relationship between the pathological score and the Pediatric End-stage Liver Disease (PELD) score. RESULTS: The median age at LT was 7 months (range 4-26 months). Inflammation and fibrosis were significantly greater in the LLS than in the RPS (P < 0.001, for both); however, there were no differences in cholestasis, ductular reaction, and hepatocellular damage (P = 0.3, 0.3, and 0.82). The same results were obtained in automated image quantitation. Moreover, the sums of the 5 pathological scores in the LLS showed a significant positive correlation with the PELD score (P = 0.016, rs = 0.3). CONCLUSIONS: More severe inflammation and fibrosis without cholestasis were observed in the LLS. The segmental atrophy may not be associated with poor bile drainage, but with etiopathogenesis of BA. Moreover, the proper site for biopsy during KP could be the LLS.

A phase II JN-I-10 efficacy study of IDRF-based surgical decisions and stepwise treatment intensification for patients with intermediate-risk neuroblastoma: a study protocol.

BACKGROUND: Few clinical trials have been reported for patients with intermediate-risk neuroblastoma because of the scarcity of the disease and the variety of clinical and biological characteristics. A multidisciplinary treatment that consists of multidrug chemotherapy and surgery is expected to lead to a good prognosis with few complications. Therefore, a clinical trial for patients with intermediate-risk tumors was designed to establish a standard treatment that reduces complications and achieves good outcomes. METHODS: We planned a prospective phase 2, single-arm study of the efficacy of image-defined risk factors (IDRF)-based surgical decision and stepwise treatment intensification for patients with intermediate-risk neuroblastomas. For the localized tumor group, IDRF evaluations will be performed after each three-course chemotherapy, and surgery will be performed when appropriate. For patients with metastatic tumors, a total of five chemotherapy courses will be performed, and primary lesions will be removed when the IDRF becomes negative. The primary endpoint is 3-year progression-free survival rate, and the secondary endpoints include 3-year progression-free survival rates and overall survival rates of the localized group and the metastasis group and the incidence of adverse events. From international results, 75% is considered an appropriate 3-year progression-free survival rate. If this trial's expected 3-year progression-free survival rate of 85% is statistically greater than 75% in the lower limit of the 95.3% confidence interval, with an accuracy 10% (85 ± 10%), both groups require more than 65 patients. DISCUSSION: This study is the first clinical trial on the efficacy of IDRF-based surgical decision and stepwise treatment intensification for patients with intermediate-risk neuroblastomas. We expect that this study will contribute to the establishment of a standard treatment for patients with intermediate-risk neuroblastoma. TRIAL REGISTRATION: UMIN000004700, jRCTs051180203; Registered on December 9, 2010.

Microvascular proliferation of the portal vein branches in the liver of biliary atresia patients at Kasai operation is associated with a better long-term clinical outcome.

AIM OF THE STUDY: We previously showed an increased number of smaller portal vein (PV) branches in the portal areas of liver biopsy specimens of biliary atresia (BA) patients. We evaluated the correlation between this histopathological feature and the prognosis. PATIENTS AND METHODS: Twenty-five consecutive patients with BA encountered between 2000 and 2012 were classified into three prognostic groups based on their postoperative outcomes: Excellent (n = 11) for native-liver survivors with a normal liver function, Good (n = 6) for native-liver survivors with liver dysfunction, and Poor (n = 8) for survivors after liver transplant or on a waiting list. Data from morphometrical analyses, including the fibrotic portal area, numbers of PVs, diameter and total area of PV branches, were statistically compared among the three groups. MAIN RESULTS: The number of PV branches per unit area of the whole-liver specimen in the poor prognostic group was significantly lower than that in the excellent group (3.1 ± 0.6 vs. 5.2 ± 2.0/mm2, p = 0.03). There were no significant differences in the other parameters. CONCLUSIONS: This is the first report on the relationships between morphometrically analyzed PV branches and the postoperative course in BA patients. The portal venous system is involved as the primary lesion in BA.

Histological significance of hepatitis-like findings in biliary atresia: An analysis of 34 Japanese cases.

BACKGROUND: Hepatocellular injury including multinuclear changes are common histological features in biliary atresia (BA), as well as in neonatal hepatitis. To date, however, no reports have examined how those findings correlate with the prognosis of BA. We clarified the clinical implications of hepatitis-related changes in BA on histological analysis. METHODS: We retrospectively reviewed 34 cases of BA treated over the past 30 years at Ibaraki Children's Hospital. Liver biopsy specimens during Kasai procedures were evaluated for hepatocyte multinuclear change, ballooning, and acidophilic body, hereby defined as hepatitis-like findings (HLF). Each finding was semi-quantitatively scored as 0-2, and their sum was defined as the HLF score, ranging from 0 to 6. We examined the correlation between HLF score and total bilirubin (T-Bil), direct bilirubin (D-Bil), and other liver function test results at the Kasai procedure, as well as 1 week, and 1, 3, and 6 months after the Kasai procedure. Subsequently, HLF score was compared between native liver survivors (NLS; n = 16) and non-NLS (n = 18) for long-term analyses. RESULTS: Hepatitis-like findings score except for aspartate aminotransferase (AST), had no correlation with the preoperative data. HLF score was positively correlated, however, with T-Bil, D-Bil, and AST at 1 week and 1 month after the Kasai procedure (1 week: P = 0.009, 0.023, and 0.019; 1 month: 0.022, 0.019, and 0.013, respectively). HLF score was not significantly different between the NLS and non-NLS groups. CONCLUSION: Higher HLF score at Kasai procedure is an indicator of poor liver function at short-term follow up.

Morphometric demonstration of portal vein stenosis and hepatic arterial medial hypertrophy in patients with biliary atresia.

PURPOSE: Portal hypertension in patients with biliary atresia (BA) is generally thought to result from portal vein (PV) narrowing secondary to hepatic fibrosis. To test the hypothesis, we morphometrically analyzed the PVs and hepatic arteries (HAs). METHODS: Morphometrical analyses of 25 BA and 26 non-BA liver biopsy specimens from patients treated from 2000 to 2014. The total specimen area, the fibrotic portal area, vessel diameter and medial thickness of the HAs were measured. RESULTS: The PV diameter in BA patients was significantly smaller than that in non-BA patients. In BA, the numbers of normal-sized PVs and capillaries were decreased and increased, respectively. The PV diameter was not significantly correlated with the degree of fibrosis. We newly found that medial hypertrophy and the HA diameter increased with the number of endothelial cells in BA. The PV diameter was not significantly correlated with the medial thickness and was positively correlated with the HA diameter in BA. CONCLUSIONS: The narrowing of the PV is unlikely to occur secondarily to liver fibrosis. The medial hypertrophy of the HA is not correlated with the decrease in the PV blood flow. These findings seem to be unique to the primary vascular lesions of BA.

Laryngo fiberscopy-guided suspension procedure for an ectopic lingual thyroid obstructing airway.

BACKGROUND: Currently, there is no consensus regarding the optimal therapeutic strategy for the management of an ectopic lingual thyroid. A surgical approach is suggested when airway obstructive symptoms cannot be tolerated at all, or when bleeding or malignancy occurs. However, for patients in whom ectopic thyroid is the only functioning thyroid tissue, complete surgical excision needs to be followed by lifelong hormone replacement therapy. We report the case of an infant with ectopic lingual thyroid obstructing the airway that was treated using our novel surgical procedure. CASE PRESENTATION: A 10-day-old male infant presented with symptoms of airway obstruction and subclinical hypothyroidism. Imaging tests revealed an ectopic lingual thyroid and the absence of a normal pretracheal thyroid gland. We administered oral levothyroxine to lower his thyroid stimulating hormone (TSH) level and reduce the volume of the lingual mass; however, his airway symptoms did not improve. Subsequently, we performed a surgical intervention when he was 2 months old. We split the hyoid bone, and then suspended the lingual thyroid by suturing it to the hyoid bone to elevate the epiglottis. We confirmed the degree of suspension using intraoperative laryngo fiberscopy. After the surgery, the symptoms of airway obstruction were resolved and the patient was clinically euthyroid on low-dose oral levothyroxine. CONCLUSIONS: Our laryngo fiberscopy-guided suspension procedure can be an effective surgical procedure for the treatment of ectopic thyroid. This relatively simple surgical procedure could completely preserve the patient's thyroid tissue and resolve airway obstruction.

Ethnic variation in the incidence of biliary atresia correlates with the frequency of the most prevalent haplotype in its population.

The cause of biliary atresia (BA) remains an enigma. However, an ethnic diversity in the incidence of BA is so unique that anthropological approach may provide some etiopathogenetic implications in the disease mechanism. We previously reported that an association of maternal microchimerism (MMc) in BA and a significant compatibility of HLA-A between the patient with BA and their mother. Across the 10 countries (Japan, South Korea, Taiwan, Philippines, New Zealand (Maori population), UK, France, Germany, Norway, and Sweden), we determined the frequency of the most prevalent HLA haplotypes of each country from Allele Frequency Database and found that it was significantly correlated with the incidence of BA of the respective country (p = 0.0126). This observation better fits the MMc theory as an etiopathogenesis, that is, maternal effector cells are likely to migrate into the fetus in a relatively homogenous population and may damage the developing bile duct structure and portal vein endothelium, depending on materno-fetal tolerance and immunity.

Unique manifestations of biliary atresia provide new immunological insight into its etiopathogenesis.

Biliary atresia (BA) is a unique cholestatic disease of newborns with a background of exaggerated immune response in the liver of unknown mechanism. Three hypotheses have been proposed; autoimmune type of cholangiopathy triggered by virus infection, graft-versus-host disease type of immune-mediated disease associated with maternal microchimerism and ductal plate malformation theory. Researchers on virus infection theory have experimentally explored immune process causing cholangiopathy on murine models of this disease, while in maternal microchimerism hypothesis were detected maternal cells in the BA patients' liver, of which roles are yet to be determined. Ductal plate malformation theory is an intriguing hypothesis in the sense that it suggests the onset of this disease is in the first trimester. This theory can be secondary to either one of these two immune-related insults. In this review, four unique points are focused; (1) the timing of onset, (2) hepatitis-like pathological picture, (3) heterogenous atrophy of the liver segments when advanced, and (4) swollen lymph nodes at the porta hepatis. These unique clinicopahtological aspects of this disease should be well explained by these hypotheses.

Aggressive gastrointestinal food allergy in neonates and its possible relationship to necrotizing enterocolitis.

INTRODUCTION: The incidence of gastrointestinal food allergy (FA) in neonates is increasing. Despite this, cases of patients with gastrointestinal FA who develop necrotizing enterocolitis (NEC) requiring laparotomy are extremely rare. PRESENTATION OF CASE: We describe two cases that presented with bloody stool with a probable diagnosis of FA as eosinophils were positive in the stool at onset. Both cases failed conservative treatment. Jejunostomy and ileostomy were performed in both cases due to secondary NEC with underlying acute FA. Post-surgery, raised peripheral blood eosinophil count, presence of cow's milk-specific IgE antibody and positive allergen-specific lymphocyte stimulation test were found. Stoma closure were performed 3 and 1 months later in both cases. Postoperative recovery was uneventful. DISCUSSION: A few reports have not identified risk factors for NEC secondary to FA. Thrombocytopenia and rise in C-reactive protein (CRP) levels 2days after the development of FA may be suggestive of FA with NEC. Methicillin-resistant Staphylococcus aureus (MRSA) was detected in the fecal culture of both patients at the time of the onset of NEC. The toxic antigen produced by MRSA may cause activation of milk-protein-primed T cells and exacerbate FA. CONCLUSION: The decrease of platelet levels and rise in CRP may indicate the development of secondary NEC in patients with FA. Additionally, MRSA detected in the fecal culture also may be a risk factor for NEC through the activation of cellular immunity reaction pathways.

The accumulation of regulatory T cells in the hepatic hilar lymph nodes in biliary atresia.

PURPOSE: A proposed etiopathogenesis of biliary atresia (BA) involves T-cell-mediated inflammatory bile duct damage and progressive hepatic fibrosis. Pediatric surgeons often observe swelling of the hepatic hilar lymph nodes during the Kasai procedure. Given the importance of regulatory mechanisms in immune responses, the present study was designed to analyze the quantitative changes of regulatory T cells (T reg cells) in the hepatic hilar lymph nodes (hepatic hilar LNs) and peripheral blood (PB) in BA. METHODS: The hepatic hilar LNs and PB obtained during the Kasai procedure were analyzed by flow cytometry. The ratios of total and active Tregs to the total CD4+ cells in the PB and the hepatic hilar LNs were compared. RESULTS: In patients with BA, the ratios of both the total and active T reg cells in the hepatic hilar LNs were higher than those in the PB (total T reg cells: PB vs. LN; P < 0.001; active T reg cells: PB vs. LN; P = 0.001). In BA patients, the increase in the ratio of active T reg cells to the CD4 + cells in the LNs in comparison to the PB was greater than that in control patients. The ratio observed in the BA patients was almost double the ratio observed in the control patients. The median LN/PB ratio in the BA patients was 3.1, while that in controls was 1.6 (P = 0.03). CONCLUSION: The present study showed that the ratios of both total T reg cells and active T reg cells were higher in the hepatic hilar lymph nodes of BA patients. This finding could shed light on the pathogenesis of BA.

Positive C4d staining of the portal vein endothelium in the liver of patients with biliary atresia: a role of humoral immunity in ongoing liver fibrosis.

PURPOSE: This study aimed to clarify the role of complement activation in fibrogenesis in BA. METHODS: In total, 27 paraffin-embedded liver biopsy samples were immunohistochemically analyzed using C4d polyclonal antibody, vascular cell adhesion molecule-1 (VCAM-1), and CD45. The liver samples were obtained from 25 patients during Kasai operation, and two additional specimens were obtained from 2 patients by needle biopsy later at the time of liver function deterioration. The degree of liver fibrosis was histologically graded 1-3. RESULTS: Among the 25 samples, 9 showed C4d-positive immunostaining localized on the endothelia of a few portal veins in the portal tract. The degree of fibrosis was correlated with C4d staining (p = 0.025). The age at Kasai operation correlated with the degree of fibrosis and the C4d positivity. Two needle biopsy samples were positive for C4d. Among 13 samples submitted for VCAM-1 staining, 2 negative samples were C4d negative and all positive C4d samples were VCAM-1 positive with CD45 mononuclear cell infiltration. CONCLUSION: These findings suggest that ongoing cirrhosis could be a result of progressive "vasculopathy" of the portal vein caused by humoral and cell-mediated immune interaction.

Maternal microchimerism in biliary atresia: are maternal cells effector cells, targets, or just bystanders?

The etiology of biliary atresia (BA) is unknown; however, the liver histology is similar to that observed in immune-mediated hepatic disorders. Liver fibrosis in BA progresses even after bile drainage has been achieved by the Kasai operation. Maternal microchimerism has been purported to play a part in the pathogenesis of BA as well as certain autoimmune diseases. However, the role of maternal cells has not yet been determined in BA. Specifically, it is unknown whether these maternal cells function as maternal effector T lymphocytes, or targets or bystanders. We currently hypothesize that the first hit is due to GvHD interaction by engrafted maternal effector T lymphocytes. Furthermore, we suggest that the secondary effects that are manifested by progressive cirrhosis are caused either by maternal chimeric effector T lymphocytes (e.g., GvHD interaction) or targets (e.g., HvGD interaction). Based on our hypothesis, mixed lymphocyte reactions between patients and their mothers might shed light on the etiopathogenesis and prognostic indicators.