Nyberg, Scott L.

The mammalian liver's regenerative ability has led researchers to engineer animals as incubators for expansion of human hepatocytes. The expansion properties of human hepatocytes in immunodeficient mice are well known. However, little has been reported about larger animals that are more scalable and practical for clinical purposes. Therefore, we engineered immunodeficient swine to support expansion of human hepatocytes and identify barriers to their clinical application. Immunodeficient swine were engineered by knockout of the recombinase-activating gene 2 ( RAG2 ) and fumarylacetoacetate hydrolase (FAH). Immature human hepatocytes (ihHCs) were injected into fetal swine by intrauterine cell transplantation (IUCT) at day 40 of gestation. Human albumin was measured as a marker of engraftment. Cytotoxicity against ihHCs was measured in transplanted piglets and control swine. We initially detected higher levels of human albumin in cord blood of newborn FAH/ RAG2 -deficient (FR) pigs compared with immunocompetent controls (196.26 ng/dL vs. 39.29 ng/dL, p = 0.008), indicating successful engraftment of ihHCs after IUCT and adaptive immunity in the fetus. Although rare hepatocytes staining positive for human albumin were observed, levels of human albumin did not rise after birth, but declined, suggesting rejection of xenografted ihHCs. Cytotoxicity against ihHCs increased after birth by 3.8% (95% CI: [2.1%–5.4%], p < 0.001) and inversely correlated with declining levels of human albumin ( p = 2.1 × 10 −5 , R 2 = 0.17). Circulating numbers of T cells and B cells were negligible in FR pigs. However, circulating natural killer (NK) cells exerted cytotoxicity against ihHCs. NK cell activity was lower in immunodeficient piglets after IUCT than in naive controls (30.4% vs. 40.1%, p = 0.011, 95% CI for difference [2.7%–16.7%]). In conclusion, ihHCs were successfully engrafted in FR swine after IUCT. NK cells were a significant barrier to expansion of hepatocytes. New approaches are needed to overcome this hurdle and allow large-scale expansion of human hepatocytes in immunodeficient swine. Impact statement There is currently a need for robust expansion of human hepatocytes. We describe an immunodeficient swine model into which we engrafted immature human hepatocytes (ihHCs). We identified the mechanism of the eventual graft rejection by the intact NK cell population, which has not been previously shown to have a significant role in xenograft rejection. By both improving engraftment and reducing NK cell-mediated cytotoxicity toward the graft through intrauterine cell transfer, we confirmed the presence of residual adaptive immunity in this model of immunodeficiency and the ability to induce hyposensitization in the NK cell population by taking advantage of the fetal microenvironment.

Cell therapies using immune cells or non-parenchymal cells of the liver have emerged as potential treatments to facilitate immunosuppression withdrawal and to induce operational tolerance in liver transplant (LT) recipients. Recent pre-clinical and clinical trials of cellular therapies including regulatory T cells, regulatory dendritic cells, and mesenchymal cells have shown promising results. Here we briefly summarize current concepts of cellular therapy for induction of operational tolerance in LT recipients.

Background. Portopulmonary hypertension (POPH), pulmonary arterial hypertension (PAH) that develops in the setting of portal hypertension, affects 5%–6% of patients with liver disease and is associated with significant morbidity and mortality. A mean pulmonary arterial pressure (mPAP) threshold of 35 mm Hg is used to stratify perioperative risk and liver transplant eligibility in treated POPH patients but does not take into account the specific factors that contribute to the pressure elevation. Methods. In this case series, we describe the characteristics and posttransplant outcomes of patients with treated POPH and an mPAP ≥35 mm Hg and pulmonary vascular resistance (PVR) <250 dynes-s-cm−5 at or just before liver transplantation (LT). We also describe the effect of PAH therapy on pulmonary hemodynamics in patients with POPH. Results. Sixteen patients were included. All patients were on PAH therapy at the time of LT. PAH therapy resulted in a decrease of mPAP (median 18.4%; interquartile range [IQR] 8.9%–27.0%) with a reduction in PVR (median 50.5%; IQR, 45.4%–70.7%), and an increase in both cardiac output (CO) (median 28.1%; IQR 5.7%–63.8%) and PAWP (median 50.0%; IQR 16.7%–108.3%) before LT. One year posttransplant survival was 69% (11/16); however, only 1 death was attributed to POPH. At 1-year posttransplant, 63.6% (7/11) of patients were weaned off all PAH therapy with clinical and echocardiographic resolution of POPH. Conclusions. In treated POPH patients with an mPAP ≥35 mm Hg and PVR < 250 dynes-s-cm−5 before LT, 1-year posttransplant survival was 69% and the majority of patients were able to discontinue PAH therapy.

Abstract The precise characterization of the lobular architecture of the liver has been subject of investigation since the earliest historical publications, but an accurate model to describe the hepatic lobular microanatomy is yet to be proposed. Our aim was to evaluate whether Voronoi diagrams can be used to describe the classic liver lobular architecture. We examined the histology of normal porcine and human livers and analyzed the geometric relationships of various microanatomic structures utilizing digital tools. The Voronoi diagram model described the organization of the hepatic classic lobules with overall accuracy nearly 90% based on known histologic landmarks. We have also designed a Voronoi-based algorithm of hepatic zonation, which also showed an overall zonal accuracy of nearly 90%. Therefore, we have presented evidence that Voronoi diagrams represent the basis of the two-dimensional organization of the normal liver and that this concept may have wide applicability in liver pathology and research.

Background. Portopulmonary hypertension (POPH) is the presence of pulmonary arterial (PA) hypertension in patients with portal hypertension and is associated with significant morbidity and mortality. In a cohort of POPH patients, we describe the clinical outcomes of POPH patients who underwent liver transplantation (LT). Methods. Retrospectively collected data from a prospectively assembled cohort of all consecutive POPH adults evaluated in 3 transplant centers from 1996 to 2019. Results. From a cohort of 228 POPH patients, 50 patients underwent LT. Significant hemodynamic improvement after PA-targeted therapy was observed, with 58% receiving only monotherapy pretransplant. After LT, 21 (42%) patients were able to discontinue and remained off PA-targeted therapy. The 1-, 3-, and 5-y unadjusted survival rates after LT were 72%, 63%, and 60%, respectively. An elevated pulmonary vascular resistance (PVR) before LT was associated with worse survival rate (HR, 1.91; 95% CI, 1.07-3.74, P = 0.04). No survival difference was observed in those granted MELD exception or transplants performed before or after the year 2010. Conclusions. Significant number of POPH patients discontinued PA-targeted therapy after LT. Higher PVR before LT was associated with worse survival, as was monotherapy use. Despite effective PA-targeted therapies, POPH survival outcomes after LT in our cohort were modest and may reflect the need for more aggressive therapy.

Background. Experience with sequential hematopoietic stem cell transplant (HSCT) and kidney transplant (KT) is limited. Methods. We conducted a retrospective observational study of adult patients who underwent both HSCT and KT at our center, with a median follow-up of 11 y. Results. In our 54 patients cohort (94% autologous HSCT), 36 (67%) patients received HSCT first followed by KT, while 18 (33%) received KT before HSCT. In both groups, AL amyloidosis represented 50% of hematologic diagnosis. Only 4 patients expired due to hematologic disease relapse (2 patients in each group) and only 3 allografts were lost due to hematologic disease recurrence (HSCT first n = 1 and KT first n = 2). Overall 1, 5, and 10 y death-censored graft survival rates were 94%, 94%, and 94%, respectively, for the HSCT first group and 89%, 89%, and 75%, respectively, for the KT first group. Overall 1, 5, and 10 y patients survival rates were 100%, 97% and 90%, respectively, for the HSCT first group and 100%, 76%, and 63%, respectively, for the KT first group. Conclusions. Our study supports safety of sequential KT and HSCT, with improved overall patient survival compared to recipients of HSCT remaining on dialysis and good long-term kidney allograft outcome.

Background. The role of MZ phenotype of α 1 antitrypsin (α1AT) deficiency as a potential cofactor in advanced liver disease arising from other primary causes is not widely understood. In the general population, MZ phenotype accounts for 2%–4% in Europe and 2%–7.1% in North America. The aim of this study was to determine the prevalence of the MZ phenotype among various causes of cirrhosis in the United States in the modern era and its impact on pulmonary function before and after liver transplantation. Methods. This retrospective study included adult patients with cirrhosis who underwent liver transplantation at Mayo Clinic. Participants’ data including pathogenesis of cirrhosis, model for end-stage liver disease-Na score, α1AT phenotype, liver decompensation events, and pulmonary outcomes was determined by retrospective review of the liver transplantation database. Results. One hundred thirty of 1341 adult patients with cirrhosis (9.7%) were α1AT MZ carriers. When comparing the distribution of protease inhibitor (PI) MZ among different pathogenesis, the prevalence of MZ was significantly increased in nonalcoholic steatohepatitis (NASH), alcoholic liver disease (ALD), and cryptogenic cirrhosis compared with other causes. Thirty-seven of 171 with NASH (22%), 37 of 187 with ALD (20%), and 9 of 39 with cryptogenic cirrhosis (23.1%) were identified as PI MZ, while in other subgroups; we detected 18 of 320 with viral hepatitis, and 11 of 339 with primary biliary cholangitis/primary sclerosing cholangitis. Also, MZ patients were more likely to develop preoperative chronic obstructive lung disease, and postoperative pulmonary hypertension and pulmonary embolism than MM patients. Conclusions. The rates of preoperative and postoperative pulmonary complications were found to be higher in PI MZ patients than in PI MM patients. The MZ phenotype was significantly enriched in NASH, ALD, and cryptogenic cirrhosis.