Hepatitis B immunoglobulin inhibits the secretion of HBV via antigen-antibody precipitation in the multivesicular body.

BACKGROUND AND AIMS: Although the main action of human hepatitis B immunoglobulin (HBIG) is to neutralize hepatitis B virus surface antigen (HBsAg) in serum, HBIG is known to be localized in the cell. However, the effect of intracellularly located HBIG is poorly understood because of the low purity of conventional plasma-derived HBIG (cHBIG). We attempted to elucidate the mechanism of action of internalized HBIG using recombinant HBIG (lenvervimab). METHODS: We used HBsAg producing cell lines, non-HBsAg cell lines and human HBsAg-producing hepatocytes. The autophagosome lysis pathway-related proteins Rab5, calnexin, giantin, and Rab7 were used to localize HBsAg and anti-HBs-IgG in the cytoplasm using Western blotting and confocal microscopy. RESULTS: Intracellular anti-HBs-IgG (lenvervimab and cHBIG) transported via Fc receptor-mediated endocytosis increased the number of autophagosomes. However, there was no change in autolysis. HBsAg and anti-HBs-IgG co-localized in the multivesicular body and precipitated in the cytoplasm. HBsAg secretion into culture medium decreased after lenvervimab treatment. Simultaneously, the amount of cellular HBsAg increased in the cell lines but decreased in human hepatocytes. Furthermore, intracellular lenvervimab is not easily removed from HBsAg cell lines. CONCLUSIONS: Lenvervimab decreases HBsAg secretion, and HBsAg antibody precipitation in the multivesicular body may play an important role.

Therapeutic Extracellular Vesicles from Tonsil-Derived Mesenchymal Stem Cells for the Treatment of Retinal Degenerative Disease.

BACKGROUND: Retinal degenerative disease (RDD), one of the most common causes of blindness, is predominantly caused by the gradual death of retinal pigment epithelial cells (RPEs) and photoreceptors due to various causes. Cell-based therapies, such as stem cell implantation, have been developed for the treatment of RDD, but potential risks, including teratogenicity and immune reactions, have hampered their clinical application. Stem cell-derived extracellular vesicles (EVs) have recently emerged as a cell-free alternative therapeutic strategy; however, additional invasiveness and low yield of the stem cell extraction process is problematic. METHODS: To overcome these limitations, we developed therapeutic EVs for the treatment of RDD which were extracted from tonsil-derived mesenchymal stem cells obtained from human tonsil tissue discarded as medical waste following tonsillectomy (T-MSC EVs). To verify the biocompatibility and cytoprotective effect of T-MSC EVs, we measured cell viability by co-culture with human RPE without or with toxic all-trans-retinal. To elucidate the cytoprotective mechanism of T-MSC EVs, we performed transcriptome sequencing using RNA extracted from RPEs. The in vivo protective effect of T-MSC EVs was evaluated using Pde6b gene knockout rats as an animal model of retinitis pigmentosa. RESULTS: T-MSC EVs showed high biocompatibility and the human pigment epithelial cells were significantly protected in the presence of T-MSC EVs from the toxic effect of all-trans-retinal. In addition, T-MSC EVs showed a dose-dependent cell death-delaying effect in real-time quantification of cell death. Transcriptome sequencing analysis revealed that the efficient ability of T-MSC EVs to regulate intracellular oxidative stress may be one of the reasons explaining their excellent cytoprotective effect. Additionally, intravitreally injected T-MSC EVs had an inhibitory effect on the destruction of the outer nuclear layer in the Pde6b gene knockout rat. CONCLUSIONS: Together, the results of this study indicate the preventive and therapeutic effects of T-MSC EVs during the initiation and development of retinal degeneration, which may be a beneficial alternative for the treatment of RDD.

Sirolimus Attenuates Calcineurin Inhibitor-Induced Epithelial-Mesenchymal Transition in Hepatocellular Carcinoma.

BACKGROUND: Calcineurin inhibitors (CNIs), which are potent immunosuppressants (ISs), increase the risk for hepatocellular carcinoma (HCC) recurrence after liver transplantation (LTx). Epithelial-mesenchymal transition (EMT) is a key process in which epithelial cancer cells lose their polarity, resulting in cancer progression and metastasis. The aim of this study was to evaluate the effect of sirolimus (SRL) individually and in combination with other ISs to reduce EMT. METHODS: HCC SK-Hep1 cells were used and various ISs (SRL, tacrolimus, cyclosporine A, or mycophenolate mofetil) were administered at 2 dosages and in combination therapies. Mice were transplanted with SK-Hep1 cells (in the liver) and were monitored after 2 weeks. RESULTS: The in vitro treatment with SRL showed a dose-dependent attenuation of cell proliferation and migration in case of the individual and IS combination treatments; further, decreased levels of pro-EMT proteins, namely, N-cadherin, transforming growth factor-ß, ZEB1, Slug, and Snail were observed. In contrast, E-cadherin expression was upregulated after both the individual and IS combination treatments. These results were also observed in the samples from mice transplanted with the SK-Hep1 cells. CONCLUSION: The present study demonstrated that SRL reduced HCC metastasis by inhibiting EMT. Thus, our findings provide a rationale for the use of SRL in combination with ISs in HCC LTx patients.

Sirolimus and MMF are insufficient immunosuppressants for regulation of the proliferation of CD133+EpCAM+ cell populations in HCC cell lines.

Studies on effective immunosuppressive strategies for the management of patients undergoing a liver transplantation (LT) due to hepatocellular carcinoma (HCC) are limited. In the present study, immunosuppressive candidates predicted to exhibit beneficial immunosuppressive and tumor-suppressive effects in patients with HCC were assessed using Huh7 and HEP3B HCC cells, which have high proportions of CD133+EpCAM+ cancer stem cell (CSC) populations. The immunosuppressants assessed were sirolimus, tacrolimus, cyclosporine A and mycophenolate mofetil (MMF), and their activities were assessed on CSCs. Sirolimus and MMF reduced the proliferation of Huh7 and HEP3B cells; however, the proportion of CD133+EpCAM+ was notably increased in treated Huh7 cells. Sirolimus treatment alone resulted in G0-G1 cell cycle arrest at all doses in all Huh7 and CD133-EpCAM- populations; however, CD133+EpCAM+ populations showed only slight G1 arrest at higher doses only. In contrast, S-phase arrest was induced at all doses in the Huh7, CD133-EpCAM- and CD133+EpCAM+ populations by MMF. Sirolimus and MMF effectively reduced the proliferation of Huh7 and HEP3B cells, but did not exert a notable effect on the CD133+EpCAM+ cells. Therefore, therapeutic strategies utilizing Sirolimus and MMF should be further studied in vivo for regulation of CSC populations in order to reduce HCC recurrence rates.

Impact of PNPLA3 (rs738409-G) polymorphism on post-transplant outcomes after liver transplantation for alcohol-related liver disease.

INTRODUCTION: We aimed to evaluate the association between PNPLA3 polymorphism and post-liver transplantation (LT) outcomes related to alcohol relapse (AR). METHOD: We retrospectively analyzed data from patients receiving LT for alcoholic liver disease (ALD) from 04/2014 to 12/2017. Liver-related clinical outcomes were assessed by the gamma-glutamyltransferase (GGT) level and alcohol-related liver failure (ARLF). Genotyping was performed using prospectively collected DNA samples in both donors and recipients. RESULTS: A total of 83 recipients were enrolled. Post-LT AR occurred in 31 patients (37.3%). Thirty-one patients (14 AR, 9 abstainers) showed elevated GGT levels, and 3 AR patients experienced ARLF. In the multivariate analysis, rs738409 G allele carrier and heavy drinking (HRAR score ≥ 4) were independent risk factors for elevated GGT levels (odds ratio [OR] = 8.69, P < .01; OR = 13.07, P = .01) and ARLF (OR = 4.52, P = .04; OR = 19.62, P = .03). Among 15 heavy AR patients, being an rs738409 G allele carrier was related to GGT elevation (P = .03) and ARLF (P = .04), but it was not related to GGT elevation in mild drinkers (n = 16) or abstainers (n = 52). CONCLUSION: PNPLA3 polymorphism of the recipient genotype can independently affect the post-LT prognosis of LT patients for ALD, especially in heavy AR patients. Therefore, strong abstinence education is recommended in patients with this single nucleotide polymorphism.

Effect of PNPLA3 I148M polymorphism on histologically proven non-alcoholic fatty liver disease in liver transplant recipients.

AIM: PNPLA3 I148M polymorphism (rs738409 C>G) is the most important and best-known polymorphism for non-alcoholic fatty liver disease (NAFLD). However, little is known about the effect of this polymorphism on NAFLD after liver transplantation (LT). We aimed to evaluate the association between this polymorphism and post-LT NAFLD. METHODS: We designed a prospective case-control study. Among adult recipients who underwent LT between April 2014 and October 2015, those whose whole blood was preoperatively collected for genotyping in both recipients and coupled donors and those who underwent protocol biopsy at 1 year post-LT were enrolled. RESULTS: A total of 32 recipients were enrolled. Histologically proven steatosis (≥5%) was present in 28.1% of patients at a mean time of 12.7 ± 2.0 months after LT. Moderate and more severe steatosis (≥33%) was present in 9.4%. One year after LT, steatosis was present in 50.0% of homozygous recipients with the rs738409-G allele. It was present in 27.3% of heterozygous recipients with the rs738409-G allele, and in 9.1% (P = 0.041) of recipients with rs738409-CC. The genotype of the donor was not significantly (P = 0.647) associated with post-LT NAFLD. When both recipient and coupled donor showed heterogeneous or homozygous genotype of the rs738409-G allele, there was significantly more post-LT NAFLD compared to that in others (47.1% vs. 6.7%; P = 0.018). In univariate and multivariate analyses, only the presence of the rs738409-G risk allele in both donor and recipient was a significant risk factor for post-LT NALFD (relative risk, 26.95; P = 0.048). CONCLUSIONS: PNPLA3 I148M polymorphism can significantly affect histologically proven NAFLD at 1 year post-LT.

Sirolimus and Metformin Synergistically Inhibits Colon Cancer In Vitro and In Vivo.

We estimated the effect of various immunosuppressants (ISs) and metformin (M) to provide theoretical background of optimal therapeutic strategy for de novo colon cancer after liver transplantation (LT). Three colon cancer cell lines (HT29, SW620, and HCT116) were used in in vitro studies. HT29 was also used in BALB/c-nude mice animal models. Following groups were used in both in vitro and in vivo studies: sirolimus (S), tacrolimus (T), cyclosporin A (CsA), M, metformin/sirolimus (Met/S), metformin/tacrolimus (Met/T), and metformin/cyclosporin A (Met/CsA). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed and western blot analyses were performed for mTOR pathway proteins, apoptosis proteins, and epithelial-mesenchymal-transition (EMT) proteins. Tumor volume was measured for 4 weeks after inoculation. MTT-assay revealed significant cell viability inhibition in all 3 colon cancer cell lines in groups of S, M, and Met/S. Of note, group Met/S showed synergistic effect compare to M or S group. Western blot analysis showed significant low levels of all investigated proteins in groups of S and Met/S in both in vitro and in vivo experiment. Tumor growth was significantly inhibited only in the Met/S group. Combination of Met and S showed the most potent inhibition in all colon cancer cell lines. This finding might have application for de novo colon cancer.