Laminin-332 promotes the invasion of oesophageal squamous cell carcinoma via PI3K activation.

Laminin-332 is major component of epithelial basement membrane, and has an important role in cell migration and tumour invasion. Recently, the phosphatidylinositol 3-kinase (PI3K) activation induced by laminin-332 during carcinogenesis or tumour invasion has been highlighted in skin squamous cell carcinoma. The expression of laminin-332 in 126 resected oesophageal squamous cell carcinoma (ESCC) specimens was immunohistochemically examined to determine its associations with the clinicopathological characteristics, and the effect of laminin-332 on the invasiveness and the PI3K activation was assessed by in vitro experiments using ESCC cell lines (ESCCs). Sections with immunostaining signals in >30% cancer cells, which were observed in 55 of 126 cases, were judged to be positive for laminin-332. The positivity was significantly correlated with pTNM stage and poor prognosis. Inactivation of the PI3K pathway by laminin-332 blocking antibody suppressed the invasiveness of TE8 cell line, which secreted laminin-332 at high level and had high PI3K activity. The addition of the purified laminin-332 activated the PI3K pathway and increased the invasiveness of TE11 cell line, which secreted laminin-332 at lower level and had low PI3K activity. The deactivation of PI3K pathway using the PI3K inhibitor decreased the invasiveness of ESCCs and the secretion of laminin-332 in vitro. The expression of laminin-332 was one of the prognostic factors of ESCC. Laminin-332 could provide the autocrine positive-feedback loop through PI3K activation, contributing the invasive ability. Therefore, the inhibitor of PI3K pathway might be useful as the anticancer therapies for ESCC.

[Evaluation of the non-effective cases of irresectable multiple liver metastasis of colorectal cancer receiving hepatic arterial infusion chemotherapy].

PURPOSE: Hepatic arterial infusion (HAI) chemotherapy is one of the suitable therapies for irresectable multiple liver metastasis from colorectal cancer, but in nearly half of such cases the therapy does not prove effective. Our goal is to clarify the characteristics of non-effective cases. METHODS: 84 cases with irresectable multiple liver metastasis from colorectal cancer were investigated clinicopathologically, and were divided into two groups; non-effective cases (N = 38) and effective cases (N = 46). All cases received continuous arterial infusion chemotherapy using 5-FU according to the following regimen; 5-FU (500 mg/day) was infused in the hepatic artery over 7 or 10 days for induction, and the infusion was maintained (250 mg/day) to the hepatic artery for 7 days every other week after the induction therapy. We evaluated the efficacy of HAI chemotherapy by Computed Tomography. RESULTS: There were statistically significant differences among these two groups in histological types. Rates of the histological type of non-effective cases were well (31.6%), mod (57.9%), por (7.9%), and muc (2.6%), respectively. Those of the effective cases were well (63.0%), mod (34.8%), por (0%), and muc (2.2%), respectively. In non-effective cases, 16 out of 38 cases (42.1%) had extra-hepatic metastasis. On the other hand, only 3 out of 46 cases (6.5%) had such metastasis in effective cases. CONCLUSION: There were non-well type cancers and extra-hepatic metastasis in a large number of non-effective cases. We thought that those cases were basically high-grade malignancies, so these were the limits of HAI chemotherapy for irresectable multiple liver metastasis of colorectal cancers.

Enhanced hepatocyte growth factor expression associated with prolonged rat hepatic allograft survival in recipients pretreated with donor-specific blood.

BACKGROUND: Pretransplantation injection of freshly heparinized donor blood (donor-specific blood transfusion, or DST) significantly prolongs the survival of hepatic allografts from ACI(RT1a) to LEW(RT1l) rats. We investigated hepatocyte growth factor (HGF) expression in rat hepatic allografts of recipients pretreated with or without DST. METHODS: The levels of HGF mRNA and protein in hepatic allografts were determined after transplantation. The localization of HGF+ cells was identified with a rat anti-HGF monoclonal antibody. RESULTS: Plasma HGF concentrations in transplanted rats treated with DST were significantly and persistently increased compared to untreated rats with hepatic allografts. The number of HGF+ cells in hepatic allografts of recipients pretreated with DST on day 14 was significantly greater than that in allografts of untreated recipients on day 7. HGF+ cells were also found in the marginal zone and red pulp of recipient spleens. Northern blot analysis revealed the presence of three HGF+ cell phenotypes: HGF+ED1+, HGF+ED2+, and HGF+ED1-ED2-. Most HGF+ cells were ED1-ED2-. In situ hybridization demonstrated HGF mRNA in the mononuclear cells in the portal and sinusoidal areas as well as the marginal zone and red pulp in both DST-treated and untreated recipient spleens. CONCLUSIONS: Enhanced HGF expression in rat hepatic allografts is associated with immunologic unresponsiveness induced by DST.

Tumor necrosis factor-beta is associated with thymic apoptosis during acute rejection.

BACKGROUND: Intrathymic events undergoing allograft rejection remain undefined. The present study investigated the role of tumor necrosis factor-beta on acute thymic involution in rat hepatic allograft recipients during rejection. METHODS: Apoptosis and cellular phenotypic changes in the thymus were studied after hepatic transplantation. RESULTS: Thymocytes in both the medulla and cortex were sparse during acute rejection. Phenotypically, CD4+CD8+ T cells decreased significantly, whereas there were relative increases in CD4-CD8-, CD4+CD8-, and CD4-CD8+ T cells in untreated allograft recipients. Additionally, thymic apoptosis was found by in situ DNA end labeling and electron microscopy. Apoptotic cells were predominantly distributed in the cortex. Biologic lymphotoxin (tumor necrosis factor-beta)/tumor necrosis factor-alpha cytotoxic activity in the serum was significantly increased in untreated hepatic allograft recipients. Tumor necrosis factor-beta mRNA was detected in untreated allograft livers, and intraperitoneal administration of recombinant human tumor necrosis factor-beta induced extensive apoptosis of thymocytes in vivo. In contrast, no significant thymic involution was observed in donor-specific blood transfusion-treated allograft and isograft recipients. Intraperitoneal administration of rabbit anti-human tumor necrosis factor-beta polyclonal antibody or recombinant human interleukin-10 inhibited thymic apoptosis in untreated hepatic allograft recipients. CONCLUSIONS: Allograft rejection, but not donor-specific transfusion-induced immunologic unresponsiveness, is associated with thymic involution, a process that may be mediated by tumor necrosis factor-beta.

Infiltrating CD45RC- T cells are associated with immunologic unresponsiveness induced by donor class I major histocompatibility complex antigens in rats.

It has previously been shown that a single intravenous injection of freshly heparinized donor-specific blood transfusion (DST) before transplantation significantly prolongs the survival of fully allogeneic ACI (RT1a)-to-LEW(RT1(1)) rat hepatic allografts. Additionally, we have shown that pretreatment of LEW rats with PVG.r1 blood, which shares only the RT1.A major histocompatibility complex (MHC) region with ACI, significantly prolongs the survival of ACI hepatic allografts. In this study, we report the cellular identity of hepatic allograft leukocyte infiltrates following transplantation. Fluorescence-activated cell sorting (FACS) analysis revealed that CD4+ T cells infiltrating liver allografts could be divided into two subsets, CD45RC- CD4+ and CD45RC+ CD4+ T cells, and that the ratio of CD45RC- CD4+/CD45RC+ CD4+ T cells was significantly higher in hepatic allografts of recipients pretreated with DST or PVG.r1 blood as compared to untreated allografts. Further, CD8+ T cells that accumulated in the liver grafts could be similarly divided into two subsets, and the ratio of CD45RC- CD8+/CD45RC+ CD8+ T cells was also significantly higher in hepatic allografts of recipients pretreated with DST or PVG.r1 blood. Reverse transcriptase polymerase chain reaction (RT-PCR) analysis revealed that CD45RC- CD4+ T cells harvested from hepatic allografts pretreated with PVG.r1 blood expressed interleukin-4 (IL-4) and interleukin-10 (IL-10), but not interleukin-2 (IL-2) or interferon-gamma (IFN-gamma). In contrast, CD45RC- CD8+ T cells from hepatic allografts pretreated with PVG.r1 blood expressed IL-4, IL-10, and IFN-lambda, but not IL-2. These results indicate that the CD45RC leukocyte common antigen could be used to differentiate CD4+ and CD8+ T cells following pretreatment with DST or PVG.r1 blood. Persistent infiltration of CD45RC- CD4+ and CD45RC- CD8+ T cells, capable of secreting Th2-type cytokines may prevent allograft rejection by causing immunologic unresponsiveness.

A significant reduction of macrophages expressing inducible nitric oxide synthase in rat hepatic allografts pretreated with donor-specific blood.

BACKGROUND: A single intravenous injection of donor-specific blood (DST) 7 days before transplantation significantly prolongs survival of hepatic allografts from fully allogeneic ACI(RT1a)-->LEW(RT1(1)) rats. The aim of this study was to investigate the kinetics of nitric oxide synthesis by macrophages in rat hepatic allografts treated with DST. METHODS: We investigated macrophages expressing inducible nitric oxide synthase in animal group I (receiving isografts), group II (hepatic allografts), and group III (hepatic allografts after donor-specific blood). RESULTS: Serum nitrite/nitrate, interferon-gamma, and tumor necrosis factor-alpha concentrations increased significantly in group II for 7 days after transplantation but were significantly much lower in groups I and III. Numbers of macrophages immunostained with an anti-macrophage nitric oxide synthase monoclonal antibody and inducible nitric oxide synthase mRNA levels in liver specimens also were much lower in groups I and III than in group II. In addition, Northern blot analysis demonstrated abundant interleukin-10 mRNA transcripts in the DST-treated hepatic allografts compared to untreated allografts. Double immunostaining revealed anti-macrophage synthase-containing cells, including both ED1+ and ED2+ cells, in liver and spleen as more numerous in group II. CONCLUSIONS: Inducible nitric oxide synthase is suppressed in immunologic unresponsiveness to grafts after donor-specific blood transfusion.

Phenotype and localization of macrophages expressing inducible nitric oxide synthase in rat hepatic allograft rejection.

BACKGROUND: We investigated the phenotype and localization of macrophages expressing inducible nitric oxide synthase (iNOS) in rat hepatic allografts, using double immunostaining with anti-macrophage iNOS (macNOS) and rat anti-macrophage (ED1 or ED2) monoclonal antibodies. METHODS: The animals were divided into three experimental groups: group 1, isografts; group 2, untreated hepatic allografts; and group 3, hepatic allografts treated with FK506. RESULTS: Plasma nitrite/nitrate concentrations in group 2 increased on day 3, peaked on day 5, and decreased thereafter. In contrast, the plasma nitrite/nitrate concentrations in group 1 increased slightly on day 3, but decreased gradually thereafter. The plasma concentrations of nitrite/nitrate did not vary in group 3. The peak nitrite/nitrate values in group 2 were significantly greater than those in groups 1 and 3. The number of macNOS+ cells peaked on day 5 in group 2. In contrast, a few macNOS+ cells were seen in the liver grafts of groups 1 and 3. Double immunostaining revealed that the macNOS+ cells consisted of macNOS+ ED1+ (80%) and macNOS+ ED2+ (40%) in the untreated hepatic allografts on day 5. In addition, a number of macNOS+ cells also were seen in the red pulp of the recipient spleen in the untreated hepatic allografts. CONCLUSIONS: These results suggest that the intense iNOS expression by the monocyte/macrophage lineage among the hepatic infiltrates and by the splenic macrophages after transplantation supports a role for nitric oxide in the immunomodulation of allogeneic responses in local and remote organs, and possibly serves as a mediator of cytotoxic graft damage.

Persistent infiltration of CD45RC- CD4+ T cells, Th2-like effector cells, in prolonging hepatic allografts in rats pretreated with a donor-specific blood transfusion.

A single intravenous injection of freshly heparinized blood from a donor-specific blood transfusion (DST) seven days before transplantation significantly prolongs the subsequent survival of hepatic allografts from ACI(RT1a) to LEW(RT1(1)) rats. We used W3/25 (anti-CD4) and OX22 (anti-CD45RC: an isoform of leukocyte-common antigen [CD45R]) monoclonal antibodies to investigate the cellular identity of hepatic allograft infiltrates following transplantation. The number of CD4+ and CD45RC+ cells in untreated allografts increased equally over time by day seven. However, in DST-treated hepatic allografts, CD4+ and CD45RC+ cells also increased over time by day 14, but the increment in the number of CD4+ cells was significantly greater than that in CD45RC+ cells. While the number of CD4+ cells remained persistently elevated in the hepatic allografts of rats pretreated with DST, they did not initiate rejection. Fluorescence-activated cell sorter (FACS) analysis revealed that the accumulated CD4+ T cells could be divided into two subsets, CD45RC- CD4+ and CD45RC+ CD4+ T cells, and that the ratio of CD45RC- CD4+/CD45RC+ CD4+ T cells in the hepatic allografts of recipients pretreated with DST was significantly greater than that in untreated allografts. Reverse-transcriptase polymerase chain reaction (RT-PCR) analysis demonstrated that CD45RC- CD4+ T cells expressed interleukin (IL)-4 and IL-10 messenger RNA (mRNA), but not IL-2 and interferon gamma (IFN-gamma). The pattern of messenger RNA expression in hepatic allograft infiltrates from animals pretreated with DST provides compelling evidence for the selective in vivo preservation of T-helper (Th2)-specific cytokines in the rat system. Our studies show that CD45RC leukocyte-common antigen expression can define different populations of hepatic infiltrating CD4+ T cells. A persistent infiltration of CD45RC- CD4+ T cells, Th2-like effector cells, is characteristic of hepatic allografts with a prolonged survival in DST-pretreated rats.

Recombinant human hepatocyte growth factor facilitates biliary transport after hepatocyte transplantation in Eisai hyperbilirubinemic rats.

Hepatocyte transplantation may offer an attractive treatment for inborn errors of liver metabolism. However, factor(s) are required as stimuli to induce proliferation of the limited number of hepatocytes transplanted. The Eisai hyperbilirubinemic rat (EHBR) is a Sprague-Dawley (SD) mutant rat with conjugated hyperbilirubinemia. EHBRs have impaired canalicular excretory transport of organic anions, bile acid glucuronide, and sulfate. Recombinant human hepatocyte growth factor (rhHGF) (100 microg/kg) was injected intravenously at 2-hr intervals for 10 hr, immediately and 35 days following the intraportal injection of 1 x 10(7) wild-type SD rat hepatocytes. Serum bilirubin concentrations decreased significantly within 35 days and were maintained at significantly reduced levels for 120 days following transplantation. Biliary excretion was demonstrated by the biliary transport of indocyanine green and sulfobromophthalein sodium into the bile. These results indicate that hepatic transport of bile acid conjugates in EHBRs can be restored by hepatocyte transplantation combined with repeated administration of exogenous rhHGF, in conjunction with functioning of the recipient's excretory biliary system.

Neutrophil elastase inhibitor reduces neutrophil chemoattractant production after ischemia-reperfusion in rat liver.

BACKGROUND & AIMS: Neutrophils are important in the development of tissue injury induced by ischemia-reperfusion. The ability of an inhibitor of neutrophil elastase (ONO-5046) to protect against ischemia-reperfusion injury in rat liver was investigated by measuring serum concentrations of cytokine-induced neutrophil chemoattractant. METHODS: Liver ischemia was induced in rats by occluding the portal vein for 30 minutes, and ONO-5046 or anticoagulants were injected intravenously 5 minutes before vascular clamping. RESULTS: Serum concentration of cytokine-induced neutrophil chemoattractant increased after reperfusion, reached a maximum at 6 hours, and then gradually decreased. However, pretreatment of animals with heparin (50 U/kg), antithrombin III (250 U/kg), or ONO-5046 (10 mg/kg) resulted in significantly smaller increases in the serum concentration of cytokine-induced neutrophil chemoattractant after reperfusion. Pretreatment with both ONO-5046 and heparin, or both ONO-5046 and antithrombin III, produced additive effects. Pretreatment of rats with both ONO-5046 and heparin or both ONO-5046 and antithrombin III also inhibited the increase in cytokine-induced neutrophil chemoattractant mRNA in liver. These combined treatments significantly reduced the increases in both the number of neutrophils accumulated in the liver and the hepatic activity of myeloperoxidase. CONCLUSIONS: Cytokine-induced neutrophil chemoattractant production after ischemia-reperfusion in the liver is mediated by neutrophil elastase and activation of coagulation within the hepatic microcirculation.

Kupffer cell production of cytokine-induced neutrophil chemoattractant following ischemia/reperfusion injury in rats.

We investigated the role of hepatic macrophages in the inflammatory response following reperfusion injury by blocking Kupffer cell phagocytosis with gadolinium chloride (GdCl3). Liver ischemia was induced in rats by occluding the portal vein for 30 minutes. A bolus of GdCl3 (7 mg/kg) was injected intravenously 1 and 2 days before surgery. The serum levels of cytokine-induced neutrophil chemoattractant (CINC) in untreated rats increased following reperfusion, peaked after 6 hours, and then gradually decreased. GdCl3 or heparin alone significantly decreased the serum levels of CINC (P < .05). In addition, pretreatment with GdCl3/heparin further inhibited the rise in the serum levels of CINC following reperfusion compared with those in untreated animals (P < .01). The in vitro production of CINC by Kupffer cells, obtained from animals pretreated with heparin or GdCl3, was significantly lower than that of cells isolated from untreated animals. Pretreatment with GdCl3/heparin further decreased CINC production by Kupffer cells compared with that of cells from animals that were pretreated with heparin or GdCl3 alone. The expression of CINC transcripts in Kupffer cells or in liver tissue peaked 3 hours after reperfusion in untreated animals. Pretreatment with heparin, GdCl3, or both significantly decreased the levels of CINC messenger RNA (mRNA) transcripts. Pretreatment with heparin, GdCl3, or GdCl3/heparin significantly decreased the number of neutrophils that accumulated in the liver 24 hours following reperfusion, compared with those in untreated animals. These results suggest that Kupffer cells release CINC and may play an important role in early neutrophil infiltration into the liver following ischemia/reperfusion.

Technique for orthotopic reduced-size hepatic transplantation combined with ex vivo liver cut down in the rat.

A technique is described for orthotopic reduced-size hepatic transplantation combined with ex vivo liver cut down in the rat. Following perfusion of the donor liver with cold heparinized saline, the portal veins, bile ducts, and hepatic arteries to the median and left lobes together were dissected in situ, encircled, and divided. After harvesting the donor liver, a hepatectomy was performed by ex vivo liver cut down of the median and left lobes. The remnant amounted to 32% of the whole liver. As a result, the suprahepatic vena cava could be well visualized with adequate exposure for vascular anastomosis. Orthotopic reduced-size hepatic transplantation was performed using the right and caudate lobes of the liver. The suprahepatic vena cava was anastomosed with a 7-0 silk running suture. A simplified cuff without processes was made with an obliquely cut polyethylene tube and used for the portal and infrahepatic caval anastomoses. A Teflon tube stent was used for the biliary anastomosis. The newly devised angled clamp and flexible arm were used for the cuff attachment and operative procedure. Transplant survival following ex vivo liver cut down was as good as that with whole liver transplantation. Reestablishment of the hepatic artery restores liver function following transplantation. The maximum hepatocyte labeling index (LI) occurs 24 hr after a 68% hepatectomy, and at 36 hr following a reduced-size hepatic transplantation with or without hepatic arterialization. Possible explanations for the slight delay in achieving the maximal LI may include damage that is induced by the operation itself, pregraft preservation, and reperfusion injuries. In conclusion, the anatomical features of the hepatic lobes in rats are well suited to successful completion of ex vivo liver cut down.

Neutrophil elastase inhibitor (ONO-5046) decreases cytokine-induced neutrophil chemoattractant after reperfusion of pancreaticoduodenal transplantation in rats.

The protective effects of a neutrophil elastase inhibitor (ONO-5046) on reperfusion injury following pancreaticoduodenal transplantation in rats were studied by measuring serum concentrations of cytokine-induced neutrophil chemoattractant (CINC). Male Wistar rats were transplanted with syngeneic pancreaticoduodenal grafts. ONO-5046 was injected intravenously 5 min before vascular clamping and immediately after reperfusion at a dose of 10 mg/kg. No significant differences were observed in the peak serum concentrations of amylase between the groups treated with and treated without ONO-5046. The serum lipase concentrations in the untreated animals increased and peaked 3 hr after reperfusion. ONO-5046 significantly decreased the peak serum lipase concentration. The serum CINC concentrations, which were determined by enzyme-linked immunosorbent assay, increased and peaked 3 hr after reperfusion, decreasing gradually thereafter. However, pretreatment with ONO-5046 significantly inhibited the rise in serum CINC concentrations after reperfusion. Expression of CICN transcripts in the pancrease grafts was evaluated by Northern blot analysis and peaked 3 hr after reperfusion in untreated animals. Pretreatment with ONO-5046 also significantly inhibited the expression of CINC mRNA transcripts in the graft. ONO-5046 significantly decreased the number of neutrophils accumulated in the pancreas graft 24 hr after transplantation. In vitro CINC production by peritoneal macrophages was increased by neutrophil elastase in dose-dependent fashion. However, ONO-5046 decreased CINC production by peritoneal macrophages in response to neutrophil elastase. These results suggest that ONO-5046 prevents early neutrophil accumulation in the pancreas following ischemia/reperfusion of pancreaticoduodenal transplantation.

Prolonged survival of rat hepatic allografts pretreated with a single donor-specific blood transfusion: the distribution of donor cells expressing class I major histocompatibility complex antigens in the recipient.

We previously reported that a pretransplant transfusion of either ACI strain rat donor blood or PVG.r1 strain blood, which shares only the RT1.A class I major histocompatibility complex (MHC) region with an ACI donor, significantly prolonged the survival of ACI-to-LEW rat hepatic allografts, suggesting that the class I MHC antigens can be immunosuppressive in rat hepatic allografts. The distribution of the donor cells expressing RT1.Aa class I MHC antigens in the recipients was investigated using a MN4-91-6 mouse anti-rat class I (RT1.Aa) MHC monoclonal antibody. The donor class I MHC-positive cells accumulated mainly in the splenic white pulp and lymph nodes at 12 and 24 hr after blood transfusion, while very few cells were seen in the thymus, liver, lungs, and kidneys. The number of cells began to decrease in the splenic white pulp and lymph nodes at 24 hr after transfusion. This may indicate the destruction of donor cells by the recipient cells. Within 48 hr after transfusion, most cells disappeared from the recipient tissue. In an attempt to study the role of the spleen in inducing immunological unresponsiveness, a splenectomy was performed at the time of transplantation and this abrogated the prolongation of hepatic allograft survival in the recipients which received the donor blood. These findings suggest that the presence of class I MHC-positive cells in the splenic white pulp, a T-dependent area, may play an important role in inducing immunological unresponsiveness.

Suppression of hepatic allograft rejection in the rat by mitomycin C-treated donor splenocytes: in situ splenic distribution of donor class I major histocompatibility complex antigen-positive cells in the recipient.

A single intravenous injection of 3 x 10(6) donor splenocytes treated with mitomycin C (MMC) 7 days before hepatic transplantation prolongs survival of hepatic allografts in the ACI(RT1a) to LEW(RT1(1)) rat combination. This effect is donor specific. The in situ distribution in the recipient of the donor cells administered preoperatively was investigated using intracellularly fluorescence-labeled donor splenocytes. The donor cells were accumulated mainly in the splenic white pulp and lymph nodes at 12 and 24 hr after injection. Contrarily, very few cells were seen in the thymus, liver, kidney, and lung. The number of cells with dull and weak fluorescence began to increase in the splenic white pulp and lymph nodes at 24 hr after injection. This may indicate the breakdown of donor cells by recipient cells. In contrast, a number of donor cells could be detected even after 48 hr and a few cells at 7 days after splenocyte injection in the LEW-to-LEW isogeneic combination. As we previously revealed the role of class I major histocompatibility complex (MHC) antigens in prolonging hepatic allograft survival in the rat, the splenic distribution of donor class I MHC-positive cells in the recipient after intravenous administration of MMC-treated donor splenocytes was studied using immunostaining with a MN4-91-6 mouse anti-rat RT1.Aa class I MHC monoclonal antibody. The donor class I-positive cells accumulated mainly in the splenic white pulp at 12 and 24 hr after injection. This is similar to that observed in the fluorescence study. Within 48 hr after injection, most cells had disappeared from the recipient tissue. These findings suggest that the splenic white pulp, a T-dependent area, may play an important role in inducing immunological unresponsiveness.