Hiroshi Yahata

Allograft transduction of IL-10 prolongs survival following orthotopic liver transplantation.

Interleukin-10 (IL-10) is an ideal candidate cytokine for suppressing the alloimmune response in transplantation. To determine whether genetic modulation of the hepatic graft with IL-10 could prolong survival following orthotopic liver transplantation, we constructed a replication-deficient adenovirus vector expressing human IL-10 (AdCMVhIL-10). Intraportal injection of this vector into a donor rat 24–48 h before grafting resulted in efficient release of IL-10 into the circulation of a recipient rat after transplantation. Moreover, levels of hIL-10 from the suprahepatic vena cava were significantly (1.48-fold) higher than those from the infrahepatic vena cava (P = 0.013), indicating local IL-10 production within the transduced hepatic graft. AdCMVhIL-10 induced a prolongation of median survival to more than 87 days, with two of five transduced grafts showing more than 100 days of ongoing survival, when compared with 11 days for grafts transduced with a control adenovirus vector carrying the E. coli β-galactosidase gene (P = 0.0021) and 11 days for untreated grafts (P = 0.0021). Pathological findings occurring in the AdCMVhIL-10-transduced hepatic grafts revealed no evidence of progressive rejection reaction resulting in graft failure. These results demonstrate that hepatic grafts modulated by IL-10 gene transfer make local and effective immunosuppression feasible in the transplantation setting.

Concise prediction models of anticancer efficacy of 8 drugs using expression data from 12 selected genes.

We developed concise, accurate prediction models of the in vitro activity for 8 anticancer drugs (5‐FU, CDDP, MMC, DOX, CPT‐11, SN‐38, TXL and TXT), along with individual clinical responses to 5‐FU using expression data of 12 genes. We first performed cDNA microarray analysis and MTT assay of 19 human cancer cell lines to sort out genes which were correlative in expression levels with cytotoxicities of the 8 drugs; we selected 13 genes with proven functional significance to drug sensitivity from a huge number of potent prediction marker genes. The correlation significance of each was confirmed using expression data quantified by real‐time RT‐PCR, and finally 12 genes (ABCB1, ABCG2, CYP2C8, CYP3A4, DPYD, GSTP1, MGMT, NQO1, POR, TOP2A, TUBB and TYMS) were selected as more reliable predictors of drug response. Using multiple regression analysis, we fixed 8 prediction formulae which embraced the variable expressions of the 12 genes and arranged them in order, to predict the efficacy of the drugs by referring to the value of Akaikes information criterion for each sample. These formulae appeared to accurately predict the in vitro efficacy of the drugs. For the first clinical application model, we fixed prediction formulae for individual clinical response to 5‐FU in the same way using 41 clinical samples obtained from 30 gastric cancer patients and found to be of predictive value in terms of survival, time to treatment failure and tumor growth. None of the 12 selected genes alone could predict such clinical responses.

Prolongation of liver allograft survival after interleukin-10 gene transduction 24-48 hours before donation.

BACKGROUND Interleukin- (IL) 10 may be a potent regulator for controlling of allograft rejection. A single administration of IL-10 is not effective for controlling graft rejection. Gene transfer is an attractive vehicle for prolonging the expression of short-lived proteins. METHODS Donor or recipient livers were transduced with 1 x 10(10) p.f.u. of replication-deficient adenovirus vectors harboring human IL-10 cDNA (AdCMVhIL-10) via the ileocecal vein before or after rat orthotopic liver transplantation. RESULTS DA allografts given AdCMVhIL-10 24-48 hr before donation survived for more than 56 days in Lewis recipients, although DA allografts given the adenovirus vector 7 days or 6 hr before, and 3 days after transplantation were rejected within 30 days in recipients. Serum levels of human IL-10 in gene-transferred rats were maximum from day 2 to 7. The serum level of human IL-10 then decreased gradually, and human IL-10 was not detected by ELISA 30 days after gene-transduction. In gene-transduced long-term surviving liver allografts, IL-10 was expressed, and the expression of IL-4 was also up-regulated on posttransplant day 3, despite the expression of Th1 cytokines (IL-2 and interferon-gamma), although in rejected liver allografts, IL-2 and interferon-gamma were expressed without expression of IL-4 and IL-10. CONCLUSIONS The prolongation of survival of IL-10 cDNA transferred liver allografts might be due to inhibition of the early phase of alloimmune-response by over expression of IL-10, despite the expression of IL-2 and interferon-gamma.

A case of choledocholithiasis with an endoclip nidus, 6 months after laparoscopic cholecystectomy

Abstract. A 69-year-old man developed obstructive jaundice 6 months after laparoscopic cholecystectomy. Endoscopic retrograde cholangiography suggested a common bile duct (CD) stone. A second operation was performed, and this revealed a CBD stone with an endoclip as a nidus. Since laparoscopic surgery has become a very common procedure, endoclips are used more frequently. Therefore, careful surveillance and strict follow-up are stressed to avoid CBD stone and various other complications caused by endoclips.

Auxiliary Heterotopic Partial Liver Transplantation in Pigs with Acute Liver Failure

Fulminant hepatic failure is usually fatal without liver transplantation; however, orthotopic liver transplantation is often difficult to perform due to the high risk of coagulopathy and the development of multiple organ failure. Auxiliary heterotopic partial liver transplantation (APLT), however, has the potential to provide an effective hepatic support system considering that the host liver is left in situ and the surgical procedure is less invasive. In this report, we describe the beneficial effects of performing 60% APLT on the hepatic function and survival of pigs with acute hepatic failure induced by hepatic artery ligation. The pigs were divided into a control group of nine animals (group 1) that had portal vein and hepatic artery ligation with a side-to-side portacaval shunt, and an APLT group of seven animals (group 2) that had portal vein and hepatic artery ligation with APLT. The two left lateral lobes of the donor liver were resected, reducing the liver weight to about 60%, and the graft was placed in the right subhepatic space. No deaths occurred intraoperatively. In group 1, eight pigs died of massive liver necrosis within 48 h and one died between 48 and 72 h (median surivival 23 h). In group 2, two pigs died within 72 h due to preservation or anesthetic problems, but five survived for more than 3 days (median survival 13.4 days), with a significant difference between the two groups (P<0.05). One animal was killed 30 days after APLT and excellent graft function was demonstrated by the synthesis of clotting factors, ammonia detoxification, and glucohomeostasis. Moreover, evidence of hepatic regeneration was found in the transplanted livers. These results indicate that APLT provides metabolic support and improves survival in animals with induced acute liver failure.

Augmentation of local antitumor immunity in liver by interleukin-2 gene transfer via portal vein.

Metastasis to the liver remains an important problem in the treatment of patients with gastrointestinal cancer. We examined the mechanism and effect on liver metastasis of in vivo interleukin-2 (IL-2) gene transfer to the liver. RCN-9 cells derived from F344 rat colon adenocarcinoma were injected into syngeneic rats via the ileocecal vein to induce liver tumors. A total of 2.5×109 pfu of adenovirus vector harboring the human IL-2 gene (AdCMVhIL-2), or 2.5×109 pfu of control vector encoding β-galactosidase was administered before RCN-9 cell challenge. On day 14, mean tumor weight was 4.0±2.4 g in the control group, whereas IL-2–transduced livers had no tumors. Survival of AdCMVhIL-2–treated rats was significantly longer than that of control rats (P<.01). Flow cytometry demonstrated that the proportion of natural killer (NK) cells had increased among sinusoidal cells collected from IL-2–transduced livers. These cells were highly cytotoxic to RCN-9 cells in vitro in the presence of a physiological high concentration of recombinant IL-2. Preventative effects of IL-2 transduction of the liver against liver metastasis were lost after depletion of NK cells by treatment with anti–asialo GM1 antibodies. Our results indicate that IL-2 gene transfer to the liver prevents liver metastasis by continuously providing physiological high concentrations of IL-2 in the liver, thereby activating sinusoidal NK cells.

Mechanism of suppression of cloned human suppressor T cells

Abstract We report the mechanism of suppression of suppressor T cell clone III‐1‐C5 using helper T cell clone III‐1‐B6, mitogen responses and rIL‐2. Clone III‐1‐C5 suppressed the mixed lymphocyte reaction (MLR) by secreting alloantigen non‐pecific, MHC non‐restricted suppressor factor(s). Clone III‐1‐C5 did not suppress mitogen (PHA, Con A, PWM) response nor proliferation by exogeneous rIL‐2. Clone III‐1‐C5 suppressed proliferation by clone III‐1‐B6, which augments proliferation by direct cell to cell contact with responder cells and not by soluble factors. These results indicated that suppressor T cells exhibit suppressive effects not only by inhibiting IL‐2 synthesis but by inhibiting the direct effects of helper T‐cells.

Induction of natural suppressor-like cells from human adult peripheral blood lymphocytes by a K562-derived factor.

K562-AC1, a subclone of the human myeloid leukemia cell line K562, secreted an inhibitor of PHA-stimulated human T cell growth into the culture supernatant (K562sup). Kinetics and absorption assays in vitro revealed that the factor acts on PBMC during the very early growth phase. PBMC gained potent suppressor activity against autologous T cell growth after preculture with K562sup for 3 days in a dose-related manner. HLA class II negative and nylon wool-nonadherent subsets possessed higher suppressor potential than unfractionated PBMC. Treatment of K562sup-precultured PBMC with mAb followed by complement-Iysing showed that the responsible suppressors belong to null cells expressing CD3-, CD4-, CD8-, CD19-, CD14-, CD11b-, but partially CD2+. Exogenous IL-2 exhibited a synergistic effect on the activity of the suppressors committed by K562sup. However, K562sup inhibited IL-2-activated lymphocytes from generating anti-K562 cytolytic activity. Macrophages inhibited the induction of the suppressors, which was restored by adding indomethacin and/or IL-2. In conclusion, K562sup has the ability to induce efficient suppressor cells from human adult PBL, which belong to null cells without NK/lym-phocyte-activated killer activity and are similar to natural suppressor cells.

A new method for laparoscopic percutaneous tube gastrostomy with a single 1-Cm-long incision for patients with esophageal obstruction

Since the introduction of percutaneous endoscopic gastrostomy (PEG) by Gauderer and Ponsky in 1981, this method of gastrostomy has been widely used because of its small degree of invasiveness. In the original technique, the gastrostomy tube was delivered from the mouth into the stomach by withdrawing the guidewire connected to the tube. Russel and colleagues modified this method and inserted the gastrostomy tube into the stomach directly from the skin over the guidewire. Both methods require the use of a gastrofiberscope, which cannot be used in patients with esophageal obstruction. Esophageal obstruction is a common symptom of a malignant tumor in the neck, and even a metallic stent cannot maintain patency of the esophagus throughout the patient’s life. For this reason, patients with advanced esophageal cancer require prophylactic PEG before complete esophageal obstruction or else they must undergo open gastrostomy, which usually requires an incision of 5 to 8 cm in length. Laparoscopic tube gastrostomy was first reported in 1990 as a method that can be used in patients with esophageal obstruction, but this method has not become popular because it is complicated and requires multiple wounds. We describe here a new method for laparoscopic percutaneous tube gastrostomy (LPTG) with a single 1-cmlong incision for patients with esophageal obstruction. METHODS Patients LPTG was performed in seven patients with esophageal obstruction caused by malignant neck tumors, such as laryngeal, thyroid, and esophageal tumors.

Inhibition of IL‐2 synthesis by donor‐pecific suppressor T cells in a renal transplant recipient

Abstract A study was conducted to elucidate the mechanism of donor‐pecific Mixed Lymphocyte Reaction (MLR and Cell Mediated Lymphotoxicity (CML) unresponsiveness in a renal transplant recipient with a long‐term well‐functioning kidney. The peripheral blood lymphocytes (PBL) of the recipient, who had not shown rejection since his transplantation 5 years previously, and those of his mother (donor), his father and two healthy third parties were examined. MLR, CML, semimicro MLR in a double chamber, interleukin‐2 (IL‐2) synthesis assay and limiting dilution assay were performed. This recipient showed donor‐pecific MLR and CML unresponsiveness. IL‐2 assay showed that the PBL of the recipient produced less IL‐2 against the donor than against the father and the third parties. The addition of exogenous recombinant IL‐2 (rIL‐2; Takeda Co.) to the priming MLR caused a recovery of CML against the donor. A limiting dilution assay indicated that cytotoxic T cell precursor (CTLp) frequencies against the donor and father did not differ. The suppressor assay in a double chamber indicated that the PBL of the recipient stimulated by the donor PBL had a non‐pecific suppressive effect on MLR, CML and IL‐2 synthesis of the PBL across the Major Histocompatibility Complex (MHC) barrier. This suppressive effect was abolished by OKT3 or OKT8 monoclonal antibody and complement. Thus, the recipient had donor‐pecific suppressor T cells that produced a humoral non‐pecific suppressive factor only when stimulated by the donor PBL, and this factor suppressed PLR and CML by inhibiting IL‐2 synthesis of the PBL.