Analysis of distribution, collection, and confirmation of capacity dependency of small extracellular vesicles toward a therapy for liver cirrhosis.

BACKGROUND: The progression of liver fibrosis leads to portal hypertension and liver dysfunction. However, no antifibrotic agents have been approved for cirrhosis to date, making them an unmet medical need. Small extracellular vesicles (sEVs) of mesenchymal stem cells (MSCs) are among these candidate agents. In this study, we investigated the effects of sEVs of MSCs, analyzed their distribution in the liver post-administration, whether their effect was dose-dependent, and whether it was possible to collect a large number of sEVs. METHODS: sEVs expressing tdTomato were generated, and their uptake into constituent liver cells was observed in vitro, as well as their sites of uptake and cells in the liver using a mouse model of liver cirrhosis. The efficiency of sEV collection using tangential flow filtration (TFF) and changes in the therapeutic effects of sEVs in a volume-dependent manner were examined. RESULTS: The sEVs of MSCs accumulated mostly in macrophages in damaged areas of the liver. In addition, the therapeutic effect of sEVs was not necessarily dose-dependent, and it reached a plateau when the dosage exceeded a certain level. Furthermore, although ultracentrifugation was commonly used to collect sEVs for research purposes, we verified that TFF could be used for efficient sEV collection and that their effectiveness is not reduced. CONCLUSION: In this study, we identified some unknown aspects regarding the dynamics, collection, and capacity dependence of sEVs. Our results provide important fundamentals for the development of therapies using sEVs and hold potential implications for the therapeutic applications of sEV-based therapies for liver cirrhosis.

Escherichia coli-derived outer-membrane vesicles induce immune activation and progression of cirrhosis in mice and humans.

BACKGROUND AND AIMS: Decompensated cirrhosis with fibrosis progression causes portal hypertension followed by an oedematous intestinal tract. These conditions weaken the barrier function against bacteria in the intestinal tract, a condition called leaky gut, resulting in invasion by bacteria and bacterial components. Here, we investigated the role of outer-membrane vesicles (OMVs) of Escherichia coli, which is the representative pathogenic gut-derived bacteria in patients with cirrhosis in the pathogenesis of cirrhosis. METHODS: We investigated the involvement of OMVs in humans using human serum and ascites samples and also investigated the involvement of OMVs from E. coli in mice using mouse liver-derived cells and a mouse cirrhosis model. RESULTS: In vitro, OMVs induced inflammatory responses to macrophages and neutrophils, including the upregulation of C-type lectin domain family 4 member E (Clec4e), and induced the suppression of albumin production in hepatocytes but had a relatively little direct effect on hepatic stellate cells. In a mouse cirrhosis model, administration of OMVs led to increased liver inflammation, especially affecting the activation of macrophages, worsening fibrosis and decreasing albumin production. Albumin administration weakened these inflammatory changes. In addition, multiple antibodies against bacterial components were increased with a progressing Child-Pugh grade, and OMVs were detected in ascites of patients with decompensated cirrhosis. CONCLUSIONS: In conclusion, OMVs induce inflammation, fibrosis and suppression of albumin production, affecting the pathogenesis of cirrhosis. We believe that our study paves the way for the future prevention and treatment of cirrhosis.

Small extracellular vesicles and liver diseases: From diagnosis to therapy.

Extracellular vesicles (EVs), especially small EVs (sEVs) derived from liver cells, have been the focus of much attention in the normal physiology and pathogenesis of various diseases affecting the liver. sEVs are approximately 100 nm in size, enclosed within lipid bilayers, and are very stable. The lipids, proteins, and nucleic acids, including miRNAs, contained within these vesicles are known to play important roles in intercellular communication. This mini-review summarizes the application of sEVs. First, liver diseases and the related diagnostic markers are described, and the current active status of miRNA research in diagnosis of hepatocellular carcinoma (HCC) is reported. Second, the biodistribution and pharmacokinetics of sEVs are described, and the liver is highlighted as the organ with the highest accumulation of sEVs. Third, the relationship between sEVs and the pathogenesis of liver disorders is described with emphesis on the current active status of miRNA research in HCC recurrence and survival. Finally, the possibility of future therapy using sEVs from mesenchymal stem (stromal) cells for cirrhosis and other diseases is described.

Synthesized HMGB1 peptide prevents the progression of inflammation, steatosis, fibrosis, and tumor occurrence in a non-alcoholic steatohepatitis mouse model.

AIM: Non-alcoholic steatohepatitis (NASH) with fibrosis eventually leads to cirrhosis and hepatocellular carcinoma. Thus, the development of therapies other than dietary restriction and exercise, particularly those that suppress steatosis and fibrosis of the liver and have a long-term beneficial effect, is necessary. We aimed to evaluate the therapeutic effects of the HMGB1 peptide synthesized from box A using the melanocortin-4 receptor-deficient (Mc4r-KO) NASH model mouse. METHODS: We performed short- and long-term administration of this peptide and evaluated the effects on steatosis, fibrosis, and carcinogenesis using Mc4r-KO mice. We also analyzed the direct effect of this peptide on macrophages and hepatic stellate cells in vitro and performed lipidomics and metabolomics techniques to evaluate the effect. RESULTS: Although this peptide did not show direct effects on macrophages and hepatic stellate cells in vitro, in the short-term administration model, we could confirm the reduction of liver damage, steatosis, and fibrosis progression. The results of lipidomics and metabolomics suggested that the peptide might ameliorate NASH by promoting lipolysis via the activation of fatty acid ß-oxidation and improving insulin resistance. In the long-term administration model, this peptide prevented progression to cirrhosis but retained the steatosis state, that is, the peptide prevents the progression to "burnt-out NASH." This peptide inhibited carcinogenesis by about one-third. CONCLUSION: This HMGB1 peptide can reduce liver damage, improve fibrosis and steatosis, and inhibit carcinogenesis, suggesting that the peptide would be a new treatment candidate for NASH and can contribute to the long-term prognosis for patients with NASH.

Therapeutic potential of mesenchymal stem cells and their exosomes in severe novel coronavirus disease 2019 (COVID-19) cases.

The novel coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019 (COVID-19) and the ensuing worldwide pandemic. The spread of the virus has had global effects such as activity restriction, economic stagnation, and collapse of healthcare infrastructure. Severe SARS-CoV-2 infection induces a cytokine storm, leading to acute respiratory distress syndrome (ARDS) and multiple organ failure, which are very serious health conditions and must be mitigated or resolved as soon as possible. Mesenchymal stem cells (MSCs) and their exosomes can affect immune cells by inducing anti-inflammatory macrophages, regulatory T and B cells, and regulatory dendritic cells, and can inactivate T cells. Hence, they are potential candidate agents for treatment of severe cases of COVID-19. In this review, we report the background of severe cases of COVID-19, basic aspects and mechanisms of action of MSCs and their exosomes, and discuss basic and clinical studies based on MSCs and exosomes for influenza-induced ARDS. Finally, we report the potential of MSC and exosome therapy in severe cases of COVID-19 in recently initiated or planned clinical trials of MSCs (33 trials) and exosomes (1 trial) registered in 13 countries on ClinicalTrials.gov.

Rare Genotype of His/His in NUDT15 Codon 139 and Thiopurine-associated Adverse Events in a Case of Ulcerative Colitis.

Thiopurine drugs are commonly used to treat immunologic diseases. However, the narrow therapeutic safety margin demands evidence-based precision medicine approaches. NUDT15 variants are associated with thiopurine-induced adverse events, particularly in Asians. We herein report a rare genotype of His/His in NUDT15 codon 139 in a case of ulcerative colitis and review the relevant literature. The patient experienced severe thiopurine-associated adverse events, including leukopenia and alopecia. There is no literature on the His/His genotype in NUDT15 codon 139, and our case suggests cautious use or the contraindication of thiopurines for patients with this genotype.

Hemorrhagic Gastric Metastasis from Hepatocellular Carcinoma Successfully Treated Using Coil Embolization of the Left Gastric Artery.

A 62-year-old man initially underwent transcatheter arterial chemoembolization for the treatment of hepatocellular carcinoma (HCC). One year after the initial treatment, he developed anemia. Upper gastrointestinal endoscopy revealed irregularly elevated tumors in the lower anterior gastric body, which were diagnosed to be metastasis from HCC. Left gastric artery coil embolization was performed to prevent sustained bleeding, and his anemia partially improved. In addition to direct invasion, hematogenous metastasis to the stomach from HCC is possible and therefore should be considered during treatment. Transcatheter arterial embolization for gastric metastasis is an effective treatment method which achieves a good degree of hemostasis in patients without any surgical indications.

Cone beam versus conventional computed tomography angiography volume measurement in partial splenic embolization.

When performing partial splenic arterial embolization (PSE), it can be difficult to determine the embolization ratio based on 2-dimensional digital subtraction angiography (DSA) image diagnosis alone. Therefore, at our department, we conduct computed tomography (CT) imaging intraoperatively and postoperatively to determine whether the planned embolization has been achieved. In recent years, developments in interventional radiology devices have enabled diagnostic imaging using cone beam CT. Here, we investigated whether the embolization ratio could be predicted from volume measurement with cone beam CT in PSE.We investigated correlations between volume measurement with conventional CT angiography (CTA) and volume measurement with cone beam CTA in 11 cases that underwent PSE with cone beam CT guidance (Allura Clarity FD20; Phillips, Amsterdam, The Netherlands) between December 2013 and May 2018.The mean subject age was 65.0 ± 5.8 years (6 men, 5 women). The subjects had underlying liver disorders of hepatitis C virus infection (4 cases), nonalcoholic steatohepatitis (4 cases), and alcohol-related disease (3 cases). A positive correlation was noted between conventional CTA and cone beam CTA, with infarction rates of 61.28 ±â€Š9.31% and 64.04 ±â€Š9.24%, respectively. The correlation coefficient between the 2 variables was .772.Because blood washout occurs rapidly in the spleen, contrast medium had to be continuously injected during imaging to enable dual-phase imaging with cone beam CT. However, we successfully performed imaging up to the second phase and volume measurement for the embolization ratio by inserting a catheter into the splenic artery and confirming the cone beam CT arrival time from the DSA images. The results were almost identical to those obtained from volume measurement with conventional CT based on CTA imaging. Thus, our results suggest that the splenic embolization ratio measurement obtained via cone beam CTA can be used to assess PSE treatment endpoints.

Efficacy and safety of repeated use of lusutrombopag prior to radiofrequency ablation in patients with recurrent hepatocellular carcinoma and thrombocytopenia.

AIMS: Thrombocytopenia is often associated with chronic liver disease. Lusutrombopag is a small molecule thrombopoietin receptor agonist designed to temporarily increase the platelet count in patients with chronic liver disease for whom elective invasive procedures are planned. In the present study, the efficacy and safety of repeated use of lusutrombopag prior to radiofrequency ablation (RFA) for recurrent hepatocellular carcinoma were examined. METHODS: Eight patients with hepatocellular carcinoma who had a platelet count <50 000/µL prior to both initial and repeat RFA at the time of recurrence received lusutrombopag (3 mg/day) orally for 7 days between March 2016 and August 2018. The following were compared: the effect of lusutrombopag to increase the platelet count as determined by the platelet count after the initial and repeated use of lusutrombopag, the rate of avoiding platelet transfusion, and the presence of any complications. RESULTS: The platelet count increased to 103 100 ± 22 800/µL 14 days after the first treatment and to 110 700 ± 17 800/µL 14 days after the repeated use. None of the patients required platelet transfusion. None of the patients developed clinical symptoms such as thrombosis, fever, rash, portal vein thrombosis, bleeding, or any other serious adverse events. CONCLUSIONS: Repeated use of lusutrombopag increased the platelet count. It did not cause any serious adverse events and led to avoidance of platelet transfusion. Radiofrequency ablation was carried out safely in all patients. Future studies with more cases of repeated use are needed to examine the long-term efficacy and safety of lusutrombopag.