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Persistent tissue reservoirs of HIV present a major barrier to cure. Defining subsets of infected cells in tissues is a major focus of HIV cure research. Herein, we describe a novel multiplexed in situ hybridization (ISH) (RNAscope) protocol to detect HIV-DNA (vDNA) and HIV-RNA (vRNA) in formalin-fixed paraffin-embedded (FFPE) human tissues in combination with immunofluorescence (IF) phenotyping of the infected cells. We show that multiplexed IF and ISH (mIFISH) is suitable for quantitative assessment of HIV vRNA and vDNA and that multiparameter IF phenotyping allows precise identification of the cellular source of the ISH signal. We also provide semi-quantitative data on the impact of various tissue fixatives on the detectability of vDNA and vRNA with RNAscope technology. Finally, we describe methods to quantitate the ISH signal on whole-slide digital images and validation of the quantitative ISH data with quantitative real-time PCR for vRNA. It is our hope that this approach will provide insight into the biology of HIV tissue reservoirs and to inform strategies aimed at curing HIV.

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Background: There is an urgent need to develop and implement low cost, high-throughput standardized methods for routine molecular assessment of transplant biopsies. Given the vast archive of formalin-fixed and paraffin-embedded (FFPE) tissue blocks in transplant centers, a reliable protocol for utilizing this tissue bank for clinical validation of target molecules as predictors of graft outcome over time, would be of great value.Methods: We designed and optimized assays to quantify 19 target genes, including previously reported set of tissue common rejection module (tCRM) genes. We interrogated their performance for their clinical utility for detection of graft rejection and inflammation by analyzing gene expression microarrays analysis of 163 renal allograft biopsies, and subsequently validated in 40 independent FFPE archived kidney transplant biopsies at a single center.Results: A QPCR (Fluidigm) and a barcoded oligo-based (NanoString) gene expression platform were compared for evaluation of amplification of gene expression signal for 19 genes from degraded RNA extracted from FFPE biopsy sections by a set protocol. Increased expression of the selected 19 genes, that reflect a combination of specific cellular infiltrates (8/19 genes) and a graft inflammation score (11/19 genes which computes the tCRM score allowed for segregation of kidney transplant biopsies with stable allograft function and normal histology from those with histologically confirmed acute rejection (AR; p = 0.0022, QPCR; p = 0.0036, barcoded assay) and many cases of histological borderline inflammation (BL). Serial biopsy shaves used for gene expression were also processed for in-situ hybridization (ISH) for a subset of genes. ISH confirmed a high degree of correlation of signal amplification and tissue localization.Conclusions: Target gene expression amplification across a custom set of genes can identify AR independent of histology, and quantify inflammation from archival kidney transplant biopsy tissue, providing a new tool for clinical correlation and outcome analysis of kidney allografts, without the need for prospective kidney biopsy biobanking efforts.

Background Risks for living-liver donors are lower in case of a left liver donation, however, due to lower graft volume, the risk for small-for-size situations in the recipients increases. This study aims to prevent small-for-size situations in recipients using an auxiliary two-staged partial resection liver transplantation (LTX) of living-donated left liver lobes. Case presentation Two patients received a two-stage auxiliary LTX using living-donated left liver lobes after left lateral liver resection. The native extended right liver was removed in a second operation after sufficient hypertrophy of the left liver graft had occurred. Neither donor developed postoperative complications. In both recipients, the graft volume increased by an average of 105% (329 ml to 641 ml), from a graft-to-body-weight ratio of 0.54 to 1.08 within 11 days after LTX, so that the remnant native right liver could be removed. No recipient developed small-for-size syndrome; graft function and overall condition is good in both recipients after a follow-up time of 25 months. Conclusions Auxiliary two-staged partial resection LTX using living-donor left lobes is technically feasible and can prevent small-for-size situation. This new technique can expand the potential living-donor pool and contributes to increase donor safety.

Liver transplantation (LTx) is often the only possible therapy for many end-stage liver diseases, but successful long-term transplant outcomes are limited by multiple factors, including ischemia reperfusion injury (IRI). This situation is aggravated by a shortage of transplantable organs, thus encouraging the use of inferior quality organs. Here, we have investigated early hepatic IRI in a retrospective, exploratory, monocentric case-control study considering organ marginality. We analyzed standard LTx biopsies from 46 patients taken at the end of cold organ preparation and two hours after reperfusion, and we showed that early IRI was present after two hours in 63% of cases. Looking at our data in general, in accordance with Eurotransplant criteria, a marginal transplant was allocated at our institution in about 54% of cases. We found that patients with a marginal-organ LTx showing evidence of IRI had a significantly worse one-year survival rate (51% vs. 75%). As we saw in our study cohort, the marginality of these livers was almost entirely due to steatosis. In contrast, survival rates in patients receiving a non-marginal transplant were not influenced by the presence or absence of IRI. Poorer outcomes in marginal organs prompted us to examine pre- and post-reperfusion biopsies, and it was revealed that transplants with IRI demonstrated significantly greater T cell infiltration. Molecular analyses showed that higher mRNA expression levels of CXCL-1, CD3 and TCRγ locus genes were found in IRI livers. We therefore conclude that the marginality of an organ, namely steatosis, exacerbates early IRI by enhancing effector immune cell infiltration. Preemptive strategies targeting immune pathways could increase the safety of using marginal organs for LTx.

This study aimed to elucidate the impact of epithelial regenerative responses and immune cell infiltration on biliary complications after liver transplantation. Bile duct (BD) damage after cold storage was quantified by a BD damage score and correlated with patient outcome in 41 patients. Bacterial infiltration was determined by fluorescence in situ hybridization (FISH). BD samples were analyzed by immunohistochemistry for E-cadherin, cytokeratin, CD56, CD14, CD4, CD8, and double-immunofluorescence for cytokine production and by messenger RNA (mRNA) microarray. Increased mRNA levels of adherens junctions (P < 0.01) were detected in damaged BDs from patients without complications compared with damaged BDs from patients with biliary complications. Immunohistochemistry showed increased expression of E-cadherin and cytokeratin in BDs without biliary complications (P = 0.03; P = 0.047). FISH analysis demonstrated translocation of bacteria in BDs. However, mRNA analysis suggested an enhanced immune response in BDs without biliary complications (P < 0.01). Regarding immune cell infiltration, CD4 and CD8 cells were significantly increased in patients without complications compared with those with complications (P = 0.02; P = 0.01). In conclusion, following BD damage during cold storage, we hypothesize that the functional regenerative capacity of biliary epithelium and enhanced local adaptive immune cell infiltration are crucial for BD recovery. Such molecular immunological BD analyses therefore could help to predict biliary complications in cases of "major" epithelial damage after cold storage.Liver Transplantation 23 1422-1432 2017 AASLD.

Background: Portal vein complications (PVCs) after pediatric liver transplantation (LT) are sometimes asymptomatic, especially in the early phase, and can threaten both the graft and patient's survival. Therefore, the purpose of this study is to analyze the risk factors for portal vein thrombosis (PVT) and portal vein stenosis (PVS) after pediatric LT.Methods: All pediatric patients (n = 115) who underwent primary LT at Regensburg University Hospital between January 2010 and April 2017 were included in this study.The pre-, intra-, and postoperative parameters of all patients were retrospectively reviewed and risk factors for both PVT and PVS were analyzed.Results: Of the 115 patients, living donor LT was performed on 57 (49.5%) patients, and biliary atresia was the primary diagnosis in 65 patients (56%). After pediatric LT, 9% of patients developed PVT, and 16.5% developed PVS. Patient weight ≤7 kg [odds ratio (OR) 9.35, 95% confidence interval (CI) 1.03-84.9, p = .04] and GRWR >3% (OR 15.4, 95% CI 1.98-129.5, p = .01) were the independent risk factors for the development of PVT and PVS, respectively upon multivariate analysis. The overall patient survival rates at 1, 3, and 5 years were 91%, 90%, and 89%, respectively, and there was no difference in patient survival among those with and without PVCs.Conclusions: Pediatric patients with body weight <7 kg and/or receiving a graft with GRWR >3% may develop PVCs and so require certain surgical modifications, close follow-up, and prophylactic anticoagulant therapy following transplant. K E Y W O R D Sliver transplant, pediatric, portal vein stenosis, portal vein thrombosis, risk factors Liver transplantation (LT) remains the treatment of choice for pediatric patients with end-stage liver diseases, acute liver failure, and certain metabolic diseases. Despite the recent advances in pediatric LT techniques, still, portal vein complications (PVCs) are serious issues that threaten graft and patient survival. 1 PVCs include portal vein thrombosis (PVT) or portal vein stenosis (PVS) and have an incidence of 3%-14% after pediatric LT which is significantly higher than their incidence in adults LT. 2,3 The clinical manifestations of PVC range from completely asymptomatic to manifestations of portal hypertension such as ascites, gastrointestinal bleeding, splenomegaly, or cytopenia up to graft failure especially in the case of early PVT. 1,4 Therefore, knowing the perioperative risk factors for PVC is crucial to improve patient and graft survival posttransplant by administration of prophylaxis anticoagulant therapy and meticulous follow-up of high-risk patients.Most of the previous studies reported the risk factors for PVT in pediatric LT and included small hypoplastic portal vein (PV), use of vascular grafts, preoperative PVT, and low body weight. 5,6 Nevertheless, few studies investigated the risk factors for PVS after pediatric LT. 7 Accordingly, this study aims to investigate the risk factors for the development of PVT and PVS after pediatric LT at our center and assess th...