The ultimate goal of transplantation is drug-free allograft acceptance, which is rarely encountered in transplant recipients. Using a novel human-to-mouse "trans vivo" delayed-type hypersensitivity assay, we assessed donor-reactive cell-mediated immune responses in kidney and liver transplant patients, four of whom discontinued all immunosuppression. One of these subjects (J.B.) rejected his graft after 7 years of stable function, while the others (D.S., R.D., M.L.) continue to have excellent graft function 5, 28, and 4 years after the cessation of immunosuppression. PBMCs from J.B. exhibited strong responses to both donor and recall antigens whereas PBMCs from patients D.S., R.D., and M.L. responded strongly to recall, but not donor, antigens. Furthermore, when donor and recall antigens were colocalized, the recall response in these three patients was inhibited. This donor antigen-linked nonresponsiveness was observed in four other patients who are still maintained on immunosuppression. The weakness of donor-reactive DTH responses in these patients is due to donor alloantigen-triggered regulation that relies on either TGF-β or IL-10. In D.S., regulation is triggered by a single donor HLA Class I antigen, either in membrane-bound or soluble form. This demonstrates that allograft acceptance in humans is associated with an immune regulation pattern, which may be useful in the diagnosis and/or monitoring of transplant patients for allograft acceptance.
The INK4a-ARF (CDKN2A)-locus on chromosome 9p21 encodes for two tumour suppressor proteins, p16 INK4a and p14 ARF , that act as upstream regulators of the Rb-CDK4 and p53 pathways. To study the contribution of each pathway in tumorigenesis of hepatocellular carcinoma (HCC), we analysed the alterations of p14 ARF , p16 INC4a and p53. After microdissection, DNA of 71 hepatocellular carcinomas was analysed for INK4-ARF inactivation and p53 mutation by DNA sequence analysis, methylation-speci®c PCR (MSP), restriction-enzyme related polymerase chain reaction (RE ± PCR), mRNA expression and immunohistochemistry. In addition, microdeletion of p14 ARF and p16 INC4a were assessed by di erential PCR. Inactivation of p14 ARF was found in 11/71 cases (15%), alterations of p16 INK4a occurred in 47/71 carcinomas (66%), which correlated with loss of mRNA transcription. Five tumours (7%) had homozygous deletions of the INK4a-ARF locus. We failed to detect speci®c mutations of both exons. P16 INK4a methylation with an unmethylated p14 ARF promotor appeared in 39 cases. Mutations of p53 were found in 30 of 71 HCC (42%), and only one of them harboured p14 ARF inactivation. We failed to establish alterations of the INK4a-ARF locus or p53 status as independent prognostic factor in these tumours. Our data indicate, that p14 ARF methylation occurs independently of p16 INK4a alterations in a subset of HCC together with wild type p53. The INK4a-ARF-/p53-pathway was disrupted in 86% of HCC, either by p53 mutations or by INK4a-ARF inactivation, and may have co-operative e ects in hepatocarcinogenesis. Oncogene (2001) 20, 7104 ± 7109.
The INK4a-ARF locus, located on chromosome 9p21, encodes two cell-cycle regulatory proteins, p16(INK4a) and p14(ARF), acting through the Rb-CDK4 and p53 pathways. To study the contribution of each pathway in the tumourigenesis of cholangiocarcinoma, the alterations of p14(ARF), p16(INK4a), p53, and pRb were analysed. After microdissection, DNAs from 51 cholangiocarcinomas were analysed by methylation-specific PCR (MSP), restriction-enzyme related polymerase chain reaction (RE-PCR), microsatellite analysis, mRNA expression, and DNA sequencing. Immunohistochemistry of p14(ARF), p16(INK4a), p53, and pRb was also performed. Promoter methylation of p14(ARF) was found in 13/51 cases (25%) and p16(INK4a) showed aberrant promoter methylation in 39/51 cases (76%) which correlated with loss of mRNA transcription. Two tumours (4%) had homozygous deletion of the INK4a-ARF locus. Specific mutations of both exons were not detected. p14(ARF) inactivation appeared in the context of an unmethylated p16(INK4a) promoter in eight of 13 cases (61%) of the carcinomas methylated at p14(ARF). Mutations of p53 were found in 19 of 51 tumours (37%), and four of them (21%) harboured p14(ARF) inactivation. The pRb protein was detected in 30/51 (59%) tumours examined. The absence of pRB protein did not correlate with any of the examined parameters. Alterations of the INK4a-ARF locus, pRB or p53 status could not be established as independent prognostic factors in these tumours. These findings indicate that the INK4a-ARF locus is frequently inactivated in cholangiocarcinoma of the liver and occurs independently of the status of p53 or pRb.
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