Prognostic significance of the prognostic nutritional index in esophageal cancer patients undergoing neoadjuvant chemotherapy.

Nutritional status is one of the most important issues faced by cancer patients. Several studies have shown that a low preoperative nutritional status is associated with a worse prognosis in patients with various types of cancer, including esophageal cancer (EC). Recently, neoadjuvant chemotherapy (NAC) and/or radiotherapy have been accepted as the standard treatment for resectable advanced EC. However, NAC has the potential to deteriorate the nutritional status of a patient. This study aimed to evaluate the prognostic significance of the nutritional status for EC patients who underwent NAC. We retrospectively reviewed 66 squamous cell EC patients who underwent NAC consisting of docetaxel, cisplatin, and 5-fluorouracil followed by subtotal esophagectomy at Nara Medical University Hospital between January 2009 and August 2015. To assess the patients' nutritional status, the prognostic nutritional index (PNI) before commencing NAC and prior to the operation was calculated as 10 × serum albumin (g/dl) + 0.005 × total lymphocyte count in the peripheral blood (per mm3). The cutoff value of the PNI was set at 45. A multivariable analysis was performed to identify prognostic factors for overall survival (OS) and relapse-free survival (RFS). The mean pre-NAC and preoperative PNI were 50.2 ± 5.7 and 48.1 ± 4.7, respectively (P = 0.005). The PNI decreased following NAC in 44 (66.7%) patients. Before initiating NAC, 9 (13.6%) patients had a low PNI, and 12 (18.2%) patients had a low PNI prior to the operation. The pre-NAC PNI and preoperative PNI were significantly associated with the OS (P = 0.013 and P = 0.004, respectively) and RFS (P = 0.036 and P = 0.005, respectively) rates. The multivariable analysis identified the preoperative PNI as an independent prognostic factor for poor OS and RFS, although the pre-NAC PNI was not an independent predictor. Our results suggest that the preoperative PNI is a useful marker for predicting the long-term outcomes of EC patients undergoing NAC and subsequent subtotal esophagectomy. Therefore, patients with a low preoperative nutritional status may be at a higher risk of EC recurrence.

Clinical importance of B7-H3 expression in human pancreatic cancer.

BACKGROUND: B7-H3 is a new member of the B7 ligand family and regulates T-cell responses in various conditions. However, the role of B7-H3 in tumour immunity is largely unknown. The purpose of this study was to evaluate the clinical significance of B7-H3 expression in human pancreatic cancer and the therapeutic potential for cancer immunotherapy. METHODS: We investigated B7-H3 expression in 59 patients with pancreatic cancer by immunohistochemistry and real-time PCR. Furthermore, we examined the anti-tumour effect of B7-H3-blocking monoclonal antibody in vivo in a murine pancreatic cancer model. RESULTS: Tumour-related B7-H3 expression was abundant in most human pancreatic cancer tissues and was significantly higher compared with that in non-cancer tissue or normal pancreas. Moreover, its expression was significantly more intense in cases with lymph node metastasis and advanced pathological stage. B7-H3 blockade promoted CD8(+) T-cell infiltration into the tumour and induced a substantial anti-tumour effect on murine pancreatic cancer. In addition, the combination of gemcitabine with B7-H3 blockade showed a synergistic anti-tumour effect without overt toxicity. CONCLUSION: Our data show for the first time that B7-H3 may have a critical role in pancreatic cancer and provide the rationale for developing a novel cancer immunotherapy against this fatal disease.

Protective effect of prostaglandin E2 receptors EP2 and EP4 in alloimmune response in vivo.

Prostaglandin E2 (PGE2) is produced during inflammatory responses mediating a variety of both innate and adaptive immune responses through 4 distinct receptors: EP1 to EP4. The use of gene-targeted mice and selective agonists/antagonists responsible for each receptor has gradually revealed that each receptor plays a unique and important role in various disease conditions. In addition, PGE2 is known to have some immunosuppressive properties. In this study, we investigated the role of PGE2 receptors by examining the therapeutic efficacy of highly selective receptor agonists on the alloimmune response in vivo. We used a fully major histocompatibility complex (MHC)-mismatched murine cardiac transplantation model. C57BL/6 cardiac allografts were heterotopically transplanted into BALB/c recipients. We treated mice with a highly selective agonist for each EP receptor. EP2 and EP4 agonists significantly prolonged allograft survival compared with controls. In particular, the EP4 agonist was more effective than the EP2 agonist in the inhibition of acute allograft rejection. In conclusion, PGE2 receptors merit further study as novel therapeutics for clinical transplantation.

A novel CCR5/CXCR3 antagonist protects intestinal ischemia/reperfusion injury.

Chemokines and chemokine receptors have been demonstrated to be critical regulators in a variety of physiologic and pathologic immune responses. In particular, CCR5 and CXCR3 have been reported to play important roles in the alloimmune response. In this study, we investigated the therapeutic efficacy of a novel small-molecule compound, TAK779, an antagonist targeting both CCR5 and CXCR3 in intestinal ischemia/reperfusion (I/R) injury. We utilized an established murine intestinal I/R injury model. TAK779 treatment significantly improved mouse survival after 60 minutes of intestinal ischemia. We then examined the local intestinal expression of several cytokines and chemokines at 2 hours after reperfusion using real-time PCR. TAK779 treatment downregulated the expression of several cytokines, including TNF-alpha, IFN-gamma, and IL-4, suggesting that the beneficial effect of TAK779 was associated with inhibition of local immune activation. We further examined the systemic response after TAK779 treatment. Lung tissue damage was significantly prevented by the treatment, as determined by lung wet-to-dry weight ratios at 4 hours after intestinal I/R injury. In addition, we observed that CCR5 expression in the lung was significantly downregulated by the treatment, suggesting that TAK779 inhibited the infiltration of CCR5-positive cells into the remote organ. Our data suggest the critical role of CCR5 and CXCR3 in intestinal I/R injury and therapeutic efficacy of a novel small compound, TAK779, for protection against the intestinal I/R injury.

In vivo engineering of metabolically active hepatic tissues in a neovascularized subcutaneous cavity.

Recent success in clinical hepatocyte transplantation therapy has encouraged further investigation into bioengineering hepatic tissues in vivo. Engineering tissues in the subcutaneous space is an attractive method; however, hepatocyte survival has been transient due to insufficient vascular network formation. To establish a vascularized cavity, we created a polyethylene terephthalate mesh device coated with poly(vinylalcohol) that allowed for the gradual release of basic fibroblast growth factor (bFGF), a potent angiogenic factor. The efficacy of the bFGF-releasing device in inducing vascular network formation in the subcutaneous space was observed in mouse and rat studies. Isolated mouse hepatocytes transplanted into newly vascularized subcutaneous cavities allowed for persistent survival up to 120 days. In the absence of a vascularized compartment, the survival of the transplanted hepatocytes was markedly diminished. Functional maintenance of the engineered hepatic tissues was confirmed by high expression of liver-specific mRNAs and proteins. These engineered hepatic tissues have the ability to take up inoculated compounds and express strong induction of drug-metabolizing enzymes, demonstrating functional relevance as a metabolic tissue. In conclusion, we have created a novel technology to engineer functionally active hepatic tissues in the subcutaneous space, which will likely facilitate hepatocyte-based therapies.

Analysis of risk factors for the development of gallstones after gastrectomy.

BACKGROUND: The incidence of gallstones is higher in people who have undergone gastrectomy than in the general population, but the cause of this is unknown. METHODS: Between January 1992 and January 2003, 749 patients underwent ultrasonography of the gallbladder after gastrectomy for gastric cancer. A total of 2327 examinations were carried out. The incidence of gallstones was compared in subgroups of patients classified according to the type of reconstruction, extent of gastrectomy, whether the duodenum was excluded and type of lymph node dissection. RESULTS: The incidence of gallstones was significantly higher after total compared with partial gastrectomy (27.9 versus 7.8 per cent at 5 years; P < 0.001). Reconstruction with duodenal exclusion was associated with a significantly higher incidence than non-exclusion (25.1 versus 8.2 per cent at 5 years; P < 0.001). Patients who had lymph node dissection in the hepatoduodenal ligament had a significantly higher incidence of gallstones than those who did not (28.2 versus 7.5 per cent at 5 years; P < 0.001). In multivariate analysis that included type of reconstruction and lymph node dissection, lymph node dissection in the hepatoduodenal ligament was identified as the most significant risk factor for gallstone development (odds ratio 3.66 (95 per cent confidence interval 2.16 to 6.22); P < 0.001). CONCLUSION: Lymph node dissection in the hepatoduodenal ligament, total gastrectomy and exclusion of the duodenum are risk factors for gallstones after gastrectomy.

Role of EP4 prostaglandin E2 receptor in the ischemic liver.

Prostaglandin E(2) (PGE(2)) mediates a variety of both innate and adaptive immunity responses through 4 distinct receptors, EP1-4. Recent studies have suggested the physiological and pathological role of EP4 in various inflammatory diseases. In this study, we investigated the importance of the EP4 receptor, and the efficacy of a selective EP4 agonist to alter hepatic ischemia/reperfusion (I/R) injury, an important cause of damage in liver resection and transplantation. We used an established murine I/R injury model, 70% partial hepatic ischemia for 90 minutes in male C57BL/6 mice. The local expression of EP4 messenger RNA (mRNA) in the naive and the ischemic liver at 2 hours after reperfusion was examined using RT-PCR analysis. Some mice received the EP4 selective agonist during I/R. Serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were measured as markers of hepatic injury. EP4 expression in the liver was significantly up-regulated at 2 hours after reperfusion. Furthermore, treatment with EP4 agonist significantly inhibited hepatic injury at 6 hours after reperfusion. Our data suggest an inhibitory role of EP4 PGE(2) receptor in hepatic I/R injury and the therapeutic efficacy of a selective EP4 agonist for liver protection.

Efficacy of auxiliary partial orthotopic liver transplantation for cure of hemophilia in a canine hemophilia A model.

Metabolic liver disease can be cured by orthotopic liver transplantation. Some successful cases of whole or partial liver transplantation have been reported. Because liver function in these recipients is normal save for the production of the responsive metabolic factor, auxiliary partial orthotopic liver transplantation (APOLT) may produce a benefit. However, no experimental model of APOLT for metabolic liver diseases has been reported. We established a canine APOLT model to evaluate the clinical feasibility and efficacy of APOLT to cure hemophilia. The donor normal beagle dog was used to establish an APOLT model. A left lobe partial liver graft taken from the donor was orthotopically transplanted to the recipient after resection of the native left lobe preserving the native right lobe. Recipients showed no atrophy and comparable blood flow in both the graft and the native liver at the time of exploration after APOLT. Thus, APOLT was performed from a normal donor to a recipient with hemophilia A. In this recipient, blood factor VIII activity markedly increased after APOLT and was maintained for 7 weeks. No episode of bleeding was seen during the observation. In conclusion, a canine APOLT model was successfully established as evidenced by sustained production of factor VIII in a hemophilia recipient. These findings suggest the clinical feasibility and efficacy of APOLT for metabolic liver diseases.

Heterotopically transplanted hepatocyte survival depends on extracellular matrix components.

The novel approach of tissue engineering to treat many forms of liver diseases using hepatocytes requires sufficient numbers and sustained survival of the transplanted cells. It has been shown that providing extracellular matrix components extracted from Engelbreth-Holm-Swarm cells (EHS-ECMs) to heterotopically transplanted hepatocytes allows significantly greater hepatocyte survival. We investigated the survival and morphology of hepatocytes and EHS-ECMs transplanted under the kidney capsule compared with hepatocytes with growth factor-reduced EHS-ECMs in mice. Both the EHS-ECMs and growth factor-reduced EHS-ECMs showed a large number of surviving hepatocytes under the kidney capsule without any intergroup differences. Histologically, transplanted hepatocytes in both groups retained their characteristic morphologies and formed small liver tissues. These data indicate that extracellular matrix components are the predominant factor in EHS-ECMs required to maintain hepatocytes at heterotopic sites.

Matrix metalloproteinase-9 and tissue inhibitor of metalloproteinase-1: expression in the lung of fetal rats with nitrofen-induced diaphragmatic hernia.

The surrounding extracellular matrix of airway wall tissues changes in response to mechanical stresses and hypoxia. The presence of matrix metalloproteinase-9 (MMP-9) and its inhibitor, tissue inhibitor of metalloproteinase-1 (TIMP-1), is correlated with collagen degradation and tissue repair in lung disorders. The aim of this study was to evaluate the expression of MMP-9 and TIMP-1 in the lung of fetal rats with nitrofen-induced congenital diaphragmatic hernia (CDH). Administering 100 mg of nitrofen dissolved in 1 ml olive oil to pregnant Wistar rats on day 9 of gestation induced left-sided CDH in fetal rats. In control animals, the same dose of olive oil was given without nitrofen. Cesarean section was performed on day 21 of gestation. The fetuses were divided into two groups: normal controls (n = 10) and nitrofen-induced left-sided CDH (n = 10). Immunoreactivity of the staining for MMP-9 and TIMP-1 in the lung tissues was semiquantitatively analyzed using the staining scores. The relative amount of MMP-9 or TIMP-1 divided by the amount of beta-actin for each lung sample was measured by using the real-time reverse-transcriptase polymerase chain reaction. The immunoreactivity of MMP-9 was significantly increased in the CDH group (n = 5) compared with the control group (n = 5) (p = 0.031). On the other hand, the immunoreactivity of TIMP-1 in the two groups was not significantly different (n = 0.134). The relative amount of MMP-9 (or TIMP-1) in the CDH group (n = 5) does not differ significantly from that in the control group (n = 5) (p = 0.059, 0.596, respectively), but the relative amount of MMP-9 is higher in the CDH group, although it is not significantly higher. On the other hand, the ratios of MMP-9 to TIMP-1 were significantly higher in the CDH group (p = 0.028). In conclusion, fetal rats with nitrofen-induced CDH, a model of respiratory disorders, manifested the excess of MMP-9 activity due to the absence of TIMP-1 that would suggest a trend toward disruption of the extracellular matrix in the CDH lung tissues.

Liver fibrosis increases the risk of intrahepatic recurrence after hepatectomy for hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) commonly develops in patients with chronic hepatitis. Intrahepatic recurrence after hepatectomy often includes nodules of new tumour in the liver remnant. The aim of this study was to examine hepatitis-related factors that might predict this type of recurrence. METHODS: The influence of various hepatitis-related factors on intrahepatic recurrence of HCC was studied by multivariate analysis in 138 patients who underwent curative resection and were followed for more than 2 years. RESULTS: The Cox proportional hazard model showed that histological evidence of fibrosis of the underlying liver was the most significant predictive factor for intrahepatic recurrence (P = 0.001). Serum albumin level was also significantly associated with recurrence (P = 0.038). The relative risks of histological fibrosis and low serum albumin levels were 8.9 and 1.7 respectively. Among tumour-related factors, only tumour size was significantly associated with recurrence (P = 0.017). Major hepatectomy was also an independent risk factor for intrahepatic recurrence (P = 0.004). CONCLUSION: Histological evidence of fibrosis and low serum albumin levels are useful predictors of intrahepatic recurrence after hepatectomy, presumably owing to metachronous multifocal tumour in the liver remnant.

Intragraft expression of p38 and activated p38 MAPK (mitogen-activated protein kinase) in rat small bowel transplantation.

Recent studies suggest that cytotoxic T-lymphocytes expressing p38 mitogen-activated protein kinase (p38MAP kinase) contribute to allograft rejection in clinical heart transplantation. Interleukin-2 (IL-2), a potent T cell mitogen, activates the p38MAP kinase pathway, resulting in phosphorylation of target transcription factors. In this study we investigated the expression of activated p38MAP kinase in intragraft cell infiltrates following rat heterotopic small bowel transplantation and examined the effects of the immunosuppressant FK506 on intragraft expression of activated p38MAP kinase and allograft rejection. Allografts receiving FK506 (0.5 mg/kg per day i. m.) for 7 days as primary anti-rejection therapy had a significant reduction in histopathological evidence of allograft rejection on Day 7, compared to allograft controls. In addition, Western blotting analysis of intragraft cell infiltrates showed a reduction in the expression of activated p38MAP kinase in allografts treated with FK506. We conclude that intragraft cell infiltrate expression of activated p38MAP kinase is an important marker of acute rejection in this animal model of small bowel transplantation, and that FK506 is an effective immunosuppressant, in this situation, that may act in part by preventing the activation of p38MAP kinase.

The association of K-ras gene mutation and vascular endothelial growth factor gene expression in pancreatic carcinoma.

BACKGROUND: Previously, the authors reported the role of the vascular endothelial growth factor (VEGF) as an angiogenic factor in 40 patients with pancreatic carcinoma. In this study, they investigated the mechanism underlying the regulation of VEGF gene expression and evaluated VEGF expression and K-ras gene status in 48 patients with pancreatic carcinoma. METHODS: The authors used quantitative reverse transcriptase-polymerase chain reaction analysis and direct sequencing techniques for a retrospective study of VEGF gene expression and K-ras gene status in tumor tissue samples from 48 patients with pancreatic carcinoma. Immunohistochemistry also was used to investigate VEGF protein expression. RESULTS: Thirty-one tumors (64.6%) were evaluated with high VEGF expression, and 17 tumors (35.4%) were evaluated with low VEGF expression. Of the 48 primary pancreatic tumors studied, 33 tumors (68.8%) contained mutations of the K-ras gene. There was a significant correlation between VEGF expression and K-ras status. Twenty-five of 33 tumors (75.8%) with mutant K-ras genes showed high VEGF expression, whereas only 6 of 15 tumors with the wild type K-ras (40.0%) showed high VEGF expression (P = 0.038). The mean (+/- standard error) VEGF conservation rate for the 33 tumors with mutant K-ras was 1.839 +/- 1.241, and that for the 15 tumors with wild type K-ras was 1.057 +/- 0.983 (P = 0.037). Furthermore, the median survival for patients with mutant K-ras was shorter than for those with wild type K-ras (10.6 months vs. 27.6 months, respectively; P = 0.026), whereas the median survival for patients with high VEGF expression was shorter compared with that for patients with low VEGF expression (9.5 months vs. 26.4 months, respectively; P = 0.002). Cox regression model analysis indicated that only the VEGF status was a significant factor for prognosis (P = 0.024). Other variables, i.e., K-ras status, histopathologic tumor grade, tumor status, lymph node status, metastatic status, gender, and age at surgery, were not significant. CONCLUSIONS: The results of this study suggest that K-ras oncogene mutation may be associated with VEGF expression and that patients with pancreatic carcinoma who have high VEGF expression are associated with a poor prognosis.

Plastic resin pellets as a transport medium for toxic chemicals in the marine environment.

Plastic resin pellets (small granules 0.1-0.5 centimeters in diameter) are widely distributed in the ocean all over the world. They are an industrial raw material for the plastic industry and are unintentionally released to the environment both during manufacturing and transport. They are sometimes ingested by seabirds and other marine organisms, and their adverse effects on organisms are a concern. In the present study, PCBs, DDE, and nonylphenols (NP) were detected in polypropylene (PP) resin pellets collected from four Japanese coasts. Concentrations of PCBs (4-117 ng/g), DDE (0.16-3.1 ng/g), and NP (0.13-16 microg/g) varied among the sampling sites. These concentrations were comparable to those for suspended particles and bottom sediments collected from the same area as the pellets. Field adsorption experiments using PP virgin pellets demonstrated significant and steady increase in PCBs and DDE concentrations throughout the six-day experiment, indicating that the source of PCBs and DDE is ambient seawater and that adsorption to pellet surfaces is the mechanism of enrichment. The major source of NP in the marine PP resin pellets was thought to be plastic additives and/or their degradation products. Comparison of PCBs and DDE concentrations in mari

Repeat liver resection for hepatocellular carcinoma.

BACKGROUND: Although hepatectomy has been accepted as a therapeutic option for the primary tumor of hepatocellular carcinoma (HCC), what role the second liver resection will play in the clinical care of patients with intrahepatic recurrence of HCC after the initial resection has not been well evaluated. STUDY DESIGN: In a retrospective review of the 6-year period between January 1991 and December 1996, records were examined of 94 patients who underwent curative liver resection for HCC. Of these, 57 patients had isolated recurrent disease to the liver; 12 of the 57 patients underwent repeat surgical resection and 45 patients received nonsurgical ablative therapy. Clinical data for these patients were reviewed for operative morbidity and mortality, survival, disease-free survival, and pattern of failure. RESULTS: There were no perioperative deaths during repeat liver resections for recurrent HCC. Operative morbidity in the second resection was comparable to the initial resection. The disease-free survival rate after the second hepatectomy was 31% at 2 years, significantly lower than that after initial hepatectomy (62%) (p = 0.009). The overall survival rate after the second hepatectomy was 90% at 2 years, in contrast to 70% after nonsurgical ablative treatment for recurrent HCC (p = 0.253). CONCLUSIONS: Although the second liver resection for recurrent HCC can be performed safely and may improve survival, the disease-free survival rate after such resection therapy is low. This likelihood of further recurrences encourages studies for the selection of patients who may benefit from repeat liver resection.

Clinical predictors of recurrence site after hepatectomy for metastatic colorectal cancer.

BACKGROUND/AIMS: Surgical resection of hepatic colorectal metastasis may produce long-term survival and cure; however, a significant proportion of patients will have intrahepatic and/or extrahepatic recurrence with a poor prognosis. The aim of this study was to define clinical predictors of recurrence site after hepatectomy in terms of stratifying patients for adequate adjuvant trials to improve the prognosis. METHODOLOGY: Clinical, pathologic, and outcome data for 70 consecutive patients undergoing hepatectomy for colorectal metastasis isolated to the liver were reviewed retrospectively, and all data were analyzed by the logistic multivariate regression model. RESULTS: Recurrence in the remnant liver was seen in 60% of patients, and recurrence in the lung was found in 34% of patients. Number of liver tumors was the only significant and independent predictor of recurrence in the remnant liver (P = 0.048). All patients with three or more tumors experienced recurrence. Location of liver tumors lying adjacent to the hepatic vein, which was confirmed by preoperative imaging techniques, was the only significant and independent predictor of recurrence in the lung (P = 0.020). CONCLUSIONS: Number and location of liver tumors would be the significant and independent clinical predictors of recurrence site after hepatectomy for metastatic colorectal cancer. This might be useful for justification and selection of effective adjuvant trials after surgery.

Pancreatic adenocarcinoma in a patient with Peutz-Jeghers syndrome: report of a case and literature review.

We present a rare case of pancreatic adenocarcinoma in a 47-year-old man with the Peutz-Jeghers syndrome. The patient underwent pancreatoduodenectomy with partial resection of the portal vein. We also review the current literature concerning Peutz-Jeghers syndrome associated with malignant tumors, especially pancreatic cancer. To our knowledge, this is the first report of a Peutz-Jeghers syndrome patient with pancreatic cancer having pancreatoduodenectomy and pathologically diagnosed with invasive ductal adenocarcinoma of the pancreas. The Peutz-Jeghers syndrome patients with pancreatic cancer were relatively young. As the pancreatic cancer in these patients was advanced and most were unresectable at diagnosis, the prognoses of these patients were extremely poor. Surgical resection offers the only chance for cure or long-term survival for Peutz-Jeghers syndrome patients, if the tumor is localized without distant metastasis. Therefore, screening even for young patients with Peutz-Jeghers syndrome is necessary for early detection of cancer.

Humoral human xenoreactivity against isolated pig pancreatic islets.

It is widely believed that the hyperacute rejection of vascularized xenografts in the pig-to-human combination is triggered by the binding of human preformed natural antibodies (PNAbs) to the Galalpha.(1,3)Gal epitope in pig endothelium and the subsequent activation of complement. However, it remains poorly defined whether xenogeneic pig pancreatic islets are damaged by antibody and complement-mediated mechanisms. We examined the expression of Galalpha(1,3)Gal on isolated adult pig islets and the presence of PNAbs in normal human sera directed against islets, using immunofluorescence staining and confocal laser scanning microscopy. The pig islets were not stained with Galalpha(1,3)Gal-specific lectin GSIB4, however, the exocrine cells reacted strongly with GSIB4, indicating that the Galalpha(1,3)Gal epitope was highly expressed on exocrine cells, but not on islets. Human sera showed weak reactivity of IgM and IgG class PNAbs to the islets, but strong reactivity to the exocrine cells. Furthermore, we investigated the cytotoxic effect of human serum on pig islets using an in vitro model of pig-to-human islet transplantation. The incubation of pig islets with normal human sera for 45 min resulted in less than 10% specific lysis despite the binding of PNAbs, whereas exposure of porcine aortic endothelial cells to the same human sera caused 56% complement-mediated lysis, determined using a MTT cytotoxic assay. These results support the view that pig islets might not undergo early antibody and complement-mediated rejection in humans.