Tokihiko Sawada

Preconditioning regimen consisting of anti-CD20 monoclonal antibody infusions, splenectomy and DFPP-enabled non-responders to undergo ABO-incompatible kidney transplantation

Abstract:  Patients undergoing ABO‐incompatible kidney transplantation must have their anti‐donor blood‐type antibody titer (ADBT) reduced to below 1:16 by using either plasma‐exchange (PEX) or double filtration plasma exchange (DFPP) before they can safely undergo a transplantation. The ADBT can be reduced to under 1:16 in most cases; however, some cases (non‐responders) do not respond to PEX or DFPP treatment. To enable kidney transplantations to be performed in non‐responders, we developed a new preconditioning regimen consisting of anti‐CD20 monoclonal antibody (rituximab) infusions, a splenectomy, and DFPP. Four non‐responders were infused with rituximab at a dose of 375 mg/m2 weekly for 3–4 wk and splenectomized 1 or 2 wk before transplantation. Four to five DFPP‐sessions were then performed after the splenectomy. Using this preconditioning regimen, the ADBT was reduced to below 1:16, enabling kidney transplantations to be successfully performed in all patients. After the kidney transplantation, no episodes of humoral rejection were observed, and only one episode of cellular rejection was encountered. The cellular rejection was associated with a reduction in immunosuppressant administration because of CMV infection that occurred 80 d after the kidney transplantation. The renal allografts were functioning well in all patients after a mean follow‐up period of 390 d. No serious complications or side effects were encountered. We have developed a new preconditioning regimen that enables PEX and DFPP non‐responders to undergo ABO‐incompatible kidney transplantations.

Lobular damage caused by cellular and humoral immunity in liver allograft rejection.

Abstract:  Backgrounds:  Diagnosis of acute liver allograft rejection (ALAR) is usually performed by the estimation of changes in portal areas. In this study, changes in the hepatic lobules were investigated retrospectively by immunohistochemistry, and compared with changes in the portal areas. Humoral immunity in ALAR was also studied by C4d‐staining.

Expression of hepcidin mRNA is uniformly suppressed in hepatocellular carcinoma

BackgroundThe present study evaluated the expression of hepcidin mRNA in hepatocellular carcinoma (HCC).MethodsSamples of cancerous and non-cancerous liver tissue were taken from 40 patients with HCC who underwent hepatectomy. Expression of hepcidin mRNA was evaluated by real-time PCR, and compared in tumors differing in their degree of differentiation, number of tumors, and vessel invasion. Correlations between hepcidin expression and the interval until HCC recurrence, and the serum concentration of hepcidin were evaluated, together with the expression of mRNAs for other iron metabolism molecules, ferroportin and transferrin receptor 2 (Trf2).ResultsHepcidin mRNA expression in non-cancerous and cancerous tissues was 1891.8 (32.3–23187.4) and 53.4 (1.9–3185.8), respectively (P < 0.0001). There were no significant differences in hepcidin expression among tumors differing in their degree of differentiation, number of tumors, or vessel invasion. There was no significant correlation between hepcidin expression and the interval until HCC recurrence. The serum concentration of hepcidin-25 was not correlated with hepcidin-mRNA expression. Finally, there were no significant differences in the expression of mRNA for ferroportin and Trf2 between cancerous and non-cancerous tissues.ConclusionExpression of hepcidin mRNA is strikingly suppressed in cancerous, but not in non-cancerous tissues, in patients with HCC, irrespective of ferroportin or Trf2 expression. Uniform suppression of hepcidin may be linked to the development of HCC.

Erythropoietin and its derivative protect the intestine from severe ischemia/reperfusion injury in the rat

OBJECTIVE To investigate the protective effect of erythropoietin (EPO) and its nonhematopoietic derivative (asialoEPO) against intestinal ischemia/reperfusion (I/R) injury in a rat model. METHODS The superior mesenteric artery of Wistar rats was clamped for 60 minutes and then released. The rats were divided into 4 groups (n = 15 in each group): sham operation (Sham), vehicle treatment (Vehicle), EPO treatment (EPO), and asialoEPO treatment (AsialoEPO). EPO and asialoEPO were administered subcutaneously at 1000 units/kg for 10 minutes before clamping, 30 minutes after the start of clamping, and just before declamping. This treatment was followed by determination of 72-hour survival rates, serum TNF-alpha and IL-6 levels, histologic evaluation of the small intestine, quantification of the number of apoptotic cells, and analysis of the antiapoptotic molecules Bcl-xL and XIAP by Western blotting. RESULTS The survival rates at 72 hours after I/R injury in the Sham, Vehicle, EPO, and AsialoEPO groups were 100%, 33%, 75%, and 83%, respectively (P < .05). Blood TNF-alpha and IL-6 were significantly more suppressed in the EPO and AsialoEPO groups than in the Vehicle group at 6 hours after I/R injury. Histologically, injury to villi in the EPO and AsialoEPO groups was significantly less than in the Vehicle group. The number of apoptotic cells in the EPO and AsialoEPO groups was significantly less than in the Vehicle group. Western blotting revealed that EPO and asialoEPO constitutively increased the expression of Bcl-xL. CONCLUSIONS EPO and asialoEPO exert a strong protective effect against intestinal I/R injury, possibly by inhibiting release of TNF-alpha and IL-6 and decreasing apoptosis.

New Antineoplastic Agent, MK615, from UME (a Variety of) Japanese Apricot Inhibits Growth of Breast Cancer Cells in vitro

Abstract:  MK615 is an extract mixture containing hydrophobic substances from Japanese apricot. In this study, the antineoplastic effects of MK615 against breast cancer cells were investigated. Two breast cancer cell lines, MDA‐MB‐468 (MDA) and MCF7, were cultured with (600, 300, and 150 μg/mL) or without MK615. After 72 hours of incubation, growth inhibition was evaluated by MTT assay. The cells were then cultured with MK615 (300 μg/mL) and morphological changes were studied by light and electron microscopy. Finally, the mechanism of the antineoplastic effect of MK615 was evaluated by cell cycle and apoptosis assay. MK615 inhibited the growth of MDA and MCF7 in a dose‐dependent manner. The percentage growth inhibition of MDA at dosages of 600, 300, and 150 μg/mL was 59.2%, 52.4%, and 23.3%, respectively, and that for MCF7 was 83.5%, 52.7%, and 16.6%, respectively. Morphological changes after MK615 treatment included massive vacuolization in the cytoplasm and apoptotic changes in the nucleus. These changes began to be apparent after at least 6 hours of incubation. Cell cycle analysis showed that MK615 increased the proportion of cells in the G2‐M phase in both MDA (7.8–11.7%) and MCF7 (8.1–18.7%), and finally both cell lines became apoptotic. The proportion of apoptotic cells increased with incubation time. MK615 effectively inhibits the growth of breast cancer cells in vitro, possibly by cell cycle modification and apotosis induction. MK615 should be further investigated as a promising anti–breast cancer agent.

Protective effect of ischemic preconditioning against liver injury after major hepatectomy using the intermittent pringle maneuver in swine.

Objective: To investigate whether ischemic preconditioning (IP) protects the liver against ischemia-reperfusion injury (I/R-I) after major hepatectomy through intermittent hepatic pedicle clamping (IC) in a swine liver resection model. Background: Although many studies have reported a protective effect of IP against continuous hepatic ischemia, it has not been elucidated whether IP protects the liver against I/R-I after hepatectomy using IC. This is the first study to evaluate the effect of IP in a swine major hepatectomy model using IC. Methods: Pigs (n = 12) were divided into 2 groups (IP or non-IP). In the IP group, livers were subjected to IP (10 min ischemia and 10 min reperfusion) before liver resection using IC (15 min ischemia and 5 min reperfusion). A left hemihepatectomy was then performed using IC in both groups. Hemodynamic changes and plasma concentrations of aspartate aminotransferase, lactate dehydrogenase, lactic acid and hyaluronic acid were measured at 60, 120 and 180 min after hepatectomy. Apoptosis (TUNEL staining and electron microscopy), plasma tumor necrosis factor-α (TNF-α) and NO2–/NO3– were evaluated for 180 min after hepatectomy. Results: There were no significant differences in body weight, blood loss, resected liver weight, Pringle time or hemodynamic changes between the 2 groups. IP significantly reduced plasma aspartate aminotransferase levels for 180 min after hepatectomy (IP: 135.8 ± 13.5 vs. non-IP: 199 ± 16.8 IU/l; p = 0.018). In the non-IP group, apoptotic changes in sinusoidal endothelial cells were observed with increased plasma TNF-α levels. IP protected liver injury from increase in plasma TNF-α (p = 0.042). Significantly fewer apoptotic cells were seen in the IP than in the non-IP group (p = 0.002). Plasma levels of lactate dehydrogenase, lactic acid and NO2–/NO3– in the IP group tended to be lower than those in the non-IP group. Conclusions: IP prior to hepatectomy with IC resulted in less hepatic injury and apoptotic cell death than in livers not subjected to IP. IP with IC has the potential to improve the clinical postoperative course of patients undergoing hepatectomy.

Hepatectomy in patients with nonuremic minimal renal failure

In this study, the perioperative management and short-term outcome of hepatectomy were evaluated in patients with nonuremic minimal renal failure to assess the safety of hepatectomy in such patients. Ninety-one patients who underwent hepatectomy were retrospectively divided into two groups based on their creatinine clearance (Ccr) values: a group with Ccr values ⩾50 but <100 ml/min (group 1; n=77) and a group with Ccr values of ⩾20 to <50 ml/min (group 2; n=14). Preoperative patient characteristics, intraoperative parameters (including operation time and blood loss), and postoperative management and complications were evaluated. The preoperative evaluation showed no differences in liver function between the two groups, and there were no statistically significant differences between the two groups in intraoperative blood loss (522 ml in group 1 and 806 ml in group 2) or intraoperative urine volume (1.01 ml/kg per hour in group 1 and 0.75 ml/kg per hour in group 2). The difference between the two groups in postoperative complications was not statistically significant. None of the patients in group 2 required dialysis therapy, and no patients died as a result of hepatectomy or hepatectomy-related causes. Adequate indications, appropriate operative procedures, and perioperative management can enable hepatectomy to be performed safely in patients with nonuremic minimal renal failure.

Asialoerythropoietin Is a Strong Modulator of Angiogenesis by Bone-Marrow Cells

The angiogenetic effect of asialoerythropoietin (asialoEPO) was investigated in a murine (BALB/c) ischemic hind limb model created by ligating the right femoral artery, and severe ischemia was created by ligating two points on the femoral artery. The mice were allocated to six groups: 1, Control (n = 15), no treatment; 2, BMC (n = 15), Injected with 1 × 106 bone marrow cells (BMC); 3, EPO (n = 15), EPO(500 U/kg for 2 weeks); 4, BMC + EPO (n = 15), BMC + erythropoietin (EPO); 5, asialoEPO (n = 15), asialoEPO (500 U/kg for 2 weeks); 6, BMC + asialoEPO (n = 15); BMC + asialoEPO. Blood flow in the ischemic and normal limbs was measured using a Doppler flowmeter on days 7 and 14. Vessel density was evaluated by immunohistochemical staining for Von Willebrand Factor (vWF). In the severe ischemia model, limb survival was investigated. Blood flow was significantly higher in the BMC + asialoEPO group than in the Control, BMC, EPO, BMC + EPO, or asialoEPO group on day 7 (p =. 007) and on day 14 (p =. 002). Vascular density was also significantly higher in the BMC + asialoEPO (0.067 ± 0.022) group than in the Control (0.026 ± 0.007), BMC (0.027 ± 0.012) EPO (0.029 ± 0.002), BMC + EPO (0.048 ± 0.015), and asialoEPO (0.031 ± 0.001) groups (p =. 006). Finally, limb survival at day 14 in the severe ischemia model was significantly better in the BMC + asialoEPO group (83.3%) than in the Control (40.0%), BMC (52.9%), EPO (44.4%), BMC + EPO (64.7%), or asialoEPO (36.4%) group (p =. 02). This provides the conclusions that asialoEPO promotes angiogenesis by BMC and that its action is significantly more potent than that of EPO.

Total Colectomy Improves Altered Distribution of Regulatory T Cells in Patients with Ulcerative Colitis

BackgroundIt is now generally believed that regulatory T cells (Tregs) play an important role in peripheral tolerance, and a defect in Tregs is considered one of the most important factors in the induction of various kinds of autoimmune disease including ulcerative colitis (UC). Here, we examined the change in frequency of Tregs phenotype in five patients with UC whose condition had not been controllable by conventional conservative therapy and who were scheduled for total colectomy.AimsThe aim of this study was to elucidate the role of Tregs in the pathogenesis of UC in a clinical setting.MethodsPeripheral blood mononuclear cells (PBMCs) were obtained from five patients with UC, and the change in frequency of Tregs was analyzed by flow cytometry before total colectomy and on postoperative days 1, 7, and 20. Tregs were defined as CD4+ CD25+ CD45RA+ T cells, and data (%) were expressed as frequency of Tregs/CD4+ T cells. Peripheral blood mononuclear cells (PBMCs) from healthy blood donors (n = 5) and from patients undergoing other types of major surgery (n = 5) were used as controls.ResultsComparison with normal subjects showed that generation of Tregs was suppressed in UC patients before they underwent total colectomy (0.95 versus 5.06; P = 0.009). The frequency of Tregs increased shortly after total colectomy. The frequency at postoperative days 1, 7, and 20 was 3.81%, 8.13%, and 3.76%, respectively. There were significant differences in the change in frequency in the period before surgery and postoperative day 1, between postoperative days 1 and 7, and between days 7 and 20.ConclusionsElimination of targeted antigens residing in the colorectal mucosa by total colectomy improved the suppressed distribution of Tregs in UC patients. The present study provides the first direct clinical evidence that Tregs play a pivotal role in the pathogenesis of UC.

Ball-valve gastric tumor associated with anomalous junction of the pancreatico-biliary ductal system and a right-sided round ligament: Report of a case

We report the case of a ball-valve gastric tumor associated with anomalous junction of the pancreatico-biliary ductal system (AJPBDS) and a right-sided round ligament, misdiagnosed preoperatively as advanced gastric cancer with pancreatic head invasion. A 72-year-old woman presented with chest pain, but laboratory data showed only anemia. Gastroscopy revealed a bleeding polypoid gastric tumor in the anterior wall of the stomach, herniating into the duodenum (ball-valve syndrome), and a Bormann type-2 tumor in the posterior wall. Ultrasonography showed gallbladder stones, dilatation of the intrahepatic bile duct and pancreatic duct, and a left-sided gallbladder (attributed to a right-sided round ligament with anomalous branches of the portal veins). Laparotomy revealed that the gastric tumors were not advanced cancer invading the pancreatic head. Intraoperative cholangiography showed an AJPBDS, causing dilatation of the intrahepatic bile duct and pancreatic duct. We performed distal gastrectomy and cholecystectomy without biliary diversion. Microscopy revealed that the polypoid tumor was a hyperplastic polyp.