Changes in the quality of life score following preoperative chemotherapy in elderly patients with esophageal cancer.

BACKGROUND: Various functions in elderly patients with esophageal cancer deteriorate easily and their quality of life can be adversely affected by treatment. The age groups covered in previous studies are wide, and the impact on the elderly individuals is unknown. This study examined changes in quality of life scores after preoperative chemotherapy to clarify aspects of physical, psychological, and social quality of life in elderly patients with esophageal cancer. METHODS: Thirty-six patients aged over 65 years, who were scheduled to undergo preoperative chemotherapy for esophageal cancer surgery, were enrolled. The survey questionnaire comprised the EORTC QLQ-C30 Japanese Language Version, EORTC QLQ-OES 18 Japanese Language Version, and G8. The surveys were conducted before chemotherapy (pre-CT) and after chemotherapy (post-CT). RESULTS: In the functional scale of QLQ-C 30, physical functioning decreased significantly, while emotional functioning increased significantly post-CT (p = 0.021, p = 0.030, respectively). Global health status was not changed. In QLQ-OES18, the mean symptom scale score decreased significantly for dysphagia, trouble swallowing saliva, choking, eating, reflux, and pain post-CT (p = 0.014, p = 0.034, p = 0.033, p = 0.022, p = 0.026, p = 0.016, respectively). The mean G8 score decreased significantly from 11.7 to 10.7 (p = 0.022) post-CT, but the proportion of patients with dysfunction decreased. CONCLUSIONS: Quality of life scores of elderly patients with esophageal cancer who received preoperative chemotherapy decreased in terms of physical function but improved in terms of esophageal cancer symptoms and mental function. Our results suggest that alleviation of symptoms contributed to the improvements in mental health.

Long-Term Outcomes of Living-Donor Liver Transplantation for Primary Biliary Cirrhosis: A Japanese Multicenter Study.

The factors that influence long-term outcomes after living-donor liver transplantation (LDLT) for primary biliary cirrhosis (PBC) are not well known. Compared with deceased-donor transplantation, LDLT has an increased likelihood of a related donor and a decreased number of human leukocyte antigen (HLA) mismatches. To clarify the effects of donor relatedness and HLA mismatch on the outcomes after LDLT, we retrospectively analyzed 444 Japanese patients. Donors were blood relatives for 332 patients, spouses for 105, and "other" for 7. The number of HLA A-B-DR mismatches was none to two in 141, three in 123, and four to six in 106 patients. The 15-year survival rate was 52.6%, and PBC recurred in 65 patients. Recipient aged 61 years or older, HLA mismatches of four or more (maximum of six), graft:recipient weight ratio less than 0.8, and husband donor were adverse indicators of patient survival. IgM 554 mg/dL or greater, donor-recipient sex mismatch, and initial immunosuppression with cyclosporine were significant risks for PBC recurrence, which did not affect patient survival. In subgroup analysis, conversion to cyclosporine from tacrolimus within 1 year diminished recurrence. Prospective studies are needed to determine the influence of pregnancy-associated sensitization and to establish an optimal immunosuppressive regimen in LDLT patients.

Practices for Supporting and Confirming Decision-Making Involved in Kidney and Liver Donation by Related Living Donors in Japan: A Nationwide Survey.

This nationwide survey investigated the actual practices for supporting and confirming the decision-making involved in related living-organ donations in Japan, focusing on organ type and program size differences. Answers to a questionnaire survey were collected from 89 of the 126 (71%) kidney and 30 of the 35 (86%) liver transplantation programs in Japan that were involved in living-donor transplantations in 2013. In 70% of the kidney and 90% of the liver transplantation programs, all donors underwent "third-party" interviews to confirm their voluntariness. The most common third parties were psychiatrists (90% and 83%, respectively). Many programs engaged in practices to support decision-making by donor candidates, including guaranteeing the right to withdraw consent to donate (70% and 100%, respectively) and prescribing a set "cooling-off period" (88% and 100%, respectively). Most donors were offered care by mental health specialists (86% and 93%, respectively). Third parties were designated by more of the larger kidney transplant programs compared with the smaller programs. In conclusion, the actual practices supporting and confirming the decision to donate a living organ varied depending on the organ concerned and the number of patients in the program.

Impact of rituximab desensitization on blood-type-incompatible adult living donor liver transplantation: a Japanese multicenter study.

We evaluated the effects of rituximab prophylaxis on outcomes of ABO-blood-type-incompatible living donor liver transplantation (ABO-I LDLT) in 381 adult patients in the Japanese registry of ABO-I LDLT. Patients underwent dual or triple immunosuppression with or without B cell desensitization therapies such as plasmapheresis, splenectomy, local infusion, intravenous immunoglobulin and rituximab. Era before 2005, intensive care unit-bound status, high Model for End-Stage Liver Disease score and absence of rituximab prophylaxis were significant risk factors for overall survival and antibody-mediated rejection (AMR) in the univariate analysis. After adjustment for era effects in the multivariate analysis, only absence of rituximab prophylaxis was a significant risk factor for AMR, and there were no significant risk factors for survival. Rituximab prophylaxis significantly decreased the incidence of AMR, especially hepatic necrosis (p < 0.001). In the rituximab group, other B cell desensitization therapies had no add-on effects. Multiple or large rituximab doses significantly increased the incidence of infection, and early administration had no advantage. In conclusion, outcomes in adult ABO-I LDLT have significantly improved in the latest era coincident with the introduction of rituximab.

Long-term outcomes of pediatric living donor liver transplantation in Japan: an analysis of more than 2200 cases listed in the registry of the Japanese Liver Transplantation Society.

The Japanese Liver Transplantation Society (JLTS) was established in 1980 in order to characterize and follow trends in patient characteristics and graft survival among all liver transplant patients in Japan. This study analyzed the comprehensive factors that may influence the outcomes of pediatric patients who undergo living donor liver transplantation (LDLT) by evaluating the largest cohort in the world. Between November 1989 and December 2010, 2224 pediatric patients underwent LDLT in Japan. There were 998 male (44.9%) and 1226 female donors (55.1%) without donor mortalities related to transplant surgery. There were 946 male (42.5%) and 1278 female (57.5%) recipients with a median age of 4.0 years (range: 13 days to 17.9 years). Cholestatic liver disease was the leading indication for LDLT (n = 1649; 76.2%), followed by metabolic disorders (n = 194; 8.7%), acute liver failure (n = 192; 8.6%) and neoplastic liver disease (n = 66; 3.0%). The 1-, 5-, 10- and 20-year patient survival rates were 88.3%, 85.4%, 82.8% and 79.6%, respectively. Blood-type incompatibility, recipient age, etiology of liver disease and transplant era were found to be significant predictors of overall survival. We are able to achieve satisfactory long-term pediatric patient survival outcomes in the JLTS series without compromising the living donors.

MicroRNA-21 induces resistance to the anti-tumour effect of interferon-α/5-fluorouracil in hepatocellular carcinoma cells.

BACKGROUND: We reported recently the clinical efficiency of interferon (IFN)-α/5-fluorouracil (5-FU) combination therapy in advanced hepatocellular carcinoma (HCC). However, prediction of the response to the combination therapy remains unsatisfactory. The aim of this study was to investigate the anti-tumour effects of microRNA (miR)-21 on the sensitivity of HCC cells to IFN-α/5-FU and whether miR-21 can be used as a predictor of the response to such therapy in HCC. METHODS: Changes in the sensitivity of HCC cells (PLC/PRF/5 and HepG2) to IFN-α/5-FU were examined after transfection with pre-miR-21 or anti-miR-21. The correlation between miR-21 expression level, evaluated by qRT-PCR, and response to the therapy was also investigated in clinical HCC specimens. RESULTS: Hepatocellular carcinoma cells transfected with pre-miR-21 were significantly resistant to IFN-α/5-FU. Annexin V assay showed that the percentage of apoptotic cells was significantly lower in cells transfected with pre-miR-21 than control cells. Transfection of anti-miR-21 rendered HCC cells sensitive to IFN-α/5-FU, and such sensitivity was weakened by transfection of siRNAs of target molecules, PETN and PDCD4. miR-21 expression in clinical HCC specimens was significantly associated with the clinical response to the IFN-α/5-FU combination therapy and survival rate. CONCLUSIONS: The miR-21 in HCC cell lines and clinical HCC samples is a significant modulator of the anti-tumour effect of IFN-α and 5-FU. This suggests that miR-21 is a potentially suitable marker for the prediction of the clinical response to the IFN-α/5-FU combination therapy.

Insulin-like growth factor-binding protein 7 alters the sensitivity to interferon-based anticancer therapy in hepatocellular carcinoma cells.

BACKGROUND: A striking efficiency of interferon (IFN)-based anticancer therapy for advanced hepatocellular carcinoma (HCC) has been reported. Because its clinical efficiency greatly depends on each patient's local response, prediction of local response is crucial. METHODS: Continuous exposure of IFN-alpha to parental PLC/PRF/5 cells (PLC-P) and a limiting dilution method resulted in the establishment of IFN-resistant cell clones (PLC-Rs). Microarray analyses of PLC-P and PLC-Rs identified insulin-like growth factor-binding protein 7 (IGFBP7) as one of the most significantly downregulated genes in PLC-Rs. Changes in anticancer effects of IFN-alpha were examined in HCC cells after genetic manipulation of IGFBP7 expression. The correlation between immunohistochemically determined IGFBP7 expression and the response to IFN-alpha/5-fluorouracil (5-FU) therapy was investigated in surgically resected HCC specimens. RESULTS: PLC-R cells showed a remarkable downregulation of IGFBP7 and resistance to IFN-alpha, compared with PLC-P. Parental PLC/PRF/5 cells transfected with short hairpin RNA against IGFBP7 showed a significant resistance to IFN-alpha relative to control cells (IC(50) fold increase=14.38 times). Insulin-like growth factor-binding protein 7 transfection into PLC-R restored sensitivity to IFN-alpha. In resected specimens, IGFBP7 expression significantly correlated with the response to IFN-alpha/5-FU therapy. CONCLUSION: IGFBP7 could be a useful predictor of the response to IFN-based therapy in advanced HCC.

Activation of Wnt/beta-catenin signalling pathway induces chemoresistance to interferon-alpha/5-fluorouracil combination therapy for hepatocellular carcinoma.

Type I IFN receptor type 2 (IFNAR2) expression correlates significantly with clinical response to interferon (IFN)-alpha/5-fluorouracil (5-FU) combination therapy for hepatocellular carcinoma (HCC). However, some IFNAR2-positive patients show no response to the therapy. This result suggests the possibility of other factors, which would be responsible for resistance to IFN-alpha/5-FU therapy. The aim of this study was to examine the mechanism of anti-proliferative effects of IFN-alpha/5-FU therapy and search for a biological marker of chemoresistance to such therapy. Gene expression profiling and molecular network analysis were used in the analysis of non-responders and responders with IFNAR2-positive HCC. The Wnt/beta-catenin signalling pathway contributed to resistance to IFN-alpha/5-FU therapy. Immunohistochemical analysis showed positive epithelial cell adhesion molecule (Ep-CAM) expression, the target molecule of Wnt/beta-catenin signalling, only in non-responders. In vitro studies showed that activation of Wnt/beta-catenin signalling by glycogen synthesis kinase-3 inhibitor (6-bromoindirubin-3'-oxime (BIO)) induced chemoresistance to IFN-alpha/5-FU. BrdU-based cell proliferation ELISA and cell cycle analysis showed that concurrent addition of BIO and IFN-alpha/5-FU significantly to hepatoma cell cultures reduced the inhibitory effects of the latter two on DNA synthesis and accumulation of cells in the S-phase. The results indicate that activation of Wnt/beta-catenin signalling pathway induces chemoresistance to IFN-alpha/5-FU therapy and suggest that Ep-CAM is a potentially useful marker for resistance to such therapy, especially in IFNAR2-positive cases.

Risk factors for graft dysfunction after adult-to-adult living donor liver transplantation.

The aim of this study was to investigate the risk factors for graft dysfunction after adult-to-adult living donor liver transplantation (LDLT). Thirty-nine adults with chronic cirrhosis underwent LDLT between 1999 and 2004. Their postoperative courses were uneventful with no vascular or bile duct complications early after LDLT, except one mild hepatic artery stenosis. The preoperative MELD scores were significantly higher in the failed graft group (n=5) than the functioning graft group (n=34; P=.004), while the graft liver weight/standard liver volume ratio was similar between these groups. We concluded that a high preoperative MELD score was associated with postoperative graft failure and that graft size had little impact on graft outcome. Although large grafts would seem intuitively more suitable for sick recipients, we did not show a benefit among this cohort; the MELD score was the best predictor, a finding that is also most consistent with donor safety.

Alpha-fetoprotein mRNA detection in peripheral blood for prediction of hepatocellular carcinoma recurrence after liver transplantation.

The aim of this study was to assess the value of alphafeto protein (AFP) mRNA-expressing cells detected in peripheral blood for predicting tumor recurrence after living donor liver transplantation (LDLT) in patients with hepatocellular carcinoma (HCC). The test group consisted of 25 patients who underwent LDLT for end-stage liver disease with HCC while the control group consisted of 37 living donors. Quantitative real-time reverse-transcriptase polymerase chain reaction was used for detection of AFP mRNA-expressing cells in peripheral blood. Nine (36%) of 25 patients developed tumor recurrences (four lung; one liver; one peritoneum; two bone; one adrenal gland) during the follow-up period. Perioperatively, AFP mRNA was positive in peripheral blood of eight patients (32.0%) but only in 1 (2.7%) of the control. Preoperative AFP mRNA was positive in three cases. Univariate analyses revealed that preoperative and perioperative AFP mRNA and microscopical vascular invasion were the significant predictors for HCC recurrence (P = .007, .037, and .005, respectively). In the patients with HCC exceeding Milan criteria (n = 15), the presence of AFP mRNA-positive cells in the peripheral blood correlated significantly with HCC recurrence (P = .033). We concluded that the presence of AFP mRNA-expressing cells could be a useful predictor of HCC recurrence in liver transplant patients.

Augmentation of antitumor activity of 5-fluorouracil by interferon alpha is associated with up-regulation of p27Kip1 in human hepatocellular carcinoma cells.

Several clinical trials have demonstrated the effectiveness of combination therapy with 5-fluorouracil (5-FU) and IFN-alpha in colon cancer, hepatocellular carcinoma (HCC), and other malignancies. In our preliminary clinical studies, we have observed outstanding effects with this combination therapy in patients with advanced HCC. However, the underlying mechanism by which IFN-alpha modulates the effects of 5-FU is unknown. We, therefore, conducted a mechanistic study using two HCC cell lines, PLC/PRF/5 and HuH7. IFN-alpha significantly enhanced the growth inhibitory effect of 5-FU in PLC/PRF/5 cells but not in HuH7 cells, and the isobolographic analysis indicated that this effect was synergistic. Flow cytometric analysis showed a delay in the progression of G0-G1 to S phase in PLC/PRF/5, and a sustained, induction of the cyclin-dependent kinase inhibitor p27-Kip1 and down-regulation of cyclin D1 was observed. Moreover, increased expression of p27Kip1 was associated with reduced CDK-2-associated kinase activity. Another difference in the two cell types was that PLC/PRF/5 expressed abundant IFN receptors, but HuH7 did not. Apoptosis assays were not helpful in explaining the mechanism. Our results suggest that the synergistic effects of 5-FU and IFN-alpha may in part be attributable to alterations in cell cycle progression via up-regulation of p27Kip1.

Genetic detection for micrometastasis in lymph node of biliary tract carcinoma.

The presence of regional lymph node metastasis is one of the most significant poor-prognosis factors in patients with biliary tract carcinoma. To establish a sensitive reverse transcription (RT)-PCR assay to detect micrometastases in lymph nodes of biliary tract carcinoma, we first investigated the optimal markers in biliary tract carcinoma. The expressions of the six candidates for a suitable RT-PCR marker [mammaglobin B, carcinoembryonic antigen (CEA), cytokeratin (CK) 20, prostate-specific antigen, and melanoma antigens (MAGE-1 and MAGE-3)] were evaluated in two bile duct cancer cell lines and human biliary tract carcinoma tissues. Of 32 carcinoma tissues, mammaglobin B, CEA, prostate-specific antigen, MAGE-1, MAGE-3, and CK 20 were expressed in 28 (88%), 26 (81%), 4 (13%), 5 (16%), 7 (22%), and 9 (28%), respectively. Mammaglobin B and CEA were considered to be good markers of the six candidates. We then examined 209 lymph nodes obtained from 15 patients with biliary tract carcinoma by RT-PCR assay using both mammaglobin B and CEA and compared the results with those of histological examination. All of 20 histologically positive lymph nodes for metastasis displayed the PCR product(s) of marker genes. Of 189 histologically negative nodes, 24 (13%) nodes expressed mammaglobin B and/or CEA mRNA, suggesting the presence of micrometastasis. Our findings suggest that mammaglobin B and CEA could be useful RT-PCR markers for the detection of lymph node micrometastases in biliary tract carcinomas. Our RT-PCR assay allows accurate clinical staging necessary for patient stratification with respect to adjuvant therapy after surgery.

Overexpression of cyclooxygenase-2 in carcinoma of the pancreas.

The level of cyclooxygenase (COX)-2 has been investigated recently in various human carcinomas. In the present study, we examined the distribution and extent of COX-2 protein in human pancreatic tumors using immunohistochemistry. A strong expression of COX-2 protein was present in 23 of 52 (44%) pancreatic carcinomas, a moderate expression was present in 24 of 52 (46%) pancreatic carcinomas, and a weak expression was present in 5 of 52 (10%) pancreatic carcinomas. In contrast, benign tumors showed weak expression or no expression of COX-2, and only islet cells displayed COX-2 expression in normal pancreatic tissues. Overexpression of COX-2 in carcinoma tissues was also confirmed by Western blot analysis. Furthermore, consistent with the results at protein levels, reverse transcription-PCR analyses indicated that COX-2 mRNA was overexpressed in 7 of 13 (54%) carcinomas, but in none of 3 benign tumors. Our findings suggest that COX-2 inhibitors might be potentially effective against pancreatic carcinomas and that COX-2 may be involved in certain biological processes in pancreatic islets.

Increased expression of COX-2 in nontumor liver tissue is associated with shorter disease-free survival in patients with hepatocellular carcinoma.

Recent studies have shown increased levels of cyclooxygenase-2 (COX-2) in a variety of human malignancies including hepatocellular carcinoma (HCC), but little is known about the prognostic value of COX-2 in HCC or its associated nontumor liver tissue. We examined the expression of COX-2 protein by immunohistochemistry in 53 patients with HCCs whose corresponding nontumor tissues were hepatitis C virus-related chronic hepatitis (n = 21) and cirrhosis (n = 32). Samples of nine histologically normal livers and eight precancerous dysplasias were also analyzed. The level of COX-2 increased from normal liver to chronic hepatitis to cirrhosis. The majority of cirrhotic livers (81%) displayed marked COX-2 expression. In dysplasias, COX-2 expression was mainly moderate or strong (88%). In HCC, 17% of samples displayed a high COX-2 expression, and 37% of samples expressed COX-2 at a moderate level. Concordant results were obtained with reverse transcription-PCR and Western blot analyses. Clinicopathological survey indicated a significant correlation between COX-2 expression and differentiated carcinoma (P = 0.019). Although there was no correlation between COX-2 expression in HCC and prognosis, a striking difference was found between COX-2 expression in nontumor tissue and shorter disease-free survival (P = 0.0132). Moreover, high COX-2 expression in nontumor tissue was significantly correlated with the presence of active inflammation (P < 0.0001). The present findings suggest that COX-2 expression in nontumor tissue may play a positive role in relapse of HCC after surgery.

Electron spin resonance signals of non-heme iron as an indicator of ischemia-reperfusion injury of the liver.

Although many different methods have been proposed to assess the viability of preserved or reperfused liver, none of them are definitive. In this study, we investigated the usefulness of electron spin resonance (ESR) spectrometry in the rat liver ischemia model. Ischemia was induced in Wistar rats weighing 250-300 g by clamping the portal triad. At 15, 30, 60, or 90 min after the clamping, the liver was reperfused by removing the clamp. Liver specimens obtained before and after the clamping and also 30 min after reperfusion were frozen with liquid nitrogen and analyzed at 140K by ESR spectrometry. Two significant signals of g-values of 2.0 and 1.96 were observed with the fresh liver. The former was thought to be a mixture of CoQ, flavin, and succinate radicals. The intensity of this signal did not change throughout the experimental period. The latter was regarded as the signal from non-heme irons of mitochondria. The intensity of this signal decreased as the ischemic time became longer (the ratio to the signal intensity of the fresh liver was 0.69 +/- 0.19, 0.22 +/- 0.08, 0.20 +/- 0.05, and 0.18 +/- 0.09 at the end of 15, 30, 60, and 90 min of ischemia, respectively). After reperfusion, each ratio recovered to 0.95 +/- 0.12, 0.77 +/- 0.06, 0.56 +/- 0.15, and 0.37 +/- 0.20, respectively. This suggests that detectable signals with Fe(II)-Fe(III) decreased and became undetectable as the reduced form of non-heme irons under the anoxic state. Then, after reperfusion, the reduced form of non-heme irons decreased and the oxidized form increased. Incomplete recovery was thought to be due to decrease in the viability or function of liver cells. ATP and energy charge had the same tendency as the non-heme iron signal observed with ESR. There was a significant correlation between the non-heme iron signal and energy charge (y = 0.73x + 0.32, r = 0.78, P < 0.001), demonstrating that the signal intensity reflects the viability or function of liver cells. This study suggests that the signal from non-heme irons detected by ESR can be a good parameter of the metabolic state of the liver in ischemia and reperfusion. This method is simple and quick and should be applicable in clinical liver transplantation.

Awareness of donor alloantigens in antiadhesion therapy induces antigen-specific unresponsiveness to islet allografts.

BACKGROUND: Antiadhesion therapy using monoclonal antibodies (mAbs) to adhesion molecules in vivo has been shown to produce significant prolongation of graft survival in various transplantation models. However, it remains unclear whether antiadhesion therapy operates by merely blocking adhesion between antigen-presenting cells and T cells physically and/or by blocking costimulatory signals while preserving signals mediated through T-cell receptors in vivo. We examined antigen-specific T-cell responses during and after antiadhesion therapy. METHODS: BALB/c islets were transplanted into the renal subcapsular space of streptozotocin-induced diabetic C57BL/6 mice given anti-lymphocyte function-associated antigen (LFA)-1 and/or anti-intercellular adhesion molecule-1 mAb treatment. The animals bearing surviving islet allografts were challenged with BALB/c or third-party islets on day 7 or more than 100 days after transplantation. RESULTS: Islet allografts were acutely rejected in untreated animals, with a mean survival time (MST) of 19+/-8 days. Administration of anti-LFA-1 mAb induced significant prolongation of graft survival with a mean survival time of 72+/-33 days, and half of the allografts showed indefinite survival. The animals given anti-LFA-1 mAb alone 7 days before transplantation showed acute rejection of BALB/c islets, whereas a significant number of animals given anti-LFA-1 mAb and the BALB/c islet allograft simultaneously accepted secondary BALB/c islets, but rejected third-party islets. Likewise, most of the animals bearing long-term functioning BALB/c allografts for more than 100 days accepted secondary BALB/c islets, but rejected C3H islets acutely. Interestingly, the spleen cells from these animals transferred unresponsiveness to BALB/c islets into the 2.5-Gy x-irradiated recipients, whereas those from naive animals induced acute rejection. CONCLUSIONS: These results indicate that anti-LFA-1 mAb treatment prevents T-cell activation leading to rejection, but results in a T-cell receptor engagement leading to antigen-specific unresponsiveness maintained by transferrable suppressor cells.

The inhibitory effect of prostaglandin E1 on oxidative stress-induced hepatocyte injury evaluated by calpain-mu activation.

BACKGROUND: Prostaglandin E1 (PGE1) is known to inhibit ischemia-reperfusion injury of the liver. The calcium-dependent neutral proteinase, calpain-mu, is involved in oxidative stress-induced hepatocyte injury. We investigated the mechanisms of cytoprotection by PGE1, focusing on the elevation of intracellular calcium ([Ca2+]i), activation of calpain-mu, and calpain-mu-mediated activation of protein kinase C-alpha (PKC-alpha). METHODS: Cultured hepatocytes were treated with various amounts of PGE1 (0, 0.1, 1.0, 10, and 100 ng/ml) for 30 min and subsequently with 0.5 mM tert-butyl hydroperoxide (TBHP). Cell injury was evaluated by the release of lactate dehydrogenase. Plasma membrane bleb formation was examined by phase contrast microscopy. Activation of calpain-mu and limited degradation of PKC-alpha was evaluated by Western blotting using antibodies that specifically recognize the amino-terminal regions of calpain-mu and PKC-alpha. [Ca2+]i was measured by confocal microscopy using Fluo-3AM. RESULTS: LDH release from cells treated with 10 ng/ml PGE1 was significantly lower than from untreated cells (135 +/- 12 vs. 258 +/- 18 IU/L, respectively; P < 0.05). Morphologically, many blebs were observed in untreated cells, but very few were seen in those treated with 10 ng/ml PGE1. Western blotting revealed that the amount of activated calpain-mu and [Ca2+]i increased up to 1,300 nM at 35 min after the addition of TBHP (0.5 mmol/L) in control experiments (without PGE1). PGE1 (10 ng/ml) delayed the rise in [Ca2+]i for about 30 min, but did not suppress it completely. PKC-alpha decreased in experiments using PGE1 (10 ng/ml). CONCLUSION: PGE1 exerts its cytoprotective effect in TBHP-induced hepatocyte injury partly by inhibiting Ca2+-calpain-mu-mediated mechanisms.

Direct antigen presentation through binding of donor intercellular adhesion molecule-1 to recipient lymphocyte function-associated antigen-1 molecules in xenograft rejection.

Cellular interactions that lead to graft rejection were examined in a rat-to-mouse xenogeneic combination using species-specific monoclonal antibodies (mAbs) against donor and recipient intercellular adhesion molecule-1 (ICAM-1) and lymphocyte function-associated antigen-1 (LFA-1) molecules, respectively. Although both mAbs displayed moderate blocking activity in an in vitro mixed lymphocyte response assay, strong suppression was observed when anti-donor (rat) ICAM-1 mAb was combined with anti-recipient (mouse) LFA-1 mAb. Likewise, significant prolongation of islet xenograft survival was observed with these mAbs. Thus, 0.05 mg of anti-mouse LFA-1 mAb and anti-rat ICAM-1 mAb given on days 0 and 1 produced significant prolongation of graft survival over the control (51+/-20 days vs. 10+/-3 days, P<0.0001), but not when anti-mouse ICAM-1 mAb was combined with anti-mouse LFA-1 mAb (13+/-3 days). In this species combination, mouse T cells were able to proliferate in the presence of rat antigen-presenting cells (APCs) in a cell number-dependent manner, but not in the presence of mouse APCs. The binding assay showed that LFA-1 molecules on mouse T cells can bind immobilized rat ICAM-1 molecules. These results suggest that rat ICAM-1 molecules on APCs can interact with mouse LFA-1 molecules on T cells across a species barrier and that this binding generates the consequent immune responses leading to rejection. mAb treatment against these adhesion molecules of recipient as well as donor is crucial for preventing rejection in a xenogeneic transplantation model.

Microchimerism in thymus is associated with up-regulated T helper type 1 cytokine transcription during cardiac allograft rejection in rats.

BACKGROUND: Intrathymic microchimerism (MC) is thought to be responsible for inducing allograft tolerance. However, the role of MC in the thymus gland after transplantation, particularly in the rejection response, is unknown. We investigated serial changes in intrathymic cytokine production associated with MC and allograft rejection. METHODS: Donor-specific cell injection (DSI) and heterotopic heart transplantation (HTx) were performed in the fully allogeneic combination using DA rats (RT1a) as donors and WS rats (RT1k)as recipients. MC was checked by polymerase chain reaction (PCR) using a donor RT1.Bbeta domain 1 region sequence-specific primers. Reverse transcription (RT)-PCR analysis of cytokine (interleukin [IL]-2, interferon-gamma, IL-4, and IL-10) profiles of the thymus was performed in animals given DSI, HTx, or DSI/HTx. RESULTS: DSI alone resulted in an immediate development of MC, detected by PCR, in various organs including the thymus, spleen, liver, and blood, of most rats, lasting for over 2 months. However, DSI-induced MC selectively disappeared in the thymus on day 7 after grafting, several days before the rejection of cardiac allograft. RT-PCR analysis of cytokine profiles showed that the levels of Th1 (IL-2 and interferon-gamma) cytokines transcribed in the thymus were higher than in the spleen. MC reappeared in the thymus on day 21 after grafting, but was not associated with elevation of Th1 cytokine transcription when allograft was replaced by fibrosis. CONCLUSIONS: Intrathymic MC does not always confer unresponsiveness to alloantigen, but can be eliminated after anti-donor response.

Pretreatment of crude pancreatic islets with mitomycin C prolongs graft survival time in xenogeneic rat-to-mouse model.

BACKGROUND: Rejection of pancreatic islet grafts is still a serious problem. We evaluated the effect of mitomycin C (MMC) on the survival of crude islets grafts after xenogeneic islet transplantation. METHODS: WS (RT1k) rat islets pretreated with various concentrations of MMC (0, 1, 3.2, 10, 32, 50, 100, 320, and 1,000 microg/ml) were transplanted into C57BL/6 mice with streptozotocin-induced diabetes. In vivo graft function was assessed by a daily measurement of nonfasting blood glucose concentration in each animal. We also examined the separate effect of MMC on purified islets and contaminants present in the crude islet preparation. RESULTS: MMC at doses of 10, 32, 50, and 100 microg/ml resulted in a significant prolongation of the mean graft survival time from a control of 12.4+/-2.5 days to 23+/-7.4, 17.5+/-5.4, 25.5+/-14.7, and 26.7+/-8.9 days, respectively. Deterioration of glucose metabolism was noted when the dose exceeded 32 microg/ml, whereas at 320 microg/ ml, MMC failed to restore normoglycemia. Prolongation of survival time of crude islets was the result of its effect on islets and contaminant components of the crude islet preparation. In vitro study showed that MMC treatment at a higher concentration than 10 microg/ml reduces the stimulatory as well as proliferative capacity of lymph node cells. CONCLUSIONS: Pretreatment of pancreatic islets with MMC at 10 microg/ml prolongs xenograft survival without deterioration of in vivo graft function. This novel treatment modality represents a new strategy for the modulation of immunity of islets and contaminants in crude islet preparations.