Sharat Varma

Retargeting of bile salt export pump and favorable outcome in children with progressive familial intrahepatic cholestasis type 2

We investigated predictors of clinical evolution in progressive familial intrahepatic cholestasis type 2 patients and how they relate to bile salt export pump (BSEP) expression and its (re)targeting. Our retrospective study included 22 children with progressive familial intrahepatic cholestasis type 2. Clinical, biochemical, and histological characteristics were reviewed on admittance and following treatment with either ursodeoxycholic acid alone (10 mg/kg thrice daily, n = 19) or partial biliary diversion (n = 3). Immunostaining of BSEP was performed in 20 patients. Response to treatment was defined as normalization of pruritus, disappearance of jaundice, and alanine aminotransferase (ALT) levels <1.5 times the upper limit of normal. Ten of 22 patients were responders, and paired biopsies were available in six. De novo or retargeted canalicular expression of BSEP occurred in four of these six, two of whom exhibited baseline intracellular expression. Twelve of 22 were nonresponders and exhibited earlier onset of jaundice (<9 months), neonatal cholestasis, and higher ALT levels. An ALT >165 IU/L produced 72% sensitivity and 55% specificity in predicting nonresponse. Seven patients were still responding at last follow‐up (median = 20 months, range 5‐67 months). Three responders relapsed after 56, 72, and 82 months, respectively. Of nine surviving responders, median relapse‐free survival time was 72 months (95% confidence interval 48‐96 months) and 5‐year relapse‐free survival was 75% (95% confidence interval 33‐100%). Intracellular BSEP at baseline was seen in six, of whom five were responders. Genetic analysis was performed in 17 of 22, confirming diagnosis in 13 (76%) and in four (24%) in whom only heterozygous mutation was identified. Conclusion: De novo or retargeted canalicular expression of BSEP occurs in treatment responders; children with late‐onset presentation, lower ALT, and intracellular BSEP expression are likely to respond, at least transiently, to nontransplant treatment. (Hepatology 2015;62:198‐206)

Biodistribution of Liver-derived Mesenchymal Stem Cells After Peripheral Injection in a Hemophilia A Patient

Background With the exception of liver transplantation, there is no cure for hemophilia, which is currently managed by preemptive replacement therapy. Liver-derived stem cells are in clinical development for inborn and acquired liver diseases and could represent a curative treatment for hemophilia A. The liver is a major factor VIII (FVIII) synthesis site, and mesenchymal stem cells have been shown to control joint bleeding in animal models of hemophilia. Adult-derived human liver stem cells (ADHLSCs) have mesenchymal characteristics and have been shown able to engraft in and repopulate both animal and human livers. Thus, the objectives were to evaluate the potency of ADHLSCs to control bleeding in a hemophilia A patient and assess the biodistribution of the cells after intravenous injection. Methods A patient suffering from hemophilia A was injected with repeated doses of ADHLSCs via a peripheral vein (35 million 111In-oxine-labeled cells, followed by 125 million cells the next day, and 3 infusions of 250 million cells every 2 weeks thereafter; total infusion period, 50 days). Results After cell therapy, we found a temporary (15 weeks) decrease in the patient’s FVIII requirements and severe bleeding complications, despite a lack of increase in circulating FVIII. The cells were safely administered to the patient via a peripheral vein. Biodistribution analysis revealed an initial temporary entrapment of the cells in the lungs, followed by homing to the liver and to a joint afflicted with hemarthrosis. Conclusion These results suggest the potential use of ADHLSCs in the treatment of hemophilia A.

The histological quantification of alpha-smooth muscle actin predicts future graft fibrosis in pediatric liver transplant recipients

Activated hepatic stellate cells express cytoplasmic ASMA prior to secreting collagen and consequent liver fibrosis. We hypothesized that quantifying ASMA could predict severity of future fibrosis after LT. For this, 32 pairs of protocol biopsies, that is, “baseline” and “follow‐up” biopsies taken at 1‐ to 2‐year intervals from 18 stable pediatric LT recipients, transplanted between 2006 and 2012 were selected. Morphometric quantification of “ASMA‐positive area percentage” was performed on the baseline biopsy. Histological and fibrosis assessment using Metavir and LAFSc was performed on all biopsies. The difference of fibrosis severity between the “baseline” and “follow‐up” was termed “prospective change in fibrosis.” Significant association was seen between extent of ASMA positivity on baseline biopsy and “prospective change in fibrosis” using Metavir (P=.02), cumulative LAFSc (P=.02), and portal LAFSc (P=.01) values. ASMA‐positive area percentage >1.05 predicted increased fibrosis on next biopsy with 90.0% specificity. Additionally, an association was observed between extent of ASMA positivity and concomitant ductular reaction (P=.06), but not with histological inflammation in the portal tract or lobular area. Hence, ASMA quantification can predict the future course of fibrosis.

Sofosbuvir/ledipasvir and ribavirin tolerability and efficacy in pediatric liver transplant recipients

The longterm results of pediatric liver transplantation (LT) have become exceptionally good, with >95% longterm survival, but hepatitis C virus (HCV) recurrence for a liver graft was an important cause of morbidity, making prevention of disease relapse after transplantation a major objective. Treating HCV in children remains challenging, but the development of oral direct-acting antivirals (DAAs) has revolutionized the modern approach for HCV treatment in adults. We describe 2 cases of pediatric HCV patients treated with sofosbuvir (SOF)/ledipasvir (LDV) and ribavirin: one treated before LT (a 1-year-old boy transplanted for biliary atresia) and the other after LT (a 16-year-old girl transplanted for Budd-Chiari syndrome and cirrhosis). The protocol used to treat these patients was approved by the local ethical committee, and informed consent was obtained for both patients. Both patients were HCV genotype 1b: the first was diagnosed during a pretransplant check-up, and the second was a recurrence 5 weeks after LT. Both received a 12-week course of SOF/LDV (Harvoni) and ribavirin, which is in accordance with recent recommendations for adults in the LT context. The first patient received 5 weeks of treatment before LT, and the remaining course was continued after LT (dosage was SOF 100mg/LDV 22.5mg once daily and ribavirin 7.5mg/kg twice daily); the second child received the full treatment course after LT. Interestingly, HCV polymerase chain reaction (PCR) on the explanted liver correlated well with the blood PCR after 5 weeks of treatment (both< 30 IU/mL) for the youngest child. Treatment response with nondetectable HCV on PCR was observed at the end of the 12 weeks of treatment, leading to a sustained virological response (SVR) 24 weeks after the end of treatment for both children. None of the commonly reported side effects were observed. For the 1-year-old, singledose and multiple-dose pharmacokinetic (PK) parameters of SOF, its predominant circulating metabolite GS-331007, and LDV were estimated using noncompartmental methods and are summarized in Table 1. Following the first-dose administration, SOF was detectable in 2 samples only; as such, the single-dose PK parameters for SOF could not be estimated reliably and are not included in the summary. The PK was repeated at day 7; mean steady-state exposure parameters for SOF were higher, whereas GS-331007 and LDV exposures were lower than the mean exposures observed in LDV/SOF adult phase 2/3 population PK analyses. Notably, all estimates remained within the ranges (minimum to maximum) of exposures in the phase 2/3 population. In conclusion, a 12-week treatment regimen with SOF/LDV/ribavirin for HCV was well tolerated and led to a SVR in 2 HCV (type 1b)– infected pediatric patients. The PK data in the 1-yearold child was within the ranges reported for adults, using a quarter of the adult DAA dosages. These first 2 successfully treated HCV patients provide us with preliminary evidence that these drugs are safe and Abbreviations: %CV, % coefficient of variation; AUC, area under the plasma concentration-time curve; Cmax, maximum plasma concentration; DAA, direct-acting antiviral; HCV, hepatitis C virus; LDV, ledipasvir; LT, liver transplantation; PCR, polymerase chain reaction; PK, pharmacokinetic; SCT, stem cell transplantation; SOF, sofosbuvir; SVR, sustained virological response.

Pnematosis Intestinalis and Portal Venous Gas in Pediatric Liver Transplant Recipient.

FIGURE 1. Abdominal ultrasound, transverse scan of the epigastric region displaying the transplanted liver. A, Air bubbles are present in the portal vein (arrow), its branches, and diffusely throughout the liver parenchyma. B, Pulsed Doppler waveform of the portal flow FIGURE 2. Abdominal ultrasound, transverse scan of the left flank: pneumatosis intestinalis in the wall of the left colon (arrows); ( 1⁄4 air in the bowel lumen).

Late graft hepatitis and fibrosis in pediatric liver allograft recipients: Current concepts and future developments.

In a recent review by Kelly et al., the authors conclude that allograft fibrosis is progressive with time in pediatric liver transplant recipients. We are opposed to this conclusion, which for us is a misleading message that may have an impact on the medical and patient community. In an important single-center series that we published early in 2016, we evaluated 281 serial protocol biopsies from 89 longterm stable recipients, with concomitant information of their human leukocyte antigen (HLA) and non-HLA antibodies. These are precisely the aspects covered in the review, and we consider that our results deserve to be mentioned because the outcomes were contradictory. We did not find allograft fibrosis being progressive with time; rather it was dynamic and could also decrease between serial biopsies from individual patients. Fibrosis evolution was observed to be an individually driven trend with no generalized patterns. This could be influenced by certain genetic and acquired characteristics. To study this, we used a mixed model of statistics that considers the temporal evolution of a characteristic in an individual as opposed to in the article, which included studies where cross-sectionally pooled data were used to determine “mean fibrosis.” That in and of itself is questionable because it is staged using an ordinal scoring system. Hence, we oppose the conclusion made by using this methodology, of allograft fibrosis being progressive with time, because we think that this message may be misleading. Additionally, the studies that have been included in the review suggest that these observations are not based on only stable recipients, nor on protocol biopsies, and expectedly, they do not reflect the natural history of an allograft in an uncomplicated recipient. The authors describe allograft hepatitis and infer that “in majority of patients with idiopathic hepatitis no etiology was found.” This is based on studies that did not evaluate the contribution of the HLA antibodies. The role of HLA antibodies in allograft inflammation is well documented as also mentioned in the review. Hence, class II HLA antibodies have to be accounted for. The review mentions the role of DQ subtype of HLA class II antibodies and that they are detected in 50%-60% of children. In our series, we have found that only donor-specific DQ antibodies with mean fluorescence intensity (MFI)> 5000 were associated with allograft inflammation and fibrosis. Using a lower cutoff of 1500 MFI, we detected class II donor-specific antibodies in 15.6%, and 11.7% had DQ subclass of >5000. Also, the HLA and non-HLA antibodies had an additive effect on causing allograft inflammation and fibrosis. In Table 1 (second line) of the review, 164 biopsies are said to be evaluated, whereas in the original article by these authors, 64 biopsies were said to be included.

The Histological Quantification of Alpha-Smooth Muscle Actin Predicts Future Graft Fibrosis in Pediatric Liver Transplant Recipients

Histological quantification of apha smooth muscle actin predicts future graft fibrosis in pediatric liver transplant recipients Varma S1, Stephenne X1, Komuta M2, Bouzin.C3, Ambroise J4, Smets F1, Reding R5, and Sokal EM1 Universite Catholique de Louvain, Cliniques Universitaires St Luc, 1Service de Gastroenterologie et Hepatologie Pediatrique and Pediatric Research Unit, 2Service de Anatomopathologie, 3 Imaging Platform (2IP) Institut de Recherche Experimentale et Clinique (IREC) , 4 Centre for Applied Molecular Technologies (CTMA), Institut de Recherche Experimentale et Clinique (IREC) 5 Unites de Chirurgie Pediatrique, Brussels, Belgium Aims: To evaluate significance of alpha smooth muscle actin (ASMA) expression on liver biopsy as predictor of future graft fibrosis in pediatric liver transplant (LT) recipients. Background: Activated hepatic stellate cells express cytoplasmic protein-alpha smooth muscle actin (ASMA) and subsequently secrete collagen leading to liver fibrosis. As activation of stellate cells precedes collagen deposition, we hypothesize that quantification of ASMA can predict the severity of future fibrosis. Patients: Stable pediatric LT recipients transplanted between 2006-20012, with two protocol biopsies less than two years apart and first being at more than 1year post LT. Patients with biliary, vascular complications, autoimmune hepatitis, hepatitis B or C infection, re-transplantation, or those with inadequate biopsy size were excluded. Methods: Metavir and liver allograft fibrosis scoring (LAFSc) used for fibrosis assessment. Automated staining for ASMA followed by digital quantification of ASMA positive area percentage was done. Fibrosis on initial biopsy specimen was labelled “current fibrosis” and on next biopsy labelled “prospective fibrosis”. The change between “current” and “prospective” fibrosis score was “prospective change in fibrosis”. Bile duct proliferation, lobular inflammation and portal tract infiltration was also evaluated. Results: 32 biopsy specimens, from 18 patients stained for ASMA and 56 evaluated for fibrosis. Significant association between the ASMA positive area percentage on initial biopsy and “prospective change in fibrosis” was seen; using Metavir (p-value = 0.02), LAFSc cumulative (p-value = 0.02) and LAFSc-portal (p-value=0.01) scores. AUROC analysis suggested ASMA positive area percentage > 1.05 to predict increased fibrosis on the next biopsy (60.0 % sensitivity, 90.0 % specificity). Conclusion: ASMA quantification on biopsy in liver transplant recipients predicts the future course of fibrosis, specifically portal fibrosis.

Allograft inflammation and fibrosis among maintenance pediatric liver transplant recipients – genetic predisposition and antibodies, connecting the missing links.

Allograft inflammation and fibrosis among maintenance pediatric liver transplant recipients – genetic predisposition and antibodies, connecting the missing links. Varma S1, Latinne D2, Komuta M3, Ambroise J4, Smets F1, Baldin P3,Reding R5 , Stephenne X1, and Sokal EM1 Universite Catholique de Louvain, Cliniques Universitaires St Luc, 1Service de Gastroenterologie et Hepatologie Pediatrique, 3Service de Anatomopathologie 4 Centre for Applied Molecular Technologies (CTMA), Institut de Recherche Experimentale et Clinique (IREC), 5 Paediatric Surgery and Transplantation Unit, Brussels, Belgium Aim: To determine impact of HLA allo-antibodies, non-HLA auto-antibodies and HLA-DRB1 genotype amongst stable, long term pediatric liver transplantation (LT) recipients; on allograft health. Background: Role of HLA allo-antibodies and non-HLA auto-antibodies in late allograft fibrosis and inflammation is unclear in pediatric LT, though inferior long-term outcomes with class II donor specific HLA antibodies (Class-II DSA) post LT are suggested. HLA DRB1*03 or 04 genotype predisposes to auto-immune hepatitis (AIH), prototype of antibody related hepatic inflammation. Patients: Stable LT recipients transplanted in 2006-2012 and with >2 protocol biopsies were included. Autoimmune hepatitis, hepatitis B or C infection as indication of LT; ABOi graft, biliary or vascular complications post LT, re-transplantation were exclusion criteria. Methods Data were collected at 1-month pre-LT and simultaneous to last protocol biopsy. HLA, ANA, SMA, LKM antibodies, HLA-DRB1 genotype of recipients and histological aspects of biopsy, were collected. Histological parameters including rejection, bile duct proliferation, lobular inflammation, and portal tract infiltration were assessed while fibrosis was evaluated using Metavir and liver allograft fibrosis scoring (LAFSc). Results 15 of 89 included children had class-II DSA post LT. Class-II DSA was associated with allograft fibrosis using Metavir and LAFSc–score (p <0.01), specifically portal fibrosis (p<0.01). Class-II DSA, non-HLA auto-antibodies were associated with portal inflammation (p <0.01) and showed additive effects. Recipient HLA-DRB1*03 or 04 genotype was associated with portal fibrosis (p <0.05) and showed additive effect with class-II DSA. Conclusion Class-II DSA, non-HLA auto-antibodies, HLA-DRB1 genotype of recipient are important factors for long term allograft health and have additive effect.