Clinical significance of mechanistic target of rapamycin expression in vessels that encapsulate tumor cluster-positive hepatocellular carcinoma patients who have undergone living donor liver transplantation.

Background: There is limited published information regarding the expression of mechanistic target of rapamycin (mTOR) in vessels that encapsulate tumor cluster (VETC)-positive hepatocellular carcinoma (HCC). The mTOR inhibitor, everolimus, has been approved as an immunosuppressant for use in HCC patients after living donor liver transplantation (LDLT). Methods: Using a database of 214 patients who underwent LDLT for HCC, we examined the mTOR protein and angiopoietin-2 (Ang-2) in VETC-positive HCC by immunohistochemical staining. The presence of VETC and mTOR expression were evaluated in both primary and recurrent HCC lesions. Results: Forty-three of the 214 patients (20.1%) were VETC-positive, and 29 of these 43 patients (67.4%) expressed mTOR. Relative Ang-2 expression was significantly higher in the mTOR-positive than in the mTOR-negative group (p = 0.037). Thirty-four of the 214 patients experienced HCC recurrence after LDLT; 20 of these were operable. The primary lesions of six of these 20 patients were VETC-positive; five of these six patients also had VETC-positive recurrent lesions (p < 0.001). The expression of mTOR was significantly higher in the VETC-positive lesions (p = 0.0018). Conclusions: We showed that mTOR expression was higher in the VETC-positive primary and recurrent lesions than in the VETC-negative ones.

Antibody titer after administration of mRNA-based vaccine against severe acute respiratory syndrome coronavirus 2 in liver transplant recipients.

Introduction: The mRNA-based vaccine was released as a COVID-19 prophylactic; however, its efficacy in organ transplant recipients is unknown. This study aimed to clarify this in liver transplant recipients. Methods: Herein, liver transplant recipients from two hospitals who received vaccines were included. Immunoglobulin-G antibodies against the spike and nucleocapsid proteins were measured chronologically after the second, third, and fourth vaccine doses. Results: Antibody levels in 125 liver transplant recipients and 20 healthy volunteers were analyzed. The median age at transplant was 35 (interquartile range 1, 53) years, and the period between transplant and the first dose was 15.2 ± 7.7 years. After the second and third doses, 89.1% and 100% of recipients displayed a positive humoral response, respectively. Anti-spike antibodies after the second dose were significantly reduced at 3 and 6 months, compared to that at 1 month (26.0 [5.4, 59.5], 14.7 [6.5, 31.4] vs. 59.7 [18.3, 164.0] AU/mL, respectively, p < 0.0001). However, a booster vaccine significantly elevated anti-spike antibodies in LT recipients (p < 0.0001) as well as in healthy controls (p < 0.0001). Additionally, the decay rate was comparable between the transplant recipients and controls (2.1 [0.8, 4.5] vs. 2.7 [1.1, 4.1] AU/mL/day, p = 0.9359). Only 4.0% of vaccinated transplant recipients were positive for anti-nucleocapsid antibodies. Conclusion: Liver transplant recipients can acquire immunity similar to that of healthy people through vaccination against SARS-CoV-2. The antibody decay rate is the same, and booster vaccinations should be administered similarly to that in healthy individuals.

Fatal disseminated mucormycosis due to Cunninghamella bertholletiae infection after ABO-incompatible living donor liver transplantation: a case report.

BACKGROUND: Fungal infection may develop because of immunosuppression after organ transplantation, in which invasive types, such as Aspergillus and Mucorales, fungi cause morbidity. We present a case of disseminated mucormycosis due to Cunninghamella bertholletiae after ABO-incompatible living donor liver transplantation (LDLT). CASE PRESENTATION: A 47-year-old man with decompensated liver cirrhosis and hepatocellular carcinoma underwent an ABO-incompatible LDLT using a graft procured from his son, who had a different blood type. Rituximab and mycophenolate mofetil were administered 3 weeks before LDLT as immunosuppressive therapy. Although liver graft function improved, mass-like infiltrates appeared in the lungs following intubation for > 1 week due to impaired consciousness. The brain magnetic resonance imaging findings were normal. Decreased ejection fraction and ST elevation were detected on echocardiography and electrocardiography, respectively. There was no dominant stenosis on coronary arteriography. The recipient underwent segmentectomy of the right lung 20 days after LDLT. C. bertholletiae was identified from a specimen using polymerase chain reaction, thus establishing a diagnosis of mucormycosis. Multiple infarctions in the brain, heart, and kidney developed within 2 weeks. Treatment with amphotericin B was ineffective. The patient developed circulatory collapse, and a temporary pacemaker and percutaneous coronary intervention were required for cardiac infarction. The recipient died of cardiac failure 27 days after the LDLT. Autopsy revealed disseminated mucormycosis involving the brain, thyroid, heart, lung, liver, gastrointestinal tract, and both kidneys. In addition, fungal endocarditis may have been responsible for septic emboli in multiple organs, resulting in multiple organ invasion. Hypothrombocytopenia was present since the pre-transplant period, and the recipient was diagnosed posthumously with myelodysplastic syndrome due to hereditary abnormalities. Multiple factors such as organ transplantation, bone marrow dysfunction, immunosuppression, and inadequate administration of antifungal reagents might have promoted mucormycosis development in our patient. CONCLUSIONS: Mucormycosis by C. bertholletiae is a fatal complication; thus, early diagnosis and treatment are warranted before multiple organ invasion.

Improved human islet preparations using glucocorticoid and exendin-4.

OBJECTIVES: The effects of glucocorticoid during culture on human islet cells have been controversial. Exendin-4 (EX) enhances the insulin secretion and significantly improves clinical outcomes in islet cell transplantation. In this study, we examined the effects of glucocorticoids and EX on human islet cells during pretransplant culture. METHODS: Methylprednisolone (MP) and/or EX were added to the standard culture medium for clinical islet cell transplantation. Islets were cultured for 24 hours with 3 different conditions (control, no additives; MP alone; and MP + EX). ß-Cell fractional viability, cellular composition, multiple cytokine/chemokine production, multiple phosphorylation proteins, and glucose-induced insulin secretion were evaluated. RESULTS: Viable ß-cell survival in MP and MP + EX group was significantly higher than in the control group. Exendin-4 prevented MP-induced reduction of insulin secretion. Methylprednisolone supplementation to the culture medium decreased cytokine and chemokine production. Moreover, extracellular signal-regulated kinase 1/2 phosphorylation was significantly increased by MP and MP + EX. CONCLUSIONS: Glucocorticoid supplementation into culture media significantly decreased the cytokine/chemokine production and increased the extracellular signal-regulated kinase 1/2 phosphorylation, resulting in the improvement of human ß-cell survival. In addition, EX maintained the insulin secretion suppressed by MP. The supplementation of MP and EX together could be a useful strategy to create suitable human islets for transplantation.

A new procedure for temporary auxiliary partial liver transplantation using living donor graft for patients with familial amyloid polyneuropathy.

To introduce duct-to-duct biliary anastomosis to conventional temporary auxiliary partial orthotopic liver transplantation (APOLT) using living donor graft for patients with familial amyloid polyneuropathy, we modified the conventional APOLT procedure in a manner characterized by the use of the recipient's common hepatic duct for biliary reconstruction and the preservation of the right posterior section alone for the certain placement of a tube into the corresponding biliary tree for external biliary drainage (modified APOLT). This procedure was performed in 3 patients without biliary complications. No complications associated with the external drainage tube occurred. Here we report the techniques and results for this new procedure.

Quantitative in situ analysis of FoxP3+ T regulatory cells on transplant tissue using laser scanning cytometry.

There is abundant evidence that immune cells infiltrating into a transplanted organ play a critical role for destructive inflammatory or regulatory immune reactions. Quantitative in situ analysis (i.e., in tissue sections) of immune cells remains challenging due to a lack of objective methodology. Laser scanning cytometry (LSC) is an imaging-based methodology that performs quantitative measurements on fluorescently and/ or chromatically stained tissue or cellular specimens at a single-cell level. In this study, we have developed a novel objective method for analysis of immune cells, including Foxp3(+) T regulatory cells (Tregs), on formalin-fixed /paraffin-embedded (FFPE) transplant biopsy sections using iCys® Research Imaging Cytometer. The development of multiple immunofluorescent staining was established using FFPE human tonsil sample. The CD4/CD8 ratio and the population of Tregs among CD4(+) cells were analyzed using iCys and compared with the results from conventional flow cytometry analysis (FCM). Our multiple immunofluorescent staining techniques allow obtaining clear staining on FFPE sections. The CD4/CD8 ratio analyzed by iCys was concordant with those obtained by FCM. This method was also applicable for liver, small intestine, kidney, pancreas, and heart transplant biopsy sections and provide an objective quantification of Tregs within the grafts.

Clinicopathological features of hepatitis C virus disease after living donor liver transplantation: relationship with in situ hybridisation data.

AIMS: Recurrent hepatitis is a significant complication after liver transplantation for hepatitis C virus (HCV) disease. To evaluate responsiveness to treatment of HCV disease after liver transplantation, in situ hybridisation (ISH) was employed. METHODS: Sense and anti-sense probes for HCV were synthesised, and ISH studies were performed on 19 liver biopsy specimens from 19 recipients who had undergone living donor liver transplantation for HCV disease. ISH positive cells and total hepatocytes were counted, and the percentage of positive cells was calculated. Other clinical findings were compared retrospectively with the ISH results. RESULTS: The subjects were divided into three groups: recurrent HCV hepatitis (RHC, n = 11), acute cellular rejection (ACR, n = 5), and recurrent HCV hepatitis with ACR (MIX, n = 3). The percentage of ISH positive cells was almost the same degree (10-20%) in the three groups. The RHC group was subdivided into two sets of patients in whom serum HCV titres decreased (group D, n = 7) or did not decrease (group ND, n = 3) after 1 month of IFN therapy. The percentage of ISH positive cells in group D was significantly lower than that in group ND (p < 0.05) CONCLUSIONS: ISH for the recipients with HCV may be useful for predicting the response to interferon therapy.

Prolactin supplementation to culture medium improves beta-cell survival.

OBJECTIVES: Recent studies demonstrated that prolactin (PRL) has beneficial effects on beta cells for islet transplantation. We examined the effect of human recombinant PRL (rhPRL) supplementation to the culture media to determine its potential use in the context of clinical islet transplantation. MATERIALS AND METHODS: Each human islet isolated from 14 deceased multiorgan donors was cultured in Miami modified media-1 supplemented with or without rhPRL (500 microg/L) for 48 hr. beta-Cell survival and proliferation (BrdU and Ki-67) were determined by laser scanning cytometry. The cytoprotective effects of rhPRL against noxious stimuli were assessed by flow cytometry (tetramethylrhodamine ethyl ester). Cytokine/chemokine and tissue factor productions were measured in vitro, and islet potency was assessed in vivo in diabetic immunodeficient mice. RESULTS: beta-Cell survival during culture was 37% higher in the rhPRL group than in control (P=0.029). rhPRL protected beta cells in vitro from cytokines, Nitric oxide donor, and H2O2. The exposure to rhPRL did not affect human beta-cell proliferation with our protocol. rhPRL treatment did not alter cytokine/chemokine and tissue factor production in vitro or affected human islet functionality in vivo: recipient mice achieved normoglycemia with a comparable tempo, whereas loss of graft function was observed in two of the seven mice in the control group and in none of the rhPRL group (p=n.s.). CONCLUSION: rhPRL supplementation to islet culture media improved human beta-cell-specific survival without altering islet quality. Addition of rhPRL to cultured islets may grant a more viable beta-cell mass in culture. The development of beta-cell cytoprotective strategies will be of assistance in improving islet transplantation outcomes.

Potential feasibility of early bone marrow cell injection into the spleen for creating functional hepatocytes.

BACKGROUND: Bone marrow cells (BMCs) are believed to have the ability to generate functional hepatocytes and have some merits as a therapeutic modality for metabolic liver diseases. However, the appearance of BMC-derived hepatocytes (BMDHs) is low. We hypothesized that early BMC injection would be feasible for creating BMDHs for two main reasons: (1) the liver is a hematopoietic organ in neonatal rats and (2) it may allow sufficient time to generate more BMDHs before severe liver injury occurs. METHODS: We used Long Evans Cinnamon (LEC) rats as recipients, a model of (1) Wilson disease and (2) liver carcinogenesis. Green fluorescent protein-expressing BMCs were injected into newborn LEC rats through the spleen. The oxidative activity of ceruloplasmin, which is low in LEC rats, was measured to evaluate the treatment. In addition, we performed fluorescence in situ hybridization to clarify the origin of the BMDHs and immunohistochemical analysis to confirm whether these BMDHs had malignant potential. RESULTS: At 18 months after injection, we found some green fluorescent protein-expressing areas macroscopically in the liver of treated LEC rats. The oxidative activity of ceruloplasmin increased in treated LEC rats (n=7) and were much higher than that in untreated LEC rats (P=0.015). Moreover, we confirmed that the BMDHs were generated by cell fusion and was not detected in any of the neoplastic lesions or cholngiofibrotic regions. CONCLUSION: Our results suggest that this novel strategy for creating BMDHs is effective and safe.

Characterization of pancreatic ductal cells in human islet preparations.

Substantial amounts of nonendocrine cells are implanted as part of human islet grafts, and a possible influence of nonendocrine cells on clinical islet transplantation outcome has been postulated. There are currently no product release criteria specific for nonendocrine cells due to lack of available methods. The aims of this study were to develop a method for the evaluation of pancreatic ductal cells (PDCs) for clinical islet transplantation and to characterize them regarding phenotype, viability, and function. We assessed 161 human islet preparations using laser scanning cytometry (LSC/iCys) for phenotypic analysis of nonendocrine cells and flow cytometry (FACS) for PDC viability. PDC and beta-cells obtained from different density fractions during the islet cell purification were compared in terms of viability. Furthermore, we examined PDC ability to produce proinflammatory cytokines/chemokines, vascular endothelial growth factor (VEGF) and tissue factor (TF) relevant to islet graft outcome. Phenotypic analysis by LSC/iCys indicated that single staining for CK19 or CA19-9 was not enough for identifying PDCs, and that double staining for amylase and CK19 or CA19-9 allowed for quantitative evaluation of acinar cells and PDC content in human islet preparation. PDC showed a significantly higher viability than beta-cells (PDC vs beta-cell: 75.5+/-13.9 and 62.7+/-18.7%; P<0.0001). Although beta-cell viability was independent of its density, that of PDCs was higher as the density from which they were recovered increased. There was no correlation between PDCs and beta-cell viability (R(2)=0.0078). PDCs sorted from high-density fractions produced significantly higher amounts of proinflammatory mediators and VEGF, but not TF. We conclude that PDCs isolated from different fractions had different viability and functions. The precise characterization and assessment of these cells in addition to beta-cells in human islet cell products may be of assistance in understanding their contribution to islet engraftment and in developing strategies to enhance islet graft function.

Anti-proinflammatory effects of sirolimus on human islet preparations.

BACKGROUND: Sirolimus plays a critical role in facilitating steroid-free immunosuppression, in conjunction with low dose tacrolimus, in current islet transplantation. Although several studies have investigated the effects of sirolimus on islet cells, conflicting results have been reported. In this study, we assessed the effects of sirolimus supplementation in culture media on human islet preparations, focusing on the anti-proinflammatory aspects. METHODS: Human islet preparations were divided into four groups: pure (purity >90%) sirolimus (30 ng/mL); pure control (0 ng/mL); impure (purity 40%-60%) sirolimus; and impure control. All groups were cultured for 3 days and assessed regarding glucose stimulated insulin release, fractional beta-cell viability, beta-cell, and macrophage content. Cytokine and chemokine production from islet preparations and sorted pancreatic ductal cells were also examined. RESULTS: Stimulated insulin release in the impure sirolimus group was significantly increased (P=0.024), as previously reported. Although fractional beta-cell viability showed no significant differences, beta-cell survival during culture significantly increased in impure sirolimus group when compared with the impure control group (P=0.015). Tumor necrosis factor-alpha, interleukin-1beta, monocyte chemotactic protein-1, and macrophage inflammatory protein-1beta production from the impure sirolimus group significantly decreased (P<0.05). Furthermore, tumor necrosis factor-alpha and macrophage inflammatory protein-1beta production from sorted ductal cells significantly decreased in the sirolimus group (P<0.05). The number of macrophages contained in islet preparations significantly decreased in the impure sirolimus group when compared with the impure control group (P<0.05). CONCLUSIONS: Sirolimus improved not only stimulated insulin release, but also beta-cell survival during culture. The antiinflammatory effects of sirolimus also appear beneficial to islet cells in culture and may be a useful strategy in improving islet transplantation outcomes.

Risk factors contributing to hepatic artery thrombosis following living-donor liver transplantation.

BACKGROUND/PURPOSE: This study was carried out to investigate the risk factors contributing to hepatic artery thrombosis in living-donor liver transplantation. METHODS: Two hundred and twenty-two recipients (113 adults and 109 children) of living-donor liver transplantation were the subjects of this study. The diagnosis of hepatic artery thrombosis was made by color-Doppler ultrasonography and/or hepatic angiography. Parameters for this study were: (1) donor sex, age, and body weight; (2) recipient sex, age, body weight, liver disease, preoperative prothrombin time, and type of arterial reconstruction; and (3) previous liver transplantation. RESULTS: Hepatic artery thrombosis occurred in 12 patients (5.4%) at 3 to 15 days posttransplant. Recipient female sex and metabolic disorder as the original disease were found to be significantly associated with hepatic artery thrombosis. The 5-year patient survival rate in recipients with hepatic artery thrombosis (58.3%) was significantly lower than that in recipients without this complication (84.4%). CONCLUSIONS: Female sex and metabolic disease may be factors contributing to hepatic artery thrombosis after living-donor liver transplantation. More intensive anticoagulation therapy for this patient population might decrease the incidence of hepatic artery thrombosis and, thus, posttransplant recipient mortality.

Expression of Fas ligand by hepatic macrophages in patients with fulminant hepatic failure.

OBJECTIVES: The mechanisms of Fas-Fas ligand (Fas-FasL)-mediated apoptosis in the pathogenesis of fulminant hepatic failure (FHF) have not been well defined. This clinical study was carried out to assess which cells expressed Fas-FasL and to determine their involvement. METHODS: The subjects were 24 patients with FHF who underwent liver transplantation at our institution. For comparison, nine chronic hepatitis (CH) patients and six living liver donors (LD) were also enrolled. Liver tissues were obtained for histological (hematoxylin-eosin, terminal deoxynucleotidyl transferase [TdT]-mediated dUTP-biotin nick-end labeling [TUNEL], immunohistochemistry, and double immunofluorescence staining) and reverse transcription PCR (RT-PCR; cytokines and chemokines) analysis. RESULTS: The numbers of TUNEL-, FasL-, and CD68-positive cells in the livers of patients with FHF were significantly larger than in those with CH or with normal livers. Double immunofluorescence staining showed that FasL was expressed predominantly on liver macrophages and rarely on CD8-positive lymphocytes. RT-PCR study showed increased expression of FasL; interferon-gamma; interleukin-18; macrophage inhibitory protein-1beta; and regulated upon activation, normal T cell expressed and secreted in the livers of patients with FHF compared with those of LD. CONCLUSIONS: Macrophages and their expression of FasL may play roles in the pathogenesis of FHF.

Life-threatening veno-occlusive disease after living-related liver transplantation.

Veno-occlusive disease (VOD) can develop in association with the administration of cytotoxic chemotherapeutic agents and irradiation. In solid-organ transplant settings, azathioprine has been implicated as a predisposing factor. VOD with fatal outcome occurred in a post liver-transplant recipient who had never been exposed to any agents that have the potential to induce VOD. At onset, the disease manifested clinically as gross ascites and progressive jaundice and was observed after clinically diagnosed acute graft rejection. The disease was confirmed by histologic examinations. Histologic studies of biopsy samples from this patient revealed that most small hepatic veins less than 300 microm in diameter were affected, exhibiting concentric intimal thickening with sparse inflammatory cells. A few of the hepatic veins exhibited active endotheliitis with occasional extension of inflammation to neighboring centrilobular areas. Despite intensified immunosuppression, the observed fibrous obliterative changes were irreversible. Although the cause of VOD in this patient is tentative, the damage to the endothelium, associated with acute rejection, is likely to be attributable. VOD deserves recognition as one of the causes for liver dysfunction and persistent ascites after liver transplantation.