Summary Overexpression of p53 correlates with neoplasia in many cytological specimens. To test the specificity of overexpressed p53 as a tumour marker for the detection of pancreatic cancer, we analysed cytological specimens of pancreatic juice samples from patients with pancreatitis or pancreatic carcinoma (n = 42) for p53 protein overexpression. p53 protein overexpression was found in 59% of patients with pancreatitis and 67% of patients with pancreatic carcinoma. Thus, Kalthoff et al, 1993), and more than 50% of pancreatic carcinomas express an altered p53 tumour-suppressor gene (Barton et al, 1991;Kalthoff et al, 1993). Deletions of cyclin kinase inhibitors pl6/MTS1 (Caldas et al, 1994) and pl5/MTS2 (Naumann et al, 1996) have been described, and a new tumour-suppressor gene, DPC4, has been found very recently (Hahn et al, 1996). In spite of this progress at the molecular level, the clinical outcome of patients with pancreatic cancer is still very poor. Thus, detection of early stages of pancreatic cancer is still crucial for a better prognosis.For many cytological specimens, the detection of p53 overexpression by immunocytochemistry strongly correlates with neoplasia (Dowell et al, 1994). Our aim was to establish whether the detection of p53 overexpression in cytological specimens from pancreatic juice samples, collected during ERCP (endoscopic retrograde cholangiopancreaticography), may be useful in detecting early stages of pancreatic carcinoma.p53 protein overexpression was detected in nearly 60% of cytological specimens from patients with pancreatitis but without any sign of pancreatic cancer for up to 5 years (median follow-up) after ERCP. This indicates that, in the case of pancreatic disease, p53 protein overexpression does not correlate with neoplasia. However p53 seems to play an important role during pancreatitis, as apoptotic cell death has been observed during chronic disease. This is in line with the function of p53 as an inductor of apoptotic cell death (Lane, 1992 MATERIALS AND METHODS Preparation of cytological specimens by cytospin of pancreatic juicePancreatic juice samples were collected during diagnostic ERCP from a total of 42 patients. One group comprised 27 patients suffering from chronic pancreatitis. The other group had 15 patients with pancreatic ductal adenocarcinomas, the vast majority of which were in stage II and III. Sample preparation was performed exactly as described previously (Schmiegel et al, 1990). Median follow-up was 5 years in the group of pancreatitis patients. During this period, no pancreatic cancer cases were observed. Antibodies and immunoperoxidase studiesThe p53-specific monoclonal antibodies PAbl801, PAb240 and PAbl620 were obtained from Oncogene Sciences (Dianova, Hamburg). The polyclonal antiserum against recombinant human p53 (CM-1) was purchased from Medac (Hamburg). Immunoperoxidase studies were performed exactly as described previously (Kalthoff et al, 1993). Samples were scored positive when at least 5% of the cells from the investigated cytospin were...

Background & Aims: Hepatitis E virus (HEV) infection is the most common cause of liver inflammation, but the pathogenic mechanisms remain largely unclear. We aim to explore whether HEV infection activates inflammasomes, the crosstalk with antiviral interferon response and potential of therapeutic targeting. Approach & Results:We measured IL-1β secretion, the hallmark of inflammasome activation, in serum of HEV-infected patients and rabbits, and in cultured macrophage cell lines and primary monocyte-derived macrophages. We found that genotypes 3 and 4 HEV infection in rabbits elevated IL-1β production. A profound increase of IL-1β secretion was further observed in HEV-infected patients (1733 pg/mL ± 1234; n = 70) compared with healthy individuals (731 pg/mL ± 701; n = 70). As macrophages are the drivers of inflammatory response, we found inoculation with infectious HEV particles robustly triggered NLRP3 inflammasome activation in primary macrophages and macrophage cell lines. We further revealed that the ORF2 capsid protein and the formed integral viral particles are responsible for activating inflammasome response. We also identified NF-κB signaling activation as a key upstream event of HEV-induced NLRP3 inflammasome response. Interestingly, inflammasome activation antagonizes interferon response to facilitate viral replication in macrophages. Pharmacological inhibitors and clinically used steroids can effectively target inflammasome activation. Combining steroids with ribavirin simultaneously inhibit HEV and inflammasome response without cross-interference. Conclusions:HEV infection strongly activates NLRP3 inflammasome activation in macrophages, which regulates host innate defense and pathogenesis. Therapeutic targeting NLRP3, in particular when combined with antiviral agents, represents a viable option for treating severe HEV infection.

Human leukocyte antigen (HLA) molecules are essential for anti-tumor immunity, as they display tumor-derived peptides to drive tumor eradication by cytotoxic T lymphocytes. HLA molecules are primarily studied as peptide-loaded complexes on cell membranes (mHLA) and much less attention is given to their secretion as soluble HLA–peptide complexes (sHLA) into bodily fluids. Yet sHLA levels are altered in various pathologies including cancer, and are thus of high interest as biomarkers. Disconcordance in results across studies, however, hampers interpretation and generalization of the relationship between sHLA levels and cancer presence, thereby impairing its use as a biomarker. Furthermore, the question remains to what extent sHLA complexes exert immunomodulatory effects and whether shifts in sHLA levels contribute to disease or are only a consequence of disease. sHLA complexes can also bear tumor-derived peptides and recent advancements in mass spectrometry now permit closer sHLA peptide cargo analysis. sHLA peptide cargo may represent a “liquid biopsy” that could facilitate the use of sHLA for cancer diagnosis and target identification for therapeutic vaccination. This review aims to outline the contradictory and unexplored aspects of sHLA and to provide direction on how the full potential of sHLA as a quantitative and qualitative biomarker can be exploited.

Immunopeptidomics is used to identify novel epitopes for (therapeutic) vaccination strategies in cancer and infectious disease. Various false discovery rates (FDRs) are applied in the field when converting liquid chromatography-tandem mass spectrometry (LC-MS/MS) spectra to peptides. Subsequently, large efforts have recently been made to rescue peptides of lower confidence. However, it remains unclear what the overall relation is between the FDR threshold and the percentage of obtained HLA-binders. We here directly evaluated the effect of varying FDR thresholds on the resulting immunopeptidomes of HLA-eluates from human cancer cell lines and primary hepatocyte isolates using HLA-binding algorithms. Additional peptides obtained using less stringent FDR-thresholds, although generally derived from poorer spectra, still contained a high amount of HLA-binders and confirmed recently developed tools that tap into this pool of otherwise ignored peptides. Most of these peptides were identified with improved confidence when cell input was increased, supporting the validity and potential of these identifications. Altogether, our data suggest that increasing the FDR threshold for peptide identification in conjunction with data filtering by HLA-binding prediction, is a valid and highly potent method to more efficient exhaustion of immunopeptidome datasets for epitope discovery and reveals the extent of peptides to be rescued by recently developed algorithms.

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