Effects of Short-Term Normothermic and Subnormothermic Perfusion After Cold Preservation on Liver Transplantation From Donors After Cardiac Death.

BACKGROUND: Liver transplantation from donors after cardiac death (DCD) could increase the pool of organs. We previously reported that oxygenated subnormothermic (20°C-25°C) ex vivo liver perfusion (SELP) improved the graft viability in rats. This study aimed to compare the effectiveness of SELP and normothermic (37°C) ex vivo liver perfusion (NELP) after cold storage (CS) in DCD liver grafts. METHODS: Male Wistar rats were used, and grafts were retrieved 30 minutes after cardiac arrest. We performed oxygenated NELP and SELP with a Krebs-Henseleit buffer for different time points and durations: Group 0, donation performed from heart-beating donors (control); Group 1 (DCD group), donation performed from DCD donors with no treatments; Group 2, NELP performed before CS (30 minutes); Group 3, NELP performed after CS (30 minutes); Group 4, SELP performed after CS (30 minutes); Group 5, SELP performed after CS (60 minutes); and Group 6, SELP performed after CS (90 minutes). After 15 minutes of incubation at room temperature, the grafts were reperfused under normothermic conditions for 60 minutes as a model of liver transplantation. RESULTS: No significant differences in body and liver weight were observed between all groups. In the SELP after CS groups, even 30 minutes of perfusion improved bile production, tumor necrosis factor-α, and interleukin-1ß significantly compared with the DCD group (P < .05), comparable with NELP groups. CONCLUSION: SELP rescued DCD livers from ischemia-reperfusion injury the same as the normothermic perfusion before or after CS groups. SELP after CS is more convenient than normothermic perfusion; hence, this technique may increase the organ pool.

Exploring Risk Factors of Patient Falls: A Retrospective Hospital Record Study in Japan.

Patient falls are common adverse medical events in hospitals. The objectives of this study were to clarify the factors of patient falls at hospitalization or transfer to another ward, which could be assumed that patients experience new environment. Patients who were hospitalized or transferred to another ward at a hospital in Japan, between January 14 and February 14, 2014 were included. We used a risk assessment sheet and applied stepwise regression analysis to identify factors of patient falls. We also investigated changes in patient conditions on the risk assessment sheet by the chi-square test. A total of 1,362 patients (53.2% female; mean age, 57.1 ± 18.0 years) were eligible for analysis, and 38 (2.8%) fell during the study period. The fallers were significantly older than the non-fallers (63.8 ± 18.0 vs. 56.9 ± 18.7 years, P = 0.03), but no significant difference was seen in sex (55.3% vs. 53.1% female). "History of falls", "Tubes inserted", "Need assistance/supervision for toileting" and "Excretion more than two times per night" were significantly related to patient falls (adjusted odds ratios [95% confidence interval]: 2.41 [1.05-5.53], 3.64 [1.57-8.43], 4.52 [2.00-10.23] and 3.92 [1.38-11.09]). Among 30 fallers, "Overestimation or non-understanding of own physical abilities" was significantly more frequent after falls (30.0%) than before falls (6.7%, P = 0.02). The factors found in this study might be useful for identifying patients at higher risk of falls.

Histopathological analysis of anaplastic thyroid carcinoma cases with long-term survival: A report from the Anaplastic Thyroid Carcinoma Research Consortium of Japan.

The aim of this study was to clarify the histopathological features of anaplastic thyroid carcinoma in patients who achieved long-term survival. We reviewed 88 anaplastic thyroid carcinoma cases in which the patient survived less than 3 months (short-term survival), and 68 anaplastic thyroid carcinoma cases in which the patient survived more than one year (long-term survival) from the database of the Anaplastic Thyroid Carcinoma Research Consortium of Japan. We examined these cases both histologically and immunohistochemically. Six (6.8%) short-term survival cases and 27 (39.7%) long-term survival cases were considered not to be anaplastic thyroid carcinoma after central review. Of these, 12 were revised to papillary carcinoma with squamous cell carcinoma. In cases without chemotherapy, long-term survival was significantly more common if there was a pre-existing tumor, epithelial growth, or lymphocytic infiltration, and short-term survival was more common if neutrophilic infiltration was present. In cases with chemotherapy, long-term survival was significantly more common if epithelial growth or a squamous cell carcinoma component was present, whereas short-term survival was more common in cases with rhabdoid cells. Immunohistochemical results were not related to survival. Some long-term survival cases showed histological findings other than those typically associated with anaplastic thyroid carcinoma. The presence of a pre-existing tumor, epithelial growth, a squamous cell carcinoma component, no neutrophilic infiltration and lymphocytic infiltration may therefore be favorable prognostic factors in anaplastic thyroid carcinoma.

Synergistic Effect of Neutral Protease and Clostripain on Rat Pancreatic Islet Isolation.

BACKGROUND: Islet isolation currently requires collagenase, neutral protease and other components. Thermolysin (TL) from Bacillus thermoproteolyticus is the gold standard neutral protease. However, we speculated that neutral protease derived from Clostridium histolyticum (Ch; ChNP) would be biologically superior for islet isolation. Tryptic-like activity has also been reported to be important. Therefore, we focused on clostripain (CP), since it is one of the main proteases in Clostridium histolyticum which possesses tryptic-like activity. We then examined the synergistic effects of highly purified ChNP and CP on rat islet isolation. METHODS: The same amount of collagenase was used in all four groups (TL, ChNP, TL+CP and ChNP+CP; n = 12/group). The efficiency was evaluated by the islet yield and function. An immunohistochemical analysis, in vitro digestion assay for each enzyme component and evaluation of the activation of endogenous exocrine proteases during islet isolation were also performed. RESULTS: The islet yield of the TL group was significantly higher than that of the ChNP group (P < 0.01). The islet yield was dose dependently increased in the ChNP+CP group, but was decreased in the TL + CP group. The islet yield in the ChNP + CP group was significantly higher than that in the TL group, but their islet function was similar. Different specificities for laminin, especially laminin-511, were observed in the TL, ChNP, and CP groups. CONCLUSIONS: Clostripain had a strong synergistic effect with ChNP, but not with TL. Therefore, ChNP and CP, in combination with collagenase derived from the same bacteria, may effectively increase the isolation efficiency without affecting the quality of islets.

Long-term survival with growth hormone replacement after liver transplantation of pediatric nonalcoholic steatohepatitis complicating acquired hypopituitarism.

Nonalcoholic steatohepatitis (NASH) is the most severe form of nonalcoholic fatty liver disease (NAFLD). In adult patients, liver transplantation (LT) is the treatment of choice for end-stage liver disease secondary to NASH. However, little information is available regarding outcomes of LT in pediatric patients with NASH. We describe here a pediatric patient with NASH associated with hypopituitarism who underwent living donor liver transplantation (LDLT). An 11-year-old boy was diagnosed with a pituitary tumor, which was removed by trans-interhemispheric approach following bifrontal craniotomy. Histopathological examination revealed a mature teratoma. Eighteen months later, magnetic resonance imaging showed recurrence of the pituitary tumor, which was found to be a germinoma. He underwent 3 months of chemoradiotherapy, with a complete response. He gradually became obese, with elevated transaminase levels. At age 15 years, he developed fatigue and dyspnea and was found to have liver cirrhosis secondary to NASH with severe hepatopulmonary syndrome. He underwent LDLT using a right liver graft from his mother. Twelve months later, abdominal computed tomography showed recurrence of NAFLD. Five years after the LDLT, transaminases were slightly elevated. Growth hormone replacement therapy was started, reducing transaminase levels to their normal ranges. Ten years after LDLT, fatty liver remains stable, although his body mass index has not been reduced. Growth hormone replacement therapy may be effective in graft maintenance. This is the first case report of a patient with maintained stable liver function 10 years after LDLT for pediatric NASH.

Renal function status in liver transplant patients in the first month post-transplant is associated with progressive chronic kidney disease.

AIM: Renal dysfunction is a common complication of liver transplantation (LT), related to hepatorenal syndrome with end-stage liver disease or calcineurin-inhibitor nephrotoxicity. Chronic kidney disease (CKD) is also a common problem in long-term survivors post-LT. This study was done to investigate the association between renal functional status soon after LT and the development of CKD. METHODS: We retrospectively evaluated 63 patients who were aged 18 years or older, and underwent LT at Tohoku University Hospital. The estimated glomerular filtration rate (eGFR) was calculated by the Modification of Diet in Renal Disease study equation for Japan. RESULTS: Before transplantation, 25 patients (39.7%) were diagnosed with CKD (eGFR, <60 mL/min per 1.73 m(2) ). The incidence of CKD was 22.4% (13/58) at 2 years, 23.2% (13/56) at 3 years and 22.7% (12/54) at 5 years. The patients with CKD at 2 years post-transplant were more likely to have a history of glomerulonephritis, and were significantly older at the time of LT, compared to those without CKD. Levels of eGFR of less than 60 mL/min per 1.73 m(2) in the first month post-transplant and a volume of intraoperative blood loss of more than 300 mL/kg were predictive factors for the development of CKD at 2 years post-transplant and thereafter. CONCLUSION: We have shown that there is an improvement of renal function in the majority of patients after LT. Regardless of the presence of pre-existing CKD, both renal function status at the first month post-transplant and a volume of intraoperative blood loss were predictive factors for the development of CKD at 2 years post-transplant and thereafter.

Water-clear cell adenoma associated with primary hyperparathyroidism: report of a case.

We report a case of water-clear cell adenoma associated with primary hyperparathyroidism. A 59-year-old woman with a history of renal stones and bone fracture was referred for investigation of hypercalcemia and an elevated serum parathyroid hormone level. Skeletal X-rays showed osteopenia and ultrasound showed enlarged tumors in both sides of the inferior thyroid region. Computed tomography demonstrated a tumor in the posterior aspect of the left thyroid lobe but no lesion in the right aspect of the neck. Grossly, we found a 500 mg left lower parathyroid gland (PTG) and a 100 mg right lower PTG. Histologically, the left lower PTG comprised mainly water-clear cells (WCCs) containing numerous vacuoles. Chief cells were dispersed among the WCCs, but the right lower PTG showed normal parathyroid tissue. Several investigators have speculated that WCCs are derived from chief cells, and we diagnosed WCC adenoma. Following this case report, we review the relevant literature.

A novel resting strategy for improving islet engraftment in the liver.

BACKGROUND: Several studies have revealed that posttransplant insulin treatment is beneficial to rest the islet grafts. However, insulin infusion per se is not enough to completely suppress the heavy workload arising caused by postprandial hyperglycemia. Therefore, the present study examined whether short-term fasting combined with insulin treatment could effectively prevent graft exhaustion after intraportal islet transplantation. METHODS: A marginal dose of syngeneic rat islet grafts were transplanted intraportally into the control, insulin-treated, and insulin+rest groups of streptozotocin-induced diabetic rats. The control group fed freely without insulin treatment, and the other groups were continuously treated with an optimal amount of insulin to maintain normoglycemia. In addition, the insulin+rest group fasted and received total parenteral nutrition during the 2 weeks after transplantation. RESULTS: The curative rate was significantly higher in both the insulin and insulin+rest groups than the control group (P<0.0001). The glucose tolerance, residual graft mass, and graft function were significantly ameliorated in the insulin+rest group, but not in the insulin group, compared to the control group (P<0.01, P=0.03, P=0.001). CONCLUSIONS: These data suggest that short-term fasting combined with insulin treatment, especially during the avascular period of the grafts, could therefore be a promising regimen for improving pancreatic islet engraftment in the liver.

Collagenase H is crucial for isolation of rat pancreatic islets.

The role(s) of collagenase G (ColG) and collagenase H (ColH) during pancreatic islet isolation remains controversial, possibly due to the enzyme blends used in the previous studies. We herein examined the role of ColG and ColH using highly pure enzyme blends of recombinant collagenase of each subtype. Rat pancreases were digested using thermolysin, together with ColG, ColH, or ColG/ColH (n = 9, respectively). No tryptic-like activity was detected in any components of the enzyme blends. The efficiency of the collagenase subtypes was evaluated by islet yield and function. Immunohistochemical analysis, in vitro collagen digestion assay, and mass spectrometry were also performed to examine the target matrix components of the crucial collagenase subtype. The islet yield was highest in the ColG/ColH group (4,101 ± 460 islet equivalents). A substantial number of functional islets (2,811 ± 581 islet equivalents) was obtained in the ColH group, whereas no islets were retrieved in the ColG group. Mass spectrometry demonstrated that ColH reacts with collagen I and III. In the immunohistochemical analysis, both collagen I and III were located in exocrine tissues, although collagen III expression was more pronounced. The collagen digestion assay showed that collagen III was more effectively digested by ColH than by ColG. The present study reveals that ColH is crucial, while ColG plays only a supporting role, in rat islet isolation. In addition, collagen III appears to be one of the key targets of ColH.

Thioredoxin-1 attenuates early graft loss after intraportal islet transplantation in mice.

AIMS: Recent studies suggest that decreasing oxidative stress is crucial to achieve successful islet transplantation. Thioredoxin-1 (TRX), which is a multifunctional redox-active protein, has been reported to suppress oxidative stress. Furthermore, it also has anti-inflammatory and anti-apoptotic effects. In this study, we investigated the effects of TRX on early graft loss after islet transplantation. METHODS: Intraportal islet transplantation was performed for two groups of streptozotocin-induced diabetic mice: a control and a TRX group. In addition, TRX-transgenic (Tg) mice were alternately used as islet donors or recipients. RESULTS: The changes in blood glucose levels were significantly lower in the TRX group compared with the TRX-Tg donor and control groups (p<0.01). Glucose tolerance and the residual graft mass were considerably better in the TRX group. TRX significantly suppressed the serum levels of interleukin-1ß (p<0.05), although neither anti-apoptotic nor anti-chemotactic effects were observed. Notably, no increase in the 8-hydroxy-2'-deoxyguanosine level was observed after islet infusion, irrespective of TRX administration. CONCLUSIONS: The present study demonstrates that overexpression of TRX on the islet grafts is not sufficient to improve engraftment. In contrast, TRX administration to the recipients exerts protective effects on transplanted islet grafts by suppressing the serum levels of interleukin-1ß. However, TRX alone appears to be insufficient to completely prevent early graft loss after islet transplantation. We therefore propose that a combination of TRX and other anti-inflammatory treatments represents a promising regimen for improving the efficacy of islet transplantation.

A human thyroid cancer cell line, DH-14-3, newly established from poorly differentiated thyroid carcinoma.

Poorly differentiated thyroid carcinoma (PDTC) is a newly recognized histological type of malignant thyroid tumor, accounting for about 2 - 13% of all thyroid carcinomas. PDTC is considered as a morphologically and biologically intermediate stage between well-differentiated thyroid carcinoma and anaplastic thyroid carcinoma. PDTC preferentially manifests bone metastases. We here established a cell line from a resected tumor specimen from a 70-year-old male patient with PDTC who presented with multiple bone metastases. This new thyroid tumor cell line was designated as DH-14-3 and was subsequently grown in culture for several years. DH-14-3 cells express thyroglobulin in the cytoplasm and thyroid transcription factor-1 in the nuclei, both proteins of which are specific markers for the thyroid gland. Importantly, triiodothyronine (T3) was detected in the cultured medium of DH-14-3 cells, in which, however, thyroxine (T4) was undetectable. Moreover, DH-14-3 cells secreted interleukin-8, transforming growth factor-ß1, vascular endothelial growth factor, matrix metalloproteinase-1 and parathyroid hormone-related protein, all of which may be responsible for the aggressiveness or bone metastasis of PDTC. Thus, the production of these proteins may reflect the metastatic potential of this cell line. DH-14-3 cells also express CXC chemokine receptor-4 and epidermal growth factor receptor, and carry a missense mutation in the p53 tumor suppressor gene. In fact, transplantation of DH-14-3 cells into the back of nude mice resulted in the formation of tumors, thereby confirming the capability of tumorigenesis. DH-14-3 cells may be useful for investigating the biological features of PDTC and will contribute to the therapeutic study of thyroid cancer.

Tacrolimus inhibits the revascularization of isolated pancreatic islets.

AIMS: Immunosuppressive drugs could be crucial factors for a poor outcome after islet allotransplantation. Unlike rapamycin, the effects of tacrolimus, the current standard immunosuppressant used in islet transplantation, on graft revascularization remain unclear. We examined the effects of tacrolimus on islet revascularization using a highly sensitive imaging system, and analyzed the gene expression in transplanted islets by introducing laser microdissection techniques. METHODS: Islets isolated from C57BL/6-Tg (CAG-EGFP) mice were transplanted into the nonmetallic dorsal skinfold chamber on the recipients. Balb/c athymic mice were used as recipients and were divided into two groups: including a control group (n = 9) and tacrolimus-treated group (n = 7). The changes in the newly-formed vessels surrounding the islet grafts were imaged and semi-quantified using multi-photon laser-scanning microscopy and a Volocity system. Gene expression in transplanted islets was analyzed by the BioMark dynamic system. RESULTS: The revascularization process was completed within 14 days after pancreatic islet transplantation at subcutaneous sites. The newly-formed vascular volume surrounding the transplanted islets in the tacrolimus-treated group was significantly less than that in the control group (p<0.05). Although the expression of Vegfa (p<0.05) and Ccnd1 (p<0.05) was significantly upregulated in the tacrolimus-treated group compared with that of the control group, no differences were observed between the groups in terms of other types of gene expression. CONCLUSIONS: The present study demonstrates that tacrolimus inhibits the revascularization of isolated pancreatic islets without affecting the characteristics of the transplanted grafts. Further refinements of this immunosuppressive regimen, especially regarding the revascularization of islet grafts, could improve the outcome of islet allotransplantation.

Ductal injection does not increase the islet yield or function after cold storage in a vascular perfusion model.

Several studies have reported that pancreatic ductal preservation greatly improved the islet yield and function after cold storage. However, these studies were devoid of appropriate controls, such as vascular perfusion, which is routinely performed to preserve organs in the clinical setting. In this study, we created a vascular perfusion model using inbred rats, and investigated the effect of ductal injection on the islet yield and function after cold storage. Rat pancreases after 10 h cold ischemia were classified as follows: without ductal/vascular perfusion; with ductal injection; with vascular perfusion; and with ductal/vascular perfusion. The islet yield, function, viability, release of inflammatory mediators, and pathological changes in the exocrine tissues were assessed in the Hanks' Balanced Salt Solution (HBSS) model. The islet yield was also assesed by introducing University of Wisconsin Solution (UWS) and Histidine-Tryptophan-Ketoglutarate solution (HTK), which are the standard clinical preservation solutions. In the HBSS model, ductal injection and vascular perfusion significantly improved the islet yield compared with the control group. However, ductal injection showed no additional effects on the islet yield, function, viability and suppressing the release of inflammatory mediators when vascular perfusion was performed. Although ductal injection significantly decreased the apoptosis of exocrine cells, no beneficial effect on vacuolation was observed. In contrast, vascular perfusion significantly suppressed vacuolation in the exocrine tissues. Likewise, in the UWS and HTK model, ductal injection and vascular perfusion improved the islet yield compared with the control group. Nevertheless, the combination group showed no additional effects. These data suggest that ductal injection has no additional effect on islet yield and function after cold storage in a vascular perfusion model. We propose that ductal injection can be an effective and simple alternative for vascular perfusion prior to pancreas harvest, but is not necessary in most cases, since vascular perfusion is routinely performed.

Contralateral glissonian pedicle occlusion in a case of portal vein tumor thrombosis.

To dissect portal vein branches directly and encircle them separately is a common procedure that is performed to control back flow bleeding during operations for hepatocellular carcinoma with portal vein tumor thrombosis. However, this technique has an increased risk of injuring contralateral portal branches and disseminating thrombosis fragments to the remnant liver. We present an alternative technique using right-sided glissonian pedicle occlusion for hepatocellular carcinoma with left portal vein tumor thrombosis due to complex anatomical vasculatures of the hepatic pedicle. This technique would be very useful for liver resection of hepatocellular carcinoma with the major type of portal vein tumor thrombosis.

Unique histopathological features of graft biopsies with liver function abnormalities in living donor liver transplant patients receiving basiliximab induction therapy.

Induction with basiliximab (BXM) has been confirmed as an effective treatment regimen for prophylaxis of acute cellular rejection (ACR). From 1991 to 2008, 116 living donor liver transplantations (LDLTs) were performed. Among these, 50 were included in this study. We compared calcineurin inhibitor plus steroid treatment without BXM (n = 14, control group) and with BXM (n = 36, BXM group). Although the rates of biopsied patients with abnormal serum biochemical tests (SBTs) were similar in the control (10/14, 71.4%) and BXM (21/36, 58.3%) groups, ACR was diagnosed in 9/10 (90.0%) patients in the control group compared with 4/21 (19.0%) patients in the BXM group. In accordance with the histopathological diagnosis, there was a significant difference in the ratios of peripheral CD4(+) CD25(+) T cells at five wk after LDLT between patients with and without ACR in the BXM group. Next, we divided the 32 patients without ACR in the BXM group into two groups: biopsied patients with abnormal SBTs and non-biopsied patients. The donor age of the biopsied patients was significantly higher than that of the non-biopsied patients. Induction with BXM reduced the incidence of ACR, and unique pathological phenomena responsible for graft dysfunction after LDLT with an increased incidence of abnormal SBTs were observed.

Brain death in combination with warm ischemic stress during isolation procedures induces the expression of crucial inflammatory mediators in the isolated islets.

Tissue factor (TF) and monocyte chemoattractant protein-1 (MCP-1) expressed on the islets have been identified as the main trigger of the instant blood-mediated inflammatory reaction (IBMIR) in islet transplantation. Because the key steps that directly induce TF and MCP-1 remain to be determined, we focused on the influence of brain death (BD) on TF and MCP-1 expression in the pancreatic tissues and isolated islets using a rodent model. TF and MCP-1 mRNA levels in the pancreatic tissues were similar between the BD and the control group. However, TF and MCP-1 mRNA in the fresh islets of the BD group were significantly higher than that of the control group (p < 0.01). BD may thus be suggested to be of great importance as an initiator of TF and MCP-1 induction in the isolated islets. Furthermore, the upregulation of crucial inflammatory mediators induced by BD could be exacerbated by warm ischemic damage during digestion procedures. In the present study, the islet yield and purity were affected by BD. However, almost no influences were observed with respect to islet viability, indicating that the expression of inflammatory mediators rather than islet viability is more susceptible to BD. According to the change in time course of TF and MCP-1 expression in the isolated islets, the selected time point for islet infusion in current clinical islet transplantation was thus shown to be at its worst level, at least with respect to the damage caused by BD and ischemic stress. In conclusion, BD in combination with warm ischemic stress during isolation procedures induces a high expression of TF and MCP-1 in the isolated islets. In order to reduce the expression of crucial inflammatory mediators in the islet grafts, the management of the pancreas from brain-dead donors with early anti-inflammatory treatments is thus warranted.

Long-term outcome of living related renal transplantation in a patient with short bowel syndrome.

Short-bowel syndrome (SBS) is defined as the malabsorptive state that occurs after extensive resection of the small intestine. In patients with SBS, oral administration of drugs usually becomes difficult because of the severity of intestinal failure. We describe a successful living related renal transplantation (LRRTx) in an 18-year-old male with SBS. Shortly after birth, the patient developed necrotizing enterocolitis requiring massive resection of the small intestine, which resulted in SBS. At seven years of age, the patient developed proteinuria and was diagnosed as focal segmental glomerulosclerosis (FSGS). His kidney function was gradually deteriorated toward the end-stage renal failure. The patient received LRRTx at age of 18 years. To evaluate the absorption capacity of the patient, we investigated pharmacokinetics of calcineurine inhibitors (tacrolimus and cyclosporine). The drug concentration, which is sufficient to provide effective immunosuppression, was achieved with cyclosporine, but not with tacrolimus. The patient therefore received a triple immunosuppressive therapy with oral cyclosporine, methyl-prednisolone and mycophenolate mofetil. To prevent both recurrent FSGS and rejection, we repeatedly analyzed the trough level and the pharmacokinetics of cyclosporine after LRRTx. The patient was successfully treated with oral immunosuppression for over 5 years, without hemodialysis. To our knowledge, this is the first report showing the long-term outcome of LRRTx treated with oral cyclosporine in a patient with SBS.