A novel split liver protocol using the subnormothermic oxygenated circuit system in a porcine model of a marginal donor procedure.

BACKGROUND: A merit of subnormothermic perfusion has been reported to preserve grafts from ischemic injury in animal models. The split liver technique is commonly performed to solve the shortage of liver grafts. However, there has been no study showing the effect of a split liver graft on subnormothermic perfusion. We herein investigated the split liver protocol using a subnormothermic oxygenated circuit system (SOCS). METHODS: Auxiliary liver transplantation was performed in a porcine marginal donor model by using a SOCS. In the SOCS group, the portal vein and hepatic artery of the graft were cannulated, and the graft was perfused by SOCS. In the cold storage (CS) group, the graft was placed in cold preservation solution. In the preservation phase, the graft was split. RESULTS: There were no significant differences in the biochemical markers between the SOCS and CS groups. In terms of the histology, the sinusoidal spaces were widened in the CS group 12 hours after implantation. CONCLUSION: We have demonstrated a possibility to use SOCS with the split liver protocol by using a porcine model. This split liver protocol using SOCS will extend the split liver criteria and rescue more patients from hepatic failure, including pediatric patients.

Impact of ex vivo administration of mesenchymal stem cells on the function of kidney grafts from cardiac death donors in rat.

BACKGROUND: Mesenchymal stem cells (MSCs) have been applied to the treatment of various diseases, and MSC administration in marginal donor grafts may help avoid the ischemia-reperfusion injury associated with solid organ transplants. Given the reports of side effects after intravenous MSC administration, local MSC administration to the target organ might be a better approach. We administered adipose tissue-derived MSCs (AT-MSCs) ex vivo to donor rat kidneys obtained after cardiac death (CD). METHODS: Using male Lewis rats (8-10 weeks), and a marginal transplant model of 1hr CD plus 1hr sub-normothermic ET-Kyoto solution preservation were conducted. AT-MSCs obtained from double-reporter (luciferase-LacZ) transgenic Lewis rats were injected either systemically (1.0 × 10(6) cells/0.5 mL) to bilaterally nephrectomized recipient rats that had received a marginal kidney graft (n = 6), or locally via the renal artery (500 µL ET-Kyoto solution containing the same number of AT-MSCs) to marginal kidney grafts, which were then preserved (1 hour; 22°C) before being transplanted into bilaterally nephrectomized recipient rats (n = 8). Serum was collected to assess the therapeutic effects of AT-MSC administration, and the recipients of rats surviving to Day 14 were separately evaluated histopathologically. Follow-up was by in vivo imaging and histological LacZ staining, and tumor formation was evaluated in MSC-injected rats at 3 months. RESULTS: Systemic injection of MSC did not improve recipient survival. In vivo imaging showed MSCs trapped in the lung that later became undetectable. Ex vivo injection of MSCs did show a benefit without adverse effects. At Day 14 after RTx, 75% of the rats in the AT-MSC-injected group (MSC[+]) had survived, whereas 50% of the rats in the AT-MSC-non-injected group (MSC[-]) had died. Renal function in the MSC(+) group was improved compared with that in the MSC(-) group at Day 4. LacZ staining revealed AT-MSCs attached to the renal tubules at 24 hours after RTx that later became undetectable. Histopathologic examination showed little difference in fibrosis between the groups at Day 14. No teratomas or other abnormalities were seen at 3 months.

Evaluation of liver preservation solutions by using rats transgenic for luciferase.

INTRODUCTION: The solution in which graft tissue is stored (that is, preservation solution) is an important component of liver transplantation technology. Its protective effect is induced by substances in the solution, including radical scavengers, buffers, and energy-giving substances. New preservation solutions have proven to be effective in preventing organ damage during cold ischemia and in extending the time limits for storage. AIM: This study determined the relationship between luminescence intensity and content of adenosine triphosphate (ATP) in liver tissue and proposes a new ex vivo screening system that uses Lewis rats transgenic for luciferase for evaluating the effectiveness of preservation solutions. METHODS: Samples (diameter, 2 mm) of liver were obtained from transgenic rats. The viability of these tissues after storage for as long as 6 hours in University of Wisconsin (UW) solution, extracellular trehalose solution of Kyoto, Euro-Collins (EC) solution, histidine-tryptophan-ketoflutarate solution, low potassium dextran solution, or normal saline was assessed by determining ATP content and luminescence intensity. RESULTS: Luminescence had a linear relationship (R = 0.88) with ATP levels. Regardless of the preservation solution used, the luminescence intensities of the liver tissue chips decreased linearly with time especially through a short span of time (0 to 2 hours; R(2) = 0.58-1.0). The luminescence of liver chip tissues maintained long term (2 to 6 hours) in UW solution tended to be higher than those of tissues stored in other solutions (P < .05; 6 hours). On the basis of luminescence intensity, EC might be preferable to the other solutions tested for ultra-short-term storage (0.5 to 2 hours). CONCLUSION: Our model, which combines the use of the bioimaging system and Lewis rats transgenic for luciferase, effectively assessed the viability of liver tissue samples. We believe that this ex vivo screening system will be an effective tool for evaluating preservation solutions for liver grafts.

Luminescence-based assay to screen preservation solutions for optimal ability to maintain viability of rat intestinal grafts.

BACKGROUND: Segmental intestinal transplantations from living, genetically related donors provide advantages compared with those from cadaveric subjects. However, successful preservation during ischemic cold storage is critical for living donor grafts. Thus, the development of preservation solutions that maintain graft viability is essential for success. Herein we have reported application of a cell-based viability assay in multiwell plates to assess the effectiveness of various solutions to preserve intestinal grafts. METHODS: Freshly isolated intestinal chips from luciferase transgenic rats were placed in 96-well tissue culture plates for incubation at 4°C for 24 hours in various preservation solutions: ET-Kyoto (ET-K), University of Wisconsin (UW) solution, Euro-Collins (EC) solution, histidine-tryptophan-ketoglutarate (HTK) solution, lactated Ringer's (LR) solution, or saline. RESULTS: As indicated by a higher level of luminescence, intestinal chips preserved in UW, HTK, or ET-K solution contained more viable cells, than those preserved in EC, LR, or saline solution. After exposure to the preservation solutions for 1 hour, the mucosal layer chips showed lower cell viability than the muscle layer chips. CONCLUSION: Our data demonstrated that ET-K and UW solutions used together with intestinal chips of Luciferase transgenic rat and in vivo imaging provided optimal viability during ischemic cold storage prior to transplantation. Further development of preservation conditions to minimize the loss of viability of intestinal grafts before clinical transplantation is essential to improve outcomes.

The application of an institutional clinical data warehouse to the assessment of adverse drug reactions (ADRs). Evaluation of aminoglycoside and cephalosporin associated nephrotoxicity.

OBJECTIVES: To apply an institutional clinical data warehouse (CDW) to the assessment of adverse drug reactions (ADRs) and demonstrate its utility through a specific example. METHODS: We modeled the process for assessing ADRs through retrospective cohort design by using CDW at the Osaka University Hospital as follows: 1) We defined a drug X, an adverse drug reaction (ADR) Y, and a laboratory measurement Z to assess Y during a given study period; 2) we excluded those whose Z value exceeded the defined criteria or were not available at the inception of the cohort; 3) we divided the patients into two groups based on exposure or non-exposure to X; 4) we matched the patient characteristics between the two groups through stratification and randomization; and 5) we compared the frequency of patients who presented Y during the study period between the two groups. Aminoglycoside and Cephalosporin associated nephrotoxicity in pediatric inpatients was used as an example to demonstrate the usefulness of this approach. RESULTS: Our evaluation indicates that there is an increased risk of nephrotoxicity for pediatric inpatients who were prescribed cephalosporin either alone or in combination with aminoglycoside; further, aminoglycoside tends to increase the cephalosporin-associated nephrotoxicity. CONCLUSIONS: Our findings are consistent with those drawn from other studies, indicating that the method of applying an institutional CDW is useful for assessing ADRs.

Streptozotocin-induced partial beta cell depletion in nude mice without hyperglycaemia induces pancreatic morphogenesis in transplanted embryonic stem cells.

AIMS/HYPOTHESIS: It appears that the adult pancreas has limited regenerative ability following beta cell destruction by streptozotocin (STZ). However, it is not clear if this limitation is due to an inability to respond to, rather than an absence of, regenerative stimuli. In this study we aimed to uncouple the regenerative signal from the regenerative response by using an exogenous stem cell source to detect regenerative stimuli produced by the STZ-injured pancreas at physiological blood glucose levels. METHOD: Adult nude mice received 150 mg/kg STZ and 1x10(6) J1 mouse embryonic stem (ES) cells by i.p. injection. Permanent beta cell depletion of 50% was estimated from the ratio of beta:alpha cells in pancreata from STZ-treated mice compared with control animals after 24 days. RESULTS: Transplanted ES cells homed to the STZ-injured pancreas and formed tumours. Immunocytochemical analysis of pancreas-associated ES tumours revealed foci containing insulin/PDX-1 double-positive and glucagon-positive/PDX-1-negative cell clusters associated with PDX-1-positive columnar lumenal epithelium and extensive alpha-amylase-positive pancreatic acini comprising approximately 0.1% of ES tumour volume. CONCLUSIONS/INTERPRETATION: These data indicate that (1) the adult pancreas produces a milieu of regenerative stimuli following beta cell destruction, and (2) this is not dependent on hyperglycaemic conditions; (3) these regenerative stimuli appear to recapitulate the signalling pathways of embryonic development, since both exocrine and endocrine lineages are produced from PDX-1-positive precursor epithelium. This model will be useful for characterising the regenerative mechanisms in the adult pancreas.

Artificial ascites technique for percutaneous radiofrequency ablation of liver cancer adjacent to the gastrointestinal tract.

BACKGROUND: Percutaneous radiofrequency ablation (RFA) of liver tumours adjacent to the gastrointestinal tract is controversial. This study assessed the value of an intraperitoneal water infusion (artificial ascites) technique for percutaneous RFA of such tumours. METHODS: Before ablation in 52 patients (55 treatments, 58 tumours), between 250 and 3000 (mean 681) ml 5 per cent glucose solution was infused into the abdominal cavity using a 14-G needle, with the aim of preventing thermal injury by separating the liver from the gastrointestinal tract. RESULTS: There were no adverse events associated with the artificial ascites technique. In 43 (78 per cent) of the 55 treatments, the liver and gastrointestinal tract were separated successfully. In the other 12 treatments, in which the separation was not confirmed by real-time ultrasonography, there was one case of perforation of the ascending colon after RFA; adhesion of the liver and colon resulting from previous laparotomy may have been related to the injury. CONCLUSION: Production of artificial ascites can be undertaken safely, making RFA safe and effective for hepatic tumours adjacent to the gastrointestinal tract. In patients with possible postoperative adhesions, confirmation of separation of the liver from surrounding organs is mandatory to avoid thermal injury.

Proposal of a new prognostic model for hepatocellular carcinoma: an analysis of 403 patients.

BACKGROUND: The prognosis of hepatocellular carcinoma (HCC) is highly dependent on tumour extension and liver function. Recently, two new prognostic scoring systems-the CLIP score, developed by Italian investigators and the BCLC score, developed in Barcelona-have been widely used to assess prognosis in patients presenting with hepatocellular carcinoma. Each system has its own relative limitations. AIMS: To create a new prognostic scoring system which is simple, easy to calculate, and suitable for estimating prognosis during radical treatment of early HCC. METHODS: A total of 403 consecutive patients with HCC treated by percutaneous ablation at the Department of Gastroenterology, University of Tokyo Hospital, between 1990 and 1997 were used as the training sample to identify prognostic factors for our patients and used to develop the Tokyo score. As a testing sample, 203 independent patients who underwent hepatectomy at the Department of Hepato-Biliary-Pancreatic Surgery were studied. Prognostic factors were analysed by univariate and multivariate Cox proportional hazard regression. RESULTS: The Tokyo score consists of four factors: serum albumin, bilirubin, and size and number of tumours. Five year survival was 78.7%, 62.1%, 40.0%, 27.7%, and 14.3% for Tokyo scores 0, 1, 2, 3, and 4-6, respectively. The discriminatory ability of the Tokyo score was internally validated by bootstrap methods. The Tokyo score, CLIP score, and BCLC staging were compared by Akaike information criterion and Harrell's c index among training and testing samples. In the testing sample, the predictive ability of the Tokyo score was equal to CLIP and better than BCLC staging. CONCLUSIONS: The Tokyo score is a simple system which provides good prediction of prognosis for Japanese patients with HCC requiring radical therapy.

S100A9 expression in invasive ductal carcinoma of the breast: S100A9 expression in adenocarcinoma is closely associated with poor tumour differentiation.

S100A9 is associated with myelomonocytic cell differentiation and is also expressed in some epithelia. However, there have been few studies on S100A9 in adenocarcinoma (AC) because the expression in normal epithelia is limited to squamous epithelia. Our previous studies on pulmonary AC and liver carcinomas suggested that S100A9 expression in carcinomas of glandular cell origin is related to poor tumour differentiation. In this study, we examined S100A9 expression in invasive breast carcinoma and evaluated the relation of the expression to the tumour differentiation in 70 cases of invasive ductal carcinoma (IDC) of the breast. S100A9 gene and protein expression was detected in MCF-7 breast carcinoma cells. The rate of S100A9 immunopositivity in IDC showed a higher correlation with poor tumour differentiation, especially in nuclear pleomorphism (P=0.0002) and mitotic activity (P=0.0001). Furthermore, transcriptional expression of S100A9 in sections of IDC could be detected in cases with a high S100A9 immunopositivity. No significant differences in the number of myelomonocytic cells expressing S100A9 were found among cases. There was no correlation between S100A9 immunopositivity and lymph node metastasis (P=0.32). S100A9 immunopositivity in non-invasive ductal carcinoma was also associated with poor tumour differentiation. No immunopositive reaction was observed in invasive lobular carcinomas with a classic cytological appearance and non-neoplastic duct cells. We conclude that S100A9 in glandular epithelial cells is newly expressed under cancerous conditions and is over-expressed in poorly differentiated AC.

Power Doppler signals after percutaneous ethanol injection therapy for hepatocellular carcinoma predict local recurrence of tumors: a prospective study using 199 consecutive patients.

BACKGROUND/AIMS: This study was prospectively conducted to elucidate the relationship between pre-/post-treatment power Doppler signals of hepatocellular carcinoma (HCC) and local recurrence. METHODS: One hundred ninety-nine consecutive patients with 359 HCC lesions receiving percutaneous ethanol injection therapy (PEIT) as a first-line option were enrolled. Arterial power Doppler signals in the tumor were found in 130 nodules, but not detected in 229. After confirmation of complete tumor necrosis on dynamic CT, Doppler signals in nodules were re-evaluated. Patients received periodical examinations to detect HCC recurrence. RESULTS: Local HCC recurrence was observed in 36 lesions; 22%(28/130) of the pretreatment signal positive lesions, in contrast to 3.5% (8/229) of the pretreatment signal negative lesions (P < 0.01). Out of 130 signal positive nodules, signals disappeared in 120 (92%) after PEIT, but were present in ten (8%). During the 25-month follow up, local recurrence was detected in 19 (16%) from the former, in contrast to nine (90%) from the latter (P < 0.001). Uni- and multivariate Cox analysis revealed that the presence of pre-/post-treatment power Doppler signals, histological differentiation and tumor number were independent factors for local recurrence. However, 3-year recurrence rate of new lesions was 51%, but no predictors were identified. CONCLUSIONS: Residual Doppler signals in tumor after PEIT were related to the local HCC recurrence.

Thoracoscopic thermal ablation therapy for hepatocellular carcinoma located beneath the diaphragm.

BACKGROUND AND STUDY AIMS: Percutaneous interstitial thermal ablation therapy effectively treats hepatocellular carcinoma (HCC) that can be visualized on percutaneous ultrasonography. However, when the tumor is located just under the top of the diaphragm, visualization can be difficult with conventional ultrasonographic examination. There are also problems concerning complete tumor ablation. We performed thoracoscopic thermal ablation therapy for HCC located just beneath the diaphragm in nine patients with advanced liver cirrhosis. PATIENTS AND METHODS: Eight patients underwent thoracoscopic microwave coagulation therapy, and one patient underwent thoracoscopic radiofrequency ablation therapy. RESULTS: Despite the poor hepatic reserve, postoperative recovery after thoracoscopic thermal ablation therapy was rapid in all patients, without deterioration of hepatic function. CONCLUSIONS: This preliminary study suggests that the new technique of thoracoscopic thermal ablation therapy is a less invasive optional therapy for HCC located in segments VII or VIII in cirrhotic patients.

Hepatocellular carcinoma depicted as hypoattenuation on CT hepatic arteriography (CTA) and hyperattenuation on CT during arterial portography (CTAP).

We report a 68-year-old man with three nodules of hepatocellular carcinoma (HCC) in a cirrhotic liver; the largest nodule was 3.0cm in diameter. The nodules showed hypoattenuation on computed tomography (CT) hepatic arteriography (CTA) and hyperattenuation on CT during arterial portography (CTAP), indicating that the dominant vascularity of the HCC nodules may have been the portal vein. A biopsy specimen obtained from the nodules showed well differentiated HCC (Edmondson-Steiner grade I). The imaging findings of the nodules on both CTA and CTAP are unusual, in spite of the rather large size, so this seemed suggestive of the hemodynamic properties of relatively large nodules of well differentiated HCC.

Percutaneous ethanol injection therapy for liver tumors.

Percutaneous ethanol injection therapy (PEIT) has been widely practiced in the treatment of liver tumors, especially of hepatocellular carcinoma (HCC). Histopathologic examinations, findings in imaging modalities and serum tumor marker levels have shown a remarkable anticancer effect of this procedure. In addition, PEIT has achieved considerably high long-term survival rates. For small HCC, PEIT has been generally accepted as an alternative to surgery. Here we will describe PEIT from the viewpoints of patient selection, technique, various evaluation procedures of efficacy, long-term results, side effects and complications, and relationship with other therapies.

Immunohistochemical investigation of S100A9 expression in pulmonary adenocarcinoma: S100A9 expression is associated with tumor differentiation.

S100 protein A9 is associated with myeloid cell differentiation and is also expressed in some epithelia. However, there have been few studies on S100A9 in specific types of carcinomas, except for squamous cell carcinoma (SCC) because the expression in normal epithelia is limited to squamous epithelia. Recently, S100A9 gene expression has been detected in cultured human adenocarcinoma (AC) cells derived from various organs. In this study, we also detected S100A9 gene expression in human pulmonary AC cell lines by reverse transcription-polymerase chain reaction. Furthermore, using the monoclonal antibody against S100A9, we carried out an immunohistochemical evaluation of S100A9 protein expression in 70 cases of resected pulmonary AC and examined the relation of S100A9 expression to tumor differentiation. S100A9 immunopositivity was 0/21 (0%) in well differentiated ACs, 12/30 (40%) in moderately differentiated ACs and 19/19 (100%) in poorly differentiated ACs, and the poorly differentiated ACs showed a significantly greater positive reaction. The immunopositivity in the moderately differentiated ACs was marked in specific cytologic subtypes. In the controls, conspicuous S100A9 immunopositivity was observed in pulmonary SCCs, regardless of the degree of differentiation, but not in adenomatous hyperplasia or normal surface epithelia. These above results suggest that the S100A9 protein is also expressed in pulmonary AC and that the expression rate in pulmonary AC shows higher correlation in poorly differentiated carcinomas, in agreement with our recent results regarding liver carcinoma. We believe S100A9 is also closely related to the differentiation of carcinomas of glandular cell origin.

Des-gamma-carboxy prothrombin as a useful predisposing factor for the development of portal venous invasion in patients with hepatocellular carcinoma: a prospective analysis of 227 patients.

BACKGROUND: Portal venous invasion (PVI) in patients with hepatocellular carcinoma (HCC) is an important factor affecting prognosis. The objective of this study was to elucidate predisposing factors for the development of PVI. METHODS: Two hundred twenty-seven patients with HCC who did not show PVI and who received percutaneous ethanol injection therapy and/or microwave coagulation therapy at the time of their first hospital admission were enrolled between 1994 and 1996. After their HCC was treated, the patients were followed for a mean of 19 months. For the detection of HCC recurrence and/or development of PVI, ultrasonography was performed every 3 months, a computed tomography (CT) scan was performed every 6 months, and the biochemical parameters of the patients were measured every month. PVI was defined as protrusion of the tumor into the first and/or second branch or into the main trunk of the portal vein. RESULTS: Of the 227 patients, 24 (11%) later developed PVI. Tabular analysis was performed on these 24 patients and indicated that tumor size, albumin, total bilirubin, prothrombin time, alpha-fetoprotein (AFP) level, and des-gamma-carboxy prothrombin (DCP) level differed significantly between the time of initial admission and the time of PVI development. A univariate analysis performed on the 227 patients indicated that an increase in the numbers of tumors, the histologic tumor grade (differentiation), the AFP level, and the DCP level at the time of initial diagnosis of HCC had a significant correlation with the later development of PVI; and a stepwise, multivariate Cox regression analysis revealed that the DCP level was the strongest predisposing factor (P < 0.0010; risk ratio = 5.65) followed by the histologic grade of tumor differentiation. CONCLUSIONS: The results suggest that the serum DCP level is the most useful predisposing clinical parameter for the development of PVI.