Autoimmune pancreatitis in MRL/Mp mice is a T cell-mediated disease responsive to cyclosporine A and rapamycin treatment.

BACKGROUND: Autoimmune pancreatitis (AIP) in humans invariably responds to steroid treatment, but little is known about the underlying pathogenesis and the benefits of alternative treatments. OBJECTIVE: To study the pathogenesis, and the efficacy of alternative immunosuppressant agents in the MRL/Mp mouse model of AIP. DESIGN: MRL/Mp mice were pretreated for 4 weeks with polyinosinic:polycytidylic acid to induce AIP. Pancreatic sections of mice genetically deleted for CTLA-4 were analysed. Blockage of CTLA-4 was achieved by intraperitoneal antibody treatment with 2 µg/g anti-mouse-CD152. Subsequent therapeutic studies were performed for a period of 4 weeks using cyclosporine A (40 µg/g), rapamycin (1 µg/g) or azathioprine (15 µg/g). RESULTS: Blockage of CTLA-4 in MRL/Mp mice suppressed regulatory T cell (Treg) function and raised the effector T cell (Teff) response with subsequent histomorphological organ destruction, indicating that AIP is a T cell-driven disease. Using an established histopathological score, we found that dexamethasone, cyclosporine A and rapamycin, but less so azathioprine, reduced pancreatic damage. However, the beneficial effects of cyclosporine A and rapamycin were achieved via different mechanisms: cyclosporine A inhibited Teff activation and proliferation whereas rapamycin led to selective expansion of Tregs which subsequently suppressed the Teff response. CONCLUSIONS: The calcineurin inhibitor cyclosporine A and the mammalian target of rapamycin (mTOR) inhibitor, rapamycin, improve the course of AIP in MRL/Mp mice via different mechanisms. These findings further support the concept of autoreactive T cells as key players in the pathogenesis of AIP and suggest that cyclosporine A and rapamycin should be considered for treatment of AIP in humans.

PTEN mutation, loss of heterozygosity, promoter methylation and expression in colorectal carcinoma: two hits on the gene?

The phosphatase and tensin homologue (PTEN) gene is considered to be a tumour-suppressor gene in various types of cancer, colorectal carcinoma among them. According to the 'two-hit' tumour-suppressor gene concept, inactivation occurs by any combination of the following three pathogenetic processes: mutation, loss of one allele [i.e. loss of heterozygosity (LOH)] or promoter methylation. To determine the frequencies of PTEN tumour-suppressor gene features in colorectal carcinoma, we used DNA from colorectal carcinoma xenografts/primary tumour cell lines (N=22) or neoplastic glands isolated by laser-capture microdissection (N=20). Sequencing exons 1-9 of the gene revealed a total of 8 somatic mutations in 5 tumours (3 with high-degree microsatellite instability). In 1 tumour, a truncating mutation of one allele was combined with two missense mutations of the other allele. Polymorphic microsatellite marker analyses (D10S5412, D10S579 and D10S1765) showed complete loss of one allele (i.e. LOH sensu stricto) in 3 tumours, but combined LOH and mutation was found only once. Promoter methylation, tested by MethyLight technology, was found in only 1 of the tumours, not combined with mutation or LOH. In contrast, by immunohistochemistry (mAb 6H2.1), reduction or even loss of PTEN expression was found in 18 tumours. Taken together, PTEN downregulation is a fairly frequent event in colorectal carcinoma, but this apparently is not usually caused by two hits on the gene.

Single nucleotide polymorphism array analysis of microsatellite-stable, diploid/near-diploid colorectal carcinomas without the CpG island methylator phenotype.

Colorectal carcinomas are considered to progress by chromosomal instability (CIN), or microsatellite instability (MSI) and/or epigenetic gene silencing; however, in previous studies we observed a small fraction of tumours without this molecular phenotype. To further investigate these 'X-type' tumours, neoplastic glands from five tumours were isolated by laser-capture microdissection and used for single nucleotide polymorphism (SNP) array analyses. DNA from our own low-passage primary colorectal carcinoma cell lines (n=9) was used for comparison. Two of these 'X-type' tumours had very low numbers of aberrations (totals of four and five, respectively), consisting of trisomies and arm amplifications. Conversely, aberrations were markedly more frequent in the control cases and three of the 'X-type' tumours (range, 11-40). These aberrations included deletions of chromosomes and chromosome arms, uniparental disomies (UPD), trisomies and arm amplifications. Recurrent microdeletions (<1 MB) were observed at 3p14.2 (FHIT), 16p13.2 (A2BP1) and 20p12.1 (MACROD2). Microsatellite analyses with polymorphic markers at five 'canonical' colorectal carcinoma loci demonstrated a complete loss of one allele in all but one case. When compared to the SNP arrays, concordant results were observed in 93% of tests; however, this was only if DNA from cell lines or laser-capture microdissections was used. In conclusion, colorectal carcinomas may develop without the classic molecular features of CIN, MSI and/or CpG island methylator phenotype (CIMP), but this is a rare event. UPD is frequent but does not define a separate molecular phenotype. Furthermore, our study supports the notion that SNP arrays are reliable for genome-wide detection of deletions and UPD, but discourages the use of microsatellite analyses to detect loss of heterozygosity with DNA from whole tissues.