A.G. Van Der Heijden

The La (SS-B) autoantigen, a key protein in RNA biogenesis, is dephosphorylated and cleaved early during apoptosis.

In the past few years, a role for apoptotic processes in the development of autoimmune diseases has been suggested. An increasing number of cellular proteins, which are modified during apoptosis, has been described, and many of these proteins have been identified as autoantigens. We have studied the effects of apoptosis on the La protein in more detail and for the first time demonstrate that this autoantigen is rapidly dephosphorylated after the induction of apoptosis. Dephosphorylation of the La protein was observed after induction of apoptosis by several initiators and in various cell types. Furthermore, we demonstrate that at least a subset of the La protein is proteolytically cleaved in vivo, generating a 45 kDa fragment. Dephosphorylation as well as cleavage of La is inhibited by ZnSO4 as well as by several tetrapeptide caspase inhibitors, indicating that these processes require the activation of caspases. Dephosphorylation of La is inhibited by low concentrations of okadaic acid, suggesting that a PP2A-like phosphatase is involved. Generation of the 45 kDa fragment is consistent with proteolytic cleavage at amino acids 371 and/or 374. The possible significance of the apoptotic changes in the La protein for autoantibody production is discussed.

REVIEW PATHOLOGY IN A DIAGNOSTIC BLADDER CANCER TRIAL: THE IMPACT OF PATIENT RISK CATEGORY

Abstract Objectives Bladder cancer pathologic features are a continuous spectrum from benign to invasive lesions, causing diagnostic difficulties. Review pathology might be an answer, but appears to be of limited value. We studied the effect of patients’ risk profile on the value of review pathology. Methods We used three Phase III multicenter studies that assessed the value of hexaminolevulinate fluorescence cystoscopy on diagnosis and management. Two studies (Europe and United States) included patients at high risk of carcinoma in situ (CIS), the third study (Europe) included all patients at risk of bladder cancer. Tumors and biopsies were examined by a local and review pathologist. Results The percentage of patients with CIS was high in the first two studies (20.6% and 15.9%) compared with the epidemiologic data (7.9%) and the third study (7.8%). The numbers of patients (specimens) in the three studies were 209 (927), 277 (986), and 142 (553). Overall conformity for both grade and stage was between 50.5% and 56.6%, comparable to published data. Although conformity was best in the high-risk study, this was predominantly because of the better conformity in low-risk tumors. Conformity in Stage T1, CIS, and invasive tumors was low. The results from Europe and the United States were comparable, although the local pathologist in the United States tended to overstage or overgrade. Conclusions Although histologic conformity was greater in the high-risk patient population, this was mainly a result of pTa tumors. The diagnosis of pT1, CIS, and invasiveness appears difficult. Because these tumors significantly influence therapy, review pathology in patients at high risk or suspicious for high risk should be considered.

Citrullination of synovial proteins in murine models of rheumatoid arthritis

OBJECTIVE Antibodies directed to citrulline-containing proteins are highly specific for rheumatoid arthritis (RA) and can be detected in up to 80% of patients with RA. Citrulline is a nonstandard amino acid that can be incorporated into proteins only by posttranslational modification of arginine by peptidylarginine deiminase (PAD) enzymes. The objective of this study was to investigate the presence of anticitrulline antibodies, PAD enzymes, and citrullinated antigens in mouse models of both acute and chronic destructive arthritis: streptococcal cell wall (SCW)-induced arthritis and collagen-induced arthritis (CIA), respectively. METHODS Synovial tissue biopsy specimens were obtained from naive mice, mice with CIA, and mice with SCW-induced arthritis. The expression of messenger RNA (mRNA) for PAD enzymes was analyzed by reverse transcriptase-polymerase chain reaction; the presence of PAD proteins and their products (citrullinated proteins) was analyzed by Western blotting and by immunolocalization. The presence of anticitrullinated protein antibodies was investigated by an anti-cyclic citrullinated peptide (anti-CCP) enzyme-linked immunosorbent assay (ELISA) and an ELISA using in vitro citrullinated fibrinogen. RESULTS In both mouse models, PAD type 2 (PAD2) mRNA was present in the synovium but was not translated into PAD2 protein. In contrast, PAD4 mRNA, although absent from healthy synovium, was readily transcribed and translated by polymorphonuclear neutrophils infiltrating the synovial tissue during inflammation. As a consequence, several synovial proteins were subjected to citrullination. One of these proteins was identified as fibrin, which has been reported to be citrullinated also in synovium of patients with RA. Although generation of citrullinated antigens during synovial inflammation in the mice was eminent, no anti-CCP antibodies could be detected. CONCLUSION Citrullination of synovial antigens is an active process during joint inflammation in both mice and humans, but the induction of autoantibodies directed to these proteins is a more specific phenomenon, detectable only in human RA patients.

Effects of hyperthermia in neutralising mechanisms of drug resistance in non-muscle-invasive bladder cancer

Abstract Non-muscle-invasive bladder cancer is a challenging disease, even given its superficial nature. It is prone to multiple recurrences and progression to muscle-invasive cancer. These features of this disease contribute significantly to reduced quality of life as well as creating significant morbidity and even mortality. Randomised trials demonstrate that when hyperthermia is added to conventional mitomycin-C treatment that local control rates and progression-free survival are substantially improved. In this review we consider how hyperthermia can exert such beneficial effects. Some of the mechanisms presented are theoretical, while others are facts. It is hoped that this review will contribute rationale for further examination of mechanisms, because an understanding of such mechanisms may lead to even better chemotherapeutic approaches, as well as potential biomarkers for predicting and monitoring treatment success.

Expression of PAD enzymes and occurrence of citrulline-containing proteins in human blood and synovial fluid cells

Antibodies directed against citrulline-containing antigens are extremely specific for RA. The amino acid citrulline is not incorporated into proteins during protein synthesis. It is generated by posttranslational modification of arginine residues by PAD (peptidylarginine deiminase) enzymes. We investigated the expression of PAD enzymes and the occurrence of citrullinated proteins in peripheral blood (PB) and synovial fluid (SF) cells. PAD types 1 and 3 were absent from the investigated cells, while PAD types 2 and 4 (also known as type 5) were present. In monocyte-derived macrophages PAD type 2 mRNA expression was at a similiar level as in monocytes, while PAD type 2 protein was increased. PAD type 4 mRNA expression was significant in monocytes and almost absent in monocyte-derived macrophages, while PAD type 4 protein levels were similar. In monocytes no citrullinated protein could be detected, while in monocyte-derived macrophages citrullinated vimentin, which is (part of) the Sa-antigen, was present. A similar pattern of mRNA and protein expression was observed in mononuclear cells in paired PB and SF samples of RA patients. These results suggest that PAD type 2 is involved in the citrullination of SF proteins during inflammation.

Reduce bladder cancer recurrence in patients treated for upper urinary tract urothelial carcinoma: The REBACARE-trial

Background Following radical nephro-ureterectomy for urothelial carcinoma of the upper urinary tract (UUT), the reported bladder recurrence rate of urothelial carcinoma is 22–47%. A single intravesical instillation of chemotherapy within 10 days following nephro-ureterectomy has the potential to decrease the risk of a bladder recurrence significantly. Despite recommendation by the European Association of Urology guideline to administer a single instillation postoperatively, the compliance rate is low because the risk of extravasation of chemotherapy. Aim To reduce the risk of bladder cancer recurrence by a single intravesical instillation of Mitomycin immediately (within 3 h) before radical nephro-ureterectomy or partial ureterectomy. Methods Adult patients (age ≥ 18 years) with a (suspicion of a) urothelial carcinoma of the UUT undergoing radical nephro-ureterectomy or partial ureterectomy will be eligible and will receive a single intravesical instillation of Mitomycin within 3 h before surgery. In total, 170 patients will be included in this prospective, observational study. Follow-up will be according to current guidelines. Results The primary endpoint is the bladder cancer recurrence rate up to two years after surgery. Secondary endpoints are: a) the compliance rate; b) oncological outcome; c) possible side-effects; d) the quality of life; e) the calculation of costs of a single neoadjuvant instillation with Mitomycin and f) molecular characterization of UUT tumors and intravesical recurrences. Conclusions A single intravesical instillation of Mitomycin before radical nephro-ureterectomy or partial ureterectomy may reduce the risk of a bladder recurrence in patients treated for UUT urothelial carcinoma and will circumvent the disadvantages of current therapy.

830 Clinical validation of a gene expression test for the non-invasive diagnosis of bladder cancer: A prospective, blinded, international and multicenter study

aDepartment and Laboratory of Urology, Hospital Clínic, Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Universitat de Barcelona, Spain; bCIBERehd, Plataforma de Bioinformática, Centro de Investigación Biomédica en red de Enfermedades Hepáticas y Digestivas, Universidad de Barcelona, Spain; cDepartment of Urology, Fundació Puigvert, Barcelona, Spain; dDepartment of Urology, Radboud University Medical Centre, Nijmegen, The Netherlands; eHospital Universitario Virgen del Rocío/CSIC/Universidad de Sevilla, Spain; fDepartment of Urology, Medical University of Vienna; gPathology Department, Hospital Clínic, Universitat de Barcelona, Spain.