Eiji Nozato

Radiation-Associated Rectal Cancer: Report of Four Cases

Background/Aims: Radiation-associated rectal cancer is a remarkable clinical entity. We demonstrate 4 patients (mean age 68 years, range 63–74) who had undergone pelvic radiotherapy for cervical cancer. We indicate some characteristics of radiation-associated rectal cancer. Results: Two patients had received intracavitary and external pelvic radiotherapy, while the remaining 2 had external pelvic radiotherapy following hysterectomy. The mean total radiation dose was 63 Gy, though radiation dose information was not available for 1 patient. Colorectal cancer developed at a mean time of 20.7 years (range 11–30) after radiation therapy. All patients presented with chronic radiation colitis, and 3 demonstrated abnormal tumor markers. Colonoscopy revealed an ulcerative, localized well-differentiated adenocarcinoma of the rectosigmoid colon in 1 patient, and diffusely infiltrating cancers of the lower rectum, one signet-ring cell carcinoma and two mucinous carcinomas in the remaining 3. One case was stage I, 2 were stage IIIa, and the remaining case was stage IV. Three patients underwent abdominoperineal resection. The remaining patient was felt to be inoperable. The colorectal wall demonstrated the changes of chronic radiation injury. Two patients died within a short time because of their advanced cancers. Conclusion: Radiation-associated rectal cancer has a tendency to be diagnosed in the advanced stage and to have a poor prognosis. A literature review and our case report suggest that since there are no reliable clinical or laboratory indicators of the presence of a curable colorectal cancer in the setting of chronic radiation proctocolitis, surveillance with a colonoscope should be done 10 years after irradiation in patients with previous pelvic radiotherapy.

Improved hepatic microcirculation by human soluble urinary thrombomodulin in the xeno-perfused porcine liver

Purpose. Both the protein C/thrombomodulin system and the heparin/anti-thrombin III system are major physiological anticoagulant systems, which may also play a major role in preserving the hepatic microcirculation in xenogeneic liver transplantation. To compensate for the functional incompatibilities of the porcine thrombomodulin (TM)-cofactor activity beyond species for human thrombin, soluble human TM protein was tested in xenogeneic perfusion of the porcine liver. Materials and Methods. The livers were harvested from adult female pigs and perfused through the portal vein (PV) and hepatic artery (HA) for 2 hr, with fresh human blood in group 1 (n=5), fresh porcine blood (10 units/ml) in group 2 (n=5), and fresh human blood with TM (50,000 units/1.5 l) in group 3 (n=5). The tissue PO2 level, tissue blood flow, PV and HA pressures were all continuously monitored. Circulating perfusate and liver tissue samples were periodically obtained for blood chemistry and histologic analyses. Results. The activated protein C (aPC) level was significantly elevated in the TM-treated group 3 (47.5%±3.5% at preperfusion and 51%±2.8% after 120 min of perfusion) in comparison to group 1 (32.3%±7.2% and 35.3±12.0%). The hepatocyte enzyme release of aspartate aminotransferase (AST) was suppressed significantly more in group 3 (238.2±107 IU/l), than in group 1 (672.3±160 IU/l) at 2 hr after reperfusion. In group 3, the tissue PO2 levels and tissue blood flow also remained significantly higher throughout the perfusion. The platelet counts in the perfusate remained significantly higher in group 3 (37.1% to 74.3% of the preperfusion level) than in group 1 (4.4% to 14.7%), after 0 to 80 min of perfusion. According to the histologic findings, the degree of interlobular hemorrhaging and congestion decreased remarkably more in group 3 than in group 1. Conclusion. These findings thus indicated that soluble thrombomodulin protein extracted from human urine remarkably improved hepatic microcirculation in the xenoperfused porcine liver. The thrombomodulin/protein C system might, thus, play an important role in restoring the physiological anticoagulant system in the xenoperfused porcine liver.

Limitations of Exogenous L-Arginine in Exerting a Cytoprotective Effect on Hepatic Ischemia/Reperfusion Injury

Abstract: To test whether or not the L -arginine/nitric oxide (NO) pathway induces a protective effect, we investigated the effect of exogenous L -arginine on hepatic ischemia/reperfusion (I/R) injury, using ex vivo perfusion of the isolated rat liver. The rat liver was removed and preserved in cold saline for 60 min, followed by 120 min of reperfusion with oxygenated perfusate at 37°C. Either 600 mg/kg of L -arginine (groups 1 and 4), D -arginine (group 2), N G -nitro- L -arginine methyl ester ( L -NAME) (group 3), or saline (group 5) were administered through the portal vein starting from 5 min before reperfusion to 5 min after reperfusion. In group 4, 600 mg/kg of L -NAME was preadministered at 10 min prior to the administration of L -arginine. The intrahepatic nitric oxide (NO) levels showed only a temporal elevation (227% ± 70% of the pre-reperfusion levels at 5 min) after reperfusion in group 1. Pretreatment with L -NAME suppressed the elevation of the NO levels immediately after reperfusion in group 4. The lactate dehydrogenase release to the effluent perfusate significantly decreased and the histological findings showed that the sinusoidal damage observed after reperfusion was mitigated in group 1 more than in the other groups. These results thus suggest that exogenous L -arginine produced a relatively small amount of NO and therefore resulted in a slight decrease of hepatic I/R injury.

Adenovirus-mediated gene transfer of triple human complement regulating proteins (DAF, MCP and CD59) in the xenogeneic porcine-to-human transplantation model

In this study, the adenovirus-mediated gene transfer of triple human complement regulating proteins was investigated in xenogeneic pig liver perfusion. The porcine liver was perfused in situ at 4 °C under a pump-driven veno-venous shunt of the portal vein and inferior vena cava, with 5 to 15×1011 plaque-forming units (pfu) of adenovirus vector (group 1: AxCALacZ; 2: AxCACD59; 3: AxCACD59 + AxCADAF; 4: AxCACD59 + AxCADAF + AxCAMCP) for 1 h (for each, n=3). The livers were harvested 24 h after gene transfer and then were reperfused ex-vivo with fresh human blood for 2 h. In immunohistochemical staining, each complement regulating protein (CRP) showed a distribution similar to that of the LacZ expression. The C3 levels in the perfusate were also maintained at higher levels in group 4 from 60 to 120 min after reperfusion (C3: 85% to 95% of the initial level) than in groups 1 to 3 (C3: 80% to 90% of the initial level) from 60 to 120 min after reperfusion. The complement deposition on the porcine liver [C3, membrane attack component (MAC)] decreased significantly more in group 4 than in groups 1 to 3. In conclusion, the adenovirus-mediated multiple gene transfer of human CRPs (hCRPs) was found to effectively suppress the complement activation in xenogeneic pig liver perfusion.

Adenovirus-mediated gene transfer of triple human complement regulating proteins (DAF, MCP and CD59) in the xenogeneic porcine-to-human transplantation model. Part I: in vitro assays using porcine aortic endothelial cells.

We assessed whether the adenovirus-mediated gene transfer of triple human complement regulating proteins (hCRPs) to the porcine aortic endothelium (PAE), could possibly exert a synergistic effect to inhibit human complement activation. Adenovirus vectors, encoding E.Coli β-galactosidase (AxCALacZ), human membrane cofactor protein (MCP) (AxCAMCP), decay-accelerating factor (DAF) (AxCADAF), and CD59 (AxCACD59) were produced by the COS-TPC method. AxCALacZ was transfected to porcine aortic endothelium cells (PAECs) under various multiplicities of infection (MOI) to determine the efficiency of adenovirus-mediated gene transfer by 5-bromo-4-chloro-3-indolyl β-D-galactopyranoside (X-gal) staining. The mRNA expressions of transfected CRPs were examined by reverse transcriptase-polymerase chain reaction (RT-PCR). Cellular damage to the PAEC was assessed by an MTT assay. PAEC was most efficiently transfected with the LacZ gene at 103 MOI/60-min incubation time (89.1%). In all samples transfected with the CRP gene, the corresponding mRNAs were detected in the RT-PCR. In the MTT assay, PAECs co-cultured with 20% human serum, showed the highest cellular viability after gene transfer of triple CRPs (117.7%), when compared with those of marker LacZ, single or double CRPs. The adenovirus-mediated multiple gene transfer of CRPs may thus be an efficient method for suppressing complement activation in the porcine-to-human model of hyperacute rejection.

Expression of anti-apoptotic protein, Bcl-2, in liver regeneration after partial hepatectomy

BACKGROUND Although Bcl-2 is well known to have anti-apoptotic activities in vitro and in vivo, the role of Bcl-2 relating to liver regeneration remains controversial. The aim of this study was to document the effect of Bcl-2 expression on liver regeneration in rats undergoing a partial hepatectomy. MATERIAL AND METHODS Adult male Wistar rats (n = 4/group) at 72 h before undergoing a 70% partial hepatectomy (PH) were administered 1 x 10(9) plaque-forming units of adenovirus vector encoding either human Bcl-2 (group 1) or LacZ (group 2) intravenously and were sacrificed at 0, 12 h, and at 1, 2, 3, 7, 14, and 21 days postoperatively. In group 3, normal saline was injected instead of adenovirus vector. Liver regeneration was monitored by measuring the restituted liver mass and proliferating cell nuclear antigen (PCNA) immunostaining. The incidence of apoptosis in the liver was analyzed by the immunohistochemical detection of single-stranded DNA at 14 and 21 days postoperatively. RESULTS The restituted liver mass showed significantly higher values in group 1 (26.1 +/- 7.2%) than in group 2 (14.7 +/- 6.8%) and 3 (13.6 +/- 5.0%) at 1 day after PH (P < 0.05). The PCNA labeling index was significantly higher in group 1 (47.2 +/- 9.9%) than in groups 2 (19.0 +/- 7.8%) and 3 (19.2 +/- 15.2%) at 1 day after a partial hepatectomy (P < 0.05). The hepatocyte growth factor (HGF) mRNA expression was significantly lower in group 1 than in group 2 at 12 h after PH (P < 0.05). The number of single-stranded DNA-positive cells decreased significantly more in group 1 (5.67 +/- 1.53 positive cells/10 fields per tissue) than those in group 2 (18.33 +/- 7.57 positive cells/10 fields per tissue) at 14 days after PH. CONCLUSIONS These results thus indicated that an overexpression of anti-apoptotic protein Bcl-2 does not necessarily have an anti-apoptotic effect on liver regeneration but appears to have a pro-proliferative effect in the early phase of liver regeneration.