Retro-pancreatic pull-through reconstruction of the hypoplastic portal vein using the autologous mesosystemic shunt vessel in adult living donor liver transplantation: a case report.

BACKGROUND: In liver transplant patients with hypoplastic portal vein (PV), when the narrowed segment is extended too deep into the dorsal side of the pancreas, it is difficult and dangerous to reconstruct the interposition graft from the upper part of the pancreas. Herein, we present a case of PV reconstruction with the autologous mesosystemic shunt vessel from the caudal side of the pancreas in a situation where the narrowed PV was deep, and we discuss the technical details. CASE PRESENTATION: A 25-year-old woman presented with cholestatic liver cirrhosis due to biliary atresia after Kasai procedure. Since her jaundice progressed, she was referred to our hospital for liver transplantation. Laboratory tests showed that her total bilirubin was elevated to 7.6 mg/dL. The Model for End-Stage Liver Disease score was 18, and the Child-Pugh score was 9 (Grade B). She underwent living donor liver transplantation (LDLT) using a right hemi-liver graft procured from her 54-year-old mother. The conventional approach from the cephalad side to the superior mesenteric vein (SMV) and splenic vein (SpV) confluence behind the pancreas was extremely difficult in this case because the confluence of SMV and SpV was close to the lower edge of the pancreas. Therefore, we decided to perform PV reconstruction from the caudal side. The main trunk of PV was documented as narrow (5 mm in diameter), for which retro-pancreatic pull-through PV reconstruction was successfully performed using her own mesosystemic shunt vessel. A contrast computed tomography (CT) scan was performed on postoperative day 5 because of an elevation of D-dimer and found a partial thrombus in the left pulmonary artery, as well as in the PV and left renal vein. Thereafter, thrombolytic therapy with low-molecular-weight heparin was started immediately and switched to a direct oral anticoagulant. The follow-up CT taken 3 months after liver transplantation revealed a patent PV without thrombus; therefore, anticoagulant therapy was discontinued. Currently, the patient has been well and active with a patent PV without anticoagulant therapy for 3 years after LDLT. CONCLUSIONS: Retro-pancreatic pull-through reconstruction of the hypoplastic PV is a feasible and effective method when conventional reconstruction is not indicated.

Outcomes of critically ill patients with liver failure who require mechanical ventilation: A retrospective, single-center study.

Background and Aims: Critically ill patients with liver failure have high mortality. Besides the management of organ-specific complications, liver transplantation constitutes a definitive treatment. However, clinicians may hesitate to introduce mechanical ventilation for patients on liver transplantation waitlists because of poor prognosis. This study investigated the outcomes of intensive care and ventilation support therapy effects in patients with liver failure. Methods: This single-center study retrospectively enrolled 32 consecutive patients with liver failure who were admitted to the intensive care unit from January 2014 to December 2020. The medical records were reviewed and analyzed retrospectively for Acute Physiologic and Chronic Health Evaluation (APACHE)-II. The model for end-stage liver disease scores, 90-day mortality, and survival was assessed using the Kaplan-Meier method. Results: The average patient age was 45.5 ± 20.1 years, and 53% of patients were women. On intensive care unit admission, APACHE-II and model for end-stage liver disease scores were 20 and 28, respectively. Among 13 patients considered for liver transplantation, 4 received transplants. Thirteen patients (40.6%) were intubated and mechanically ventilated in the intensive care unit. The 90-day mortality rate of patients with and without mechanical ventilation in the intensive care unit (13, 61.5% vs. 19, 47.4%, p = 0.4905) was similar. APACHE-II score >21 was an independent predictor of mechanical ventilation requirement in patients with liver failure during intensive care unit stay. Conclusion: Although critically ill patients with liver failure are at risk of multiorgan failure with poor outcomes, mechanical ventilation did not negatively affect the 90-day mortality or performance rates of liver transplantation. Clinicians should consider mechanical ventilation-based life support in critically ill patients with liver failure who are awaiting liver transplantation.

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.

Long-term Catch-up Growth and Risk Factors for Short Adult Height After Pediatric Liver Transplantation: A Retrospective Study.

BACKGROUND: Children requiring liver transplantation generally have severe growth retardation. Recipients experience posttransplantation catch-up growth, although some show short adult heights. We aimed to determine decades-long catch-up growth trends and risk factors for short adult height following liver transplantation. METHODS: We analyzed long-term height Z scores and risk factors for short adult height in a single-center retrospective cohort of 117 pediatric liver transplantation recipients who survived >5 y, with 75 of them reaching adult height. RESULTS: Median age at transplantation was 1.3 y, and the most common primary diagnosis was biliary atresia (76.9%). Mean height Z scores pretransplantation and 1, 3, and 8 y after transplantation were -2.26, -1.59, -0.91, and -0.59, respectively. The data then plateaued until 20 y posttransplantation when mean adult height Z score became -0.88, with a median follow-up of 18.6 y. Nineteen recipients did not show any catch-up growth, and one quarter of recipients had short adult height (<5th percentile of the healthy population). Multivariate analysis identified old age (odds ratio, 1.22 by 1 y; P = 0.002), low height Z scores at transplantation (odds ratio, 0.46 by 1 point; P < 0.001), and posttransplantation hospital stay ≥60 d (odds ratio, 4.95; P = 0.015) as risk factors for short adult height. In contrast, prolonged steroid use after transplantation was not considered a significant risk factor. CONCLUSIONS: Although tremendous posttransplantation catch-up growth was observed, final adult height remained inadequate. For healthy physical growth, liver transplantation should be performed as early as possible, before growth retardation becomes severe.

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.

Innovative formulae for the estimation of standard liver volume in the era of widespread imaging analysis software.

BACKGROUND: Various formulae have been used for the estimation of standard liver volume (SLV) in preparation for living donor liver transplantation (LDLT). However, these formulae have the disadvantage of being constructed using parameters that are substantially affected by the patient's condition. Here, we aimed to establish more precise formulae that are less affected by the general condition of the patient. METHODS: We analyzed the liver volumes of LDLT donors and patients with normal livers (total: n = 213) using the SYNAPSE VINCENT imaging analysis system, to develop new formulae. The accuracy of the new formulae were compared with those of existing formulae in a separate validation group of healthy patients (n = 200). The new formulae were also validated using 81 LDLT recipients to assess their utility for graft selection in LDLT. RESULTS: Body surface area (BSA) and skeletal muscle mass index (SMI) independently affected total liver volume (TLV). We produced new formulae for SLV incorporating SMI: SLV = 32.2 × L3-SMI-466.9 for men, with R2 .92, and 25.7 × L3-SMI-55.97 for women, with R2 .79 (alongside a BSA formula with R2 .57), which provided the most accurate predictions of TLV in the validation group. A graft volume (GV)/SLV <.35, calculated using the new formulae, predicted the postoperative prognosis, including the development of small-for-size syndrome, sepsis, or acute rejection, significantly more effectively than GV/SLV using the previous formulae. CONCLUSIONS: The newly developed L3-SMI-based formula is more accurate for the estimation of SLV than previously reported formulae, and may thus help to improve the safety of LDLT.

Late-onset Chronic Kidney Disease Over 2 Decades After Pediatric Liver Transplantation: A Single-center, Retrospective Study.

BACKGROUND: Although chronic kidney disease (CKD) after liver transplantation (LTx) is a common complication in adults, its long-term significance after pediatric LTx remains unclear. We examined the decades-long transition of renal function and revealed the risk factors for late-onset CKD after pediatric LTx in a single-center retrospective cohort of 117 pediatric LTx recipients who survived >5 y. METHODS: The estimated glomerular filtration rate (eGFR) and CKD stages were calculated using serum creatinine. Risk factor analysis for late-onset CKD was performed in 41 patients whose eGFR could be evaluated at >20 y after LTx. RESULTS: The median age at LTx was 1.3 y, and most primary diagnoses were biliary atresia (77%). The mean pre-LTx and 1, 5, 10, 20, and >20 y post-LTx eGFRs were 180, 135, 131, 121, 106, and 95 mL/min/1.73 m 2 , respectively, with a median renal follow-up period of 15 y. The eGFR declined by 47% at >20 y after LTx ( P < 0.001). CKD was observed in 8%, 19%, and 39% of cases at 10, 20, and >20 y after LTx, respectively. In patients receiving cyclosporine, trough levels were 1.5 times higher in those with CKD up to 10 y after LTx. The multivariate analysis showed that older age at LTx (odds ratio, 1.3 by 1 y; P = 0.008) and episodes of repeated/refractory rejection (odds ratio, 16.2; P = 0.002) were independent risk factors of CKD >20 y after LTx. CONCLUSIONS: In conclusion, renal function deteriorates slowly yet steadily after pediatric LTx. Long-term careful surveillance is essential after pediatric LTx, especially in repeated/refractory rejection or long-term high trough-level use of cyclosporine cases.

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.

Small-for-size syndrome in liver transplantation: Definition, pathophysiology and management.

BACKGROUND: Since the first success in an adult patient, living donor liver transplantation (LDLT) has become an universally used procedure. Small-for-size syndrome (SFSS) is a well-known complication after partial LT, especially in cases of adult-to-adult LDLT. The definition of SFSS slightly varies among transplant physicians. The use of a partial liver graft has risks of SFSS development. Persistent portal vein (PV) hypertension and PV hyper-perfusion after LT were identified as the main factors. Hence, various approaches were explored to modulate PV flow and decrease PV pressure in order to alleviate this syndrome. Herein, the definition, clinical symptoms, pathophysiology, basic research, as well as preventive and treatment strategies for SFSS are reviewed based on an extensive review of the literature and on our own experiences. DATA SOURCES: The articles were collected through PubMed using search terms "liver transplantation", "living donor liver transplantation", "living liver donation", "partial graft", "small-for-size graft", "small-for-size syndrome", "graft volume", "remnant liver", "standard liver volume", "graft to recipient body weight ratio", "sarcopenia", "porcine", "swine", and "rat". English publications published before March 31, 2020 were included in this review. RESULTS: Many transplant surgeons performed PV flow modulation, including portocaval shunt, splenic artery ligation and splenectomy. With these techniques, patient outcome has been improved even when using a "small" graft. Other factors, such as preoperative recipients' nutritional and skeletal muscle status, graft congestion, and donor factors, were also identified as risk factors which all have been addressed using various strategies. CONCLUSIONS: The surgical approach controlling PV flow and pressure could help to prevent SFSS especially in severely ill recipients. In the absence of efficacious medications to resolve SFSS, conservative treatments, including aggressive fluid balance correction for massive ascites, anti-microbiological therapy to prevent or control sepsis and intensive nutritional therapy, are all required if SFSS could not be prevented.

Successful recovery from critical COVID-19 pneumonia with extracorporeal membrane oxygenation: A case report.

A public health emergency of current international concern is the outbreak of a severe respiratory illness, that is, coronavirus disease (COVID-19). The disease initially started in Wuhan, China, and it rapidly spread to most regions of the world. Herein, we report a case of critical COVID-19 pneumonia treated with extracorporeal membrane oxygenation from symptom onset day 19 (SOD#19) to SOD#30. We describe the patient's clinical course, from mild symptoms at the time of illness onset to symptoms of severe pneumonia as the illness progressed. We provide important information regarding our clinical experience for further understanding of management discrepancies, as treatment with extracorporeal membrane oxygenation or pharmacotherapy (e.g., antivirals, immunomodulators, and glucocorticoids) is often dependent on the severity of symptoms.

Divergent antioxidant capacity of human islet cell subsets: A potential cause of beta-cell vulnerability in diabetes and islet transplantation.

BACKGROUND: Type 1 and Type 2 diabetes mellitus (T1DM and T2DM) are caused by beta(ß)-cell loss and functional impairment. Identification of mechanisms of ß-cell death and therapeutic interventions to enhance ß-cell survival are essential for prevention and treatment of diabetes. Oxidative stress is a common feature of both T1DM and T2DM; elevated biomarkers of oxidative stress are detected in blood, urine and tissues including pancreas of patients with DM. Islet transplantation is a promising treatment for diabetes. However, exposure to stress (chemical and mechanical) and ischemia-reperfusion during isolation and transplantation causes islet loss by generation of reactive oxygen species (ROS). Human intracellular antioxidant enzymes and related molecules are essential defenses against ROS. Antioxidant enzyme levels including superoxide dismutase (SOD), catalase, and glutathione peroxidase (GPX) have been shown to be low in islet cells. However, little is known about the expression and function of antioxidant enzymes within islet cell subsets. We evaluated the expression of the key antioxidant enzymes in ß- and alpha(α)-cell and accessed effects of oxidative stress, islet isolation and transplantation on ß/α-cell ratio and viability in human islets. METHODS: Human pancreata from T1DM, T2DM and non-diabetic deceased donors were obtained and analyzed by confocal microscopy. Isolated islets were (I) transplanted in the renal sub-capsular space of streptozotocin-induced diabetic nude mice (in vivo bioassay), or (II) exposed to oxidative (H2O2) and nitrosative (NO donor) stress for 24 hrs in vitro. The ratio, % viability and death of ß- and α-cells, and DNA damage (8OHdG) were measured. RESULTS AND CONCLUSIONS: Catalase and GPX expression was much lower in ß- than α-cells. The ß/α-cell ratio fells significantly following islet isolation and transplantation. Exposure to oxidative stress caused a significantly lower survival and viability, with higher DNA damage in ß- than α-cells. These findings identified the weakness of ß-cell antioxidant capacity as a main cause of vulnerability to oxidative stress. Potential strategies to enhance ß-cell antioxidant capacity might be effective in prevention/treatment of diabetes.

Successful living donor liver transplantation for acute liver failure after acetylsalicylic acid overdose.

A 20-year-old woman was admitted to an emergency hospital after ingesting 66 g of acetylsalicylic acid in a suicide attempt. Although she was treated with gastric lavage, oral activated charcoal, and intravenous hydration with sodium bicarbonate, her hepatic and renal function gradually deteriorated and serum amylase levels increased. Steroid pulse therapy, plasma exchange, and continuous hemodiafiltration did not yield any improvement in her hepatic or renal function, and she was transferred to our hospital for living donor liver transplantation. Nine days after drug ingestion, she developed hepatic encephalopathy: thus, we diagnosed the patient with acute liver failure with hepatic coma accompanied by acute pancreatitis due to the overdose of acetylsalicylic acid. Living donor liver transplantation was immediately performed using a left lobe graft from the patient's mother. Following transplantation, the patient's renal and hepatic function and consciousness improved, and she was discharged. In this report, we describe a rare case of acetylsalicylic acid-induced acute liver failure with acute hepatic coma and concomitant acute pancreatitis and acute renal failure, which were treated successfully with emergency living donor liver transplantation.

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.