Alexander J. Bondoc

Cholangiocyte expression of α2β1-integrin confers susceptibility to rotavirus-induced experimental biliary atresia

Inoculation of BALB/c mice with rhesus rotavirus (RRV) in the newborn period results in biliary epithelial cell (cholangiocyte) infection and the murine model of biliary atresia. Rotavirus infection of a cell requires attachment, which is governed in part by cell-surface expression of integrins such as alpha2beta1. We hypothesized that cholangiocytes were susceptible to RRV infection because they express alpha2beta1. RRV attachment and replication was measured in cell lines derived from cholangiocytes and hepatocytes. Flow cytometry was performed on these cell lines to determine whether alpha2beta1 was present. Cholangiocytes were blocked with natural ligands, a monoclonal antibody, or small interfering RNA against the alpha2-subunit and were infected with RRV. The extrahepatic biliary tract of newborn mice was screened for the expression of the alpha2beta1-integrin. Newborn mice were pretreated with a monoclonal antibody against the alpha2-subunit and were inoculated with RRV. RRV attached and replicated significantly better in cholangiocytes than in hepatocytes. Cholangiocytes, but not hepatocytes, expressed alpha2beta1 in vitro and in vivo. Blocking assays led to a significant reduction in attachment and yield of virus in RRV-infected cholangiocytes. Pretreatment of newborn pups with an anti-alpha2 monoclonal antibody reduced the ability of RRV to cause biliary atresia in mice. Cell-surface expression of the alpha2beta1-integrin plays a role in the mechanism that confers cholangiocyte susceptibility to RRV infection.

Cholangiocyte secretion of chemokines in experimental biliary atresia

UNLABELLED Biliary atresia (BA) is a disease of the newborn that results in obstruction of the biliary tree. The cause of BA remains unknown; however, recent studies using the murine model of biliary atresia have found that rotavirus infection of the biliary epithelial cell (cholangiocyte) triggers an inflammatory response. We hypothesized that rotavirus infection of cholangiocytes results in the release of chemokines, important mediators of the host immune response. METHODS In vivo, Balb/c pups were injected with rhesus rotavirus (RRV) or saline, and, their extrahepatic bile ducts were microdissected 2, 5, 7, and 14 days after injection. Next, an immortalized cholangiocyte cell line (mCl) was incubated with RRV or serum-free media. Qualitative and quantitative chemokine assessment was performed using enzyme-linked immunosorbent assay, polymerase chain reaction, and immunohistochemistry. RESULTS In vivo, increased levels of the chemokines macrophage inflammatory protein 2, monocyte chemotactic protein 1, KC and Regulated upon Activation, Normal T Expressed and Secreted were found in RRV-infected murine bile ducts. In vitro, infected mCl cells produced increasing amounts of these same chemokines in relation to dose and time. CONCLUSION These novel results suggest that chemokine expression by RRV-infected cholangiocytes may trigger a host inflammatory process that causes bile duct obstruction. Understanding how viral infection initiates this response may shed light on the pathogenesis of biliary atresia.

The Rhesus Rotavirus Gene Encoding VP4 Is a Major Determinant in the Pathogenesis of Biliary Atresia in Newborn Mice

ABSTRACT Biliary atresia (BA) is a devastating disease of childhood for which increasing evidence supports a viral component in pathogenesis. The murine model of BA is induced by perinatal infection with rhesus rotavirus (RRV) but not with other strains of rotavirus, such as TUCH. To determine which RRV gene segment(s) is responsible for pathogenesis, we used the RRV and TUCH strains to generate a complete set of single-gene reassortants. Eleven single-gene “loss-of-function” reassortants in which a TUCH gene replaced its RRV equivalent and 11 single-gene “gain-of-function” reassortants in which an RRV gene replaced its TUCH equivalent were generated. Newborn BALB/c mice were inoculated with the reassortants and were monitored for biliary obstruction and mortality. In vitro, the ability to bind to and replicate within cholangiocytes was analyzed. Infection of mice with the “loss-of-function” reassortant RT(VP4), where gene 4 from TUCH was placed on an RRV background, eliminated the ability of RRV to cause murine BA. In a reciprocal fashion, the “gain-of-function” reassortant TR(VP4) resulted in murine BA with 88% mortality. Compared with those for RRV, RT(VP4) binding and titers in cholangiocytes were significantly attenuated, while TR(VP4) binding and titers were significantly increased over those for TUCH. Reassortants RT(VP3) and TR(VP3) induced an intermediate phenotype. RRV gene segment 4 plays a significant role in governing tropism for the cholangiocyte and the ability to induce murine BA. Gene segment 3 did not affect RRV infectivity in vitro but altered its in vivo effect.

Prevention of the murine model of biliary atresia after live rotavirus vaccination of dams

PURPOSE Biliary atresia (BA) is a neonatal disease that results in the obliteration of the biliary tree. The murine model of BA has been established where rhesus rotavirus (RRV) infection of newborn mice leads to an obstructive cholangiopathy. We determined whether maternal postconception rotavirus vaccination could prevent the murine model of BA. MATERIALS AND METHODS Female mice were mated and injected intraperitoneally with one of the following materials: purified rotavirus strains RRV or Wa, high or low-dose Rotateq (Merck and Co Inc, Whitehouse Station, NJ) (a pentavalent rotavirus vaccine [PRV]), purified recombinant viral antigens of rotavirus (VP6) or influenza (NP), or saline. B-cell-deficient females also underwent postconception PRV injection. RESULTS Maternal vaccination with PRV improves survival of pups infected with RRV. Serum rotavirus IgG, but not IgA, levels were increased in pups delivered from dams who received RRV, Wa, PRV, or VP6, but in the case of the Wa, PRV, and VP6 groups, these antibodies were not neutralizing. Postconception injection of high-dose PRV did not improve survival of pups born to B-cell-deficient dams. CONCLUSION Maternal vaccination against RRV can prevent the rotavirus-induced murine model of BA in newborn mouse pups.

The beneficial impact of revision of Kasai portoenterostomy for biliary atresia: an institutional study.

Objective:To determine whether portoenterostomy (PE) revision in patients afflicted with biliary atresia (BA) is a viable treatment option and, if so, identify which patients may benefit. Background:BA, the most common cause of neonatal liver disease, results in biliary tract obstruction and hepatic fibrosis. Kasai PE is the initial surgical intervention performed and, if successful, restores drainage and preserves the native liver. Portoenterostomy failure warrants liver transplantation, but because of complications related to transplantation, treatment strategies to salvage the native liver may be beneficial. Using uniformly applied criteria, we have revised PEs to delay or avoid transplantation. Methods:A retrospective review of medical records of patients diagnosed with BA since 1983 was performed. Patient demographics, symptoms, indications for revision, laboratory values, and outcomes were recorded. A cohort of patients who underwent revision after initial PE was identified. Survival rates were assessed using the Kaplan-Meier method. For patients who required transplantation, operative data from the revised PE cohort were compared with those from the unrevised PE cohort. A Cox proportional hazards model was used to determine covariates predictive of a favorable outcome. Results:Of 181 children who underwent PE, 24 underwent revision. Adequate biliary drainage, as evidenced by normalized conjugated bilirubin levels, was achieved in 75% of revised patients. Overall survival in patients who underwent revision, regardless of transplantation, was 87%. Among patients who underwent PE revision, 46% have survived with their native liver. Conclusion:Experience at our center suggests that with appropriate patient selection, PE revision may delay the need for liver transplanation yielding encouraging patient outcomes.

Rotavirus Replication in the Cholangiocyte Mediates the Temporal Dependence of Murine Biliary Atresia

Biliary atresia (BA) is a neonatal disease that results in obliteration of the biliary tree. The murine model of BA, which mirrors the human disease, is based upon infection of newborn mice with rhesus rotavirus (RRV), leading to an obstructive cholangiopathy. The purpose of this study was to characterize the temporal relationship between viral infection and the induction of this model. BALB/c mice were infected with RRV on day of life (DOL) 0, 3, 5, and 7. Groups were characterized as early-infection (infection by DOL 3) or late-infection (infection after DOL 5). Early RRV infection induced symptoms in 95% of pups with a mortality rate of 80%. In contrast, late infection caused symptoms in only 50% of mice, and 100% of pups survived. The clinical findings correlated with histological analysis of extrahepatic biliary trees, cytokine expression, and viral titers. Primary murine cholangiocytes isolated, cultured, and infected with RRV yielded higher titers of infectious virus in those harvested from DOL 2 versus DOL 9 mice. Less interferon alpha and beta was produced in DOL 2 versus DOL 9 RRV infected primary cholangiocytes. Injection of BALB/c interferon alpha/beta receptor knockout (IFN-αβR−/−) pups at DOL 7 showed increased symptoms (79%) and mortality (46%) when compared to late infected wild type mice. In conclusion, the degree of injury sustained by relatively immature cholangiocytes due to more robust RRV replication correlated with more severe clinical manifestations of cholangiopathy and higher mortality. Interferon alpha production by cholangiocytes appears to play a regulatory role. These findings confirm a temporal dependence of RRV infection in murine BA and begin to define a pathophysiologic role of the maturing cholangiocyte.

Surgical management of children and adolescents with upfront completely resected hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is an aggressive malignant neoplasm that is often chemoresistant. Complete surgical resection remains the mainstay of therapy. The role of liver transplantation (LT) in pediatric HCC is in evolution, as is the role of adjuvant chemotherapy for stage I disease.

Esophageal dysmotility: An intrinsic feature of megacystis, microcolon, hypoperistalsis syndrome (MMIHS)

OBJECTIVES Megacystis-microcolon-hypoperistalsis syndrome (MMIHS) also called Berdons Syndrome, is a smooth muscle myopathy that results in an enlarged bladder, microcolon, and small bowel hypoperistalsis. In our series of six patients with this disorder, all had disordered swallowing. Therefore, we prospectively characterized esophageal structure and function in all. METHODS Diagnoses had been established by contrast radiography, small bowel manometry, and urodynamic studies. To investigate the esophagus, we endoscoped and biopsied the esophagus of each patient on multiple occasions. All patients also underwent water soluble contrast esophagography and esophageal manometry. RESULTS Upon careful questioning, all patients had swallowing dysfunction, and the majority of their enteral intake was via gastrostomy or gastrojejunostomy. All took some oral alimentation, but eating was slow and none could aliment themselves completely by the oral route, receiving 50% or less of their calories by mouth. Four had megaesophagus whereas the esophagus of the two youngest was of normal caliber. All had eosinophilic esophagitis and/or esophageal Candidiasis from time to time, but successful treatment of these findings failed to improve their symptoms. Manometry revealed normal lower esophageal sphincter (LES) resting tone and normal LES relaxation, but for all, peristalsis was absent in the esophageal body. CONCLUSIONS This series expands the spectrum of findings in MMIHS, to include a primary motility disorder of the esophageal body. As patients age, the esophageal caliber appears to increase. Successful treatment of neither esophageal eosinophilia nor Candidiasis is effective in ameliorating the motility disorder. If our findings are confirmed in more patients with MMIHS, this disorder should be renamed, megacystis-microcolon-intestinal-and esophageal hypoperistalsis syndrome. TYPE OF STUDY Prognosis study, Level IV (case series).

Pediatric pancreas transplantation, including total pancreatectomy with islet autotransplantation

Unlike other solid-organ transplants, whole pancreas transplantation in children is relatively rare, and it occurs more frequently in the context of multivisceral or composite organ transplantation. Because children only infrequently suffer severe sequelae of type 1 diabetes mellitus, pancreas transplantation is rarely indicated in the pediatric population. More commonly, pediatric pancreas transplant occurs in the setting of incapacitating acute recurrent or chronic pancreatitis, specifically islet autotransplantation after total pancreatectomy. In this clinical scenario, total pancreatectomy removes the nidus of chronic pain and debilitation, while autologous islet transplantation aims to preserve endocrine function. The published experiences with pediatric total pancreatectomy with islet autotransplantation (TPIAT) in children has demonstrated excellent outcomes including liberation from chronic opioid use, as well as improved mental and physical quality of life with good glycemic control. Given the complexity of the operation, risk of postoperative complication, and long-term physiologic changes, appropriate patient selection and comprehensive multidisciplinary care teams are critical to ensuring optimal outcomes.

Hepatic Artery Pseudoaneurysm After Liver Transplantation

Hepatic artery pseudoaneurysms (HAPs) are rare after liver transplantation with reported incidence of 0.5–2.0 %. They can lead to life threatening hemorrhage. HAP usually occurs at the arterial anastomosis and is often caused by infection and/or technical failure. Early diagnosis and aggressive intervention are essential. Several treatment modalities were described and included excision with ligation, retransplantation, excision with arterial reconstruction, balloon angioplasty with stenting, endovascular embolization, and percutaneous thrombin injection. We are presenting a case of HAP following liver transplantation, which was successfully treated by surgical resection and primary arterial reconstruction of the hepatic artery. A 56-year-old female with prior history of gastric bypass and open cholecystectomy developed end-stage liver disease (ESLD) secondary to primary biliary cirrhosis and underwent uncomplicated orthotopic liver transplantation in April of 2015. Her preoperative MELD score was 20 and she was intubated prior to liver transplantation because of encephalopathy. The donor was a 35-year-old male DCD with history of hypertension, heroin abuse, BMI of 26, and normal liver function tests. Due to DCD, tPAwas injected into the donor hepatic artery prior to the performing of the portal vein anastomosis. Arterial anastomosis was performed between donor common hepatic artery and recipient common hepatic artery with 6-0 Prolene in a running fashion. Intraoperative Doppler ultrasound confirmed a good pulse in the hepatic artery and good flow in the liver. Duct-to-duct biliary anastomosis was performed with longitudinal ductoplasty on the donor site. Cold ischemia time was 406 min, cold ischemia time was 36 min. Immunosuppression included tacrolimus, cellcept, and prednisolone. Steroids continued for 3 months after transplantation. Patient presented 6 months after surgery with right upper quadrant pain. All liver function tests were normal. A computed tomography (CT) scan of the abdomen showed focal aneurysmal dilation of the common hepatic artery. Subsequent angiogram showed a 1 cm pseudoaneurysm arising from a highly tortuous segment of the common hepatic artery, with a short segment of narrowing proximal to the pseudoaneurysm (Fig. 1). Due to the angulation and tortuosity of the hepatic artery and pseudoaneurysm, it was felt that endovascular approach was not possible. After careful discussion of risks and benefits of surgery with the patient, the decision was made to proceed with operation which included resection of HPAwith reconstruction. We used the previous transplant incision to enter intraabdominal cavity. After all adhesions were taken down, porta hepatis was exposed. After hepatic artery aneurysm was dissected out, distal and proximal control was obtained. The pseudoaneurysmwas larger than appeared on imaging studies, reaching 2×1.5 cm in size (Fig. 2). There was no evidence of infection around pseudoaneurysm. The pseudoaneurysm was resected (Fig. 3). Direct anastomosis of the common hepatic artery of the donor and of the common hepatic artery of the recipient was performed with 7-0 Prolene in an interrupted * Aleksandr A. Reznichenko reznicaa@ucmail.uc.edu