Treatment of High-grade Non-muscle-invasive Bladder Carcinoma by Standard Number and Dose of BCG Instillations Versus Reduced Number and Standard Dose of BCG Instillations: Results of the European Association of Urology Research Foundation Randomised Phase III Clinical Trial "NIMBUS".

BACKGROUND: Intravesical instillation of bacillus Calmette-Guérin (BCG) is an accepted strategy to prevent recurrence of non-muscle-invasive bladder cancer (NMIBC) but associated with significant toxicity. OBJECTIVE: NIMBUS assessed whether a reduced number of standard-dose BCG instillations are noninferior to the standard number and dose in patients with high-grade NMIBC. DESIGN, SETTING, AND PARTICIPANTS: A total of 345 patients from 51 sites were randomised between December 2013 and July 2019. We report results after a data review and safety analysis by the Independent Data Monitoring Committee based on the cut-off date of July 1, 2019. INTERVENTION: The standard BCG schedule was 6 wk of induction followed by 3 wk of maintenance at 3, 6, and 12 mo (15 instillations). The reduced frequency BCG schedule was induction at wks 1, 2, and 6 followed by 2 wk (wks 1 and 3) of maintenance at 3, 6, and 12 mo (nine instillations). OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: The primary endpoint was time to first recurrence. Secondary endpoints included progression to ≥ T2 and toxicity. RESULTS AND LIMITATIONS: In total, 170 patients were randomised to reduced frequency and 175 to standard BCG. Prognostic factors at initial resection were as follows: Ta/T1: 46/54%; primary/recurrent: 92/8%; single/multiple: 57/43%; and concomitant carcinoma in situ: 27%. After 12 mo of median follow-up, the intention-to-treat analysis showed a safety-relevant difference in recurrences between treatment arms: 46/170 (reduced frequency) versus 21/175 patients (standard). Additional safety analyses showed a hazard ratio of 0.40 with the upper part of the one-sided 97.5% confidence interval of 0.68, meeting a predefined stopping criterion for inferiority. CONCLUSIONS: The reduced frequency schedule was inferior to the standard schedule regarding the time to first recurrence. Further recruitment of patients was stopped immediately to avoid harm in the reduced frequency BCG arm. PATIENT SUMMARY: After surgical removal of the tumour, patients with high-grade non-muscle-invasive bladder cancer are treated with bacillus Calmette-Guérin to prevent recurrence and progression. This is associated with significant side effects. We report the results of a clinical trial showing a reduction in the number of instillations (from 15 to nine in total) being inferior to the standard protocol. From today's perspective, complete tumour resection and a standard number of instillations remain the standard of care.

Adjuvant recMAGE-A3 Immunotherapy After Cystectomy for Muscle-invasive Bladder Cancer: Lessons Learned from the Phase 2 MAGNOLIA Clinical Trial.

The MAGNOLIA study, investigating the concept of perioperative immunotherapy in muscle- invasive bladder cancer, was prematurely terminated. The lessons learned that should be considered before initiating and conducting future clinical trials in this field are highlighted.

Proteins and peptides in parotid saliva of irradiated patients compared to that of healthy controls using SELDI-TOF-MS.

BACKGROUND: Radiotherapy to the head and neck area damages the salivary glands. As a consequence hyposalivation may occur, but also the protein composition of saliva may be affected possibly compromising oral health. The aim of our study was to compare the relative abundance of proteins and peptides in parotid saliva of irradiated patients to that of healthy controls. METHODS: Using Lashley cups and citric acid, saliva from the parotid glands was collected from nine irradiated patients and ten healthy controls. The samples were analyzed with SELDI-TOF-MS using a NP20 and IMAC-30 chip in the molecular weight range of 1-30 kDa. RESULTS: On the NP20 chip 61 (out of 217) and on the IMAC-30 chip 32 (out of 218) peaks differed significantly in intensity between the saliva of the irradiated patients and healthy controls. 55 % of the significant peaks showed higher intensity and 45 % showed lower intensity in the saliva of irradiated patients. The peaks may represent, amongst others, the salivary proteins lysozyme, histatins, cystatin, protein S100 and PRP's. CONCLUSIONS: Large differences were found in the relative abundance of a wide range of proteins and peptides in the parotid saliva of irradiated patients compared to healthy controls.

Perioperative chemotherapy in muscle-invasive bladder cancer: overview and the unmet clinical need for alternative adjuvant therapy as studied in the MAGNOLIA trial.

The European Association of Urology Research Foundation has proposed that alternatives to perioperative chemotherapy should be evaluated. The MAGNOLIA study represents a unique opportunity to investigate the concept of immunotherapy in muscle-invasive bladder cancer.

Identification of salivary proteins at oil-water interfaces stabilized by lysozyme and beta-lactoglobulin.

In this research, we investigated the interaction occurring between oil-in-water emulsion droplets, stabilized by different emulsifiers, i.e. lysozyme and beta-lactoglobulin (beta-lg), and salivary proteins (SPs) with a molecular mass (M(r)) above about 10kDa. Different techniques, i.e. infrared spectroscopy, Western blotting, PAS staining and SDS-PAGE coupled to MS, were employed for this purpose. This study demonstrated the interaction between several salivary proteins and the emulsifiers at the oil-water interfaces. In particular, results show that the high M(r) mucin MUC5B was strongly bound to lysozyme stabilized emulsions, whereas beta-lg stabilized emulsions associated with MUC7 and, moderately, with MUC5B. Furthermore, we observed that salivary proteins in the range M(r) 10-100kDa associated differently with emulsion droplets. A large majority of SPs was found to interact with lysozyme stabilized emulsion droplets whilst in case of beta-lg stabilized emulsions, the SPs distribute more evenly between the fraction associated and non-associated with the droplets. A clear example is alpha-amylase (M(r) approximately 55kDa) which predominantly associates with lysozyme stabilized emulsion droplets, but not with beta-lg emulsion droplets. To conclude, our findings indicate that adsorption/association of salivary protein components onto the emulsion droplets is related to the type of emulsifying proteins at the oil-water interfaces and it is probably driven by the overall net charge at the droplet's oil-water interfaces, i.e. positive for lysozyme stabilized emulsions and negative for beta-lactoglobulin stabilized emulsion at neutral pH.

Mucin-bacterial interactions in the human oral cavity and digestive tract.

Mucins are a family of heavily glycosylated proteins that are the major organic components of the mucus layer, the protective layer covering the epithelial cells in many human and animal organs, including the entire gastro-intestinal tract. Microbes that can associate with mucins benefit from this interaction since they can get available nutrients, experience physico-chemical protection and adhere, resulting in increased residence time. Mucin-degrading microorganisms, which often are found in consortia, have not been extensively characterized as mucins are high molecular weight glycoproteins that are hard to study because of their size, complexity and heterogeneity. The purpose of this review is to discuss how advances in mucus and mucin research, and insight in the microbial ecology promoted our understanding of mucin degradation. Recent insight is presented in mucin structure and organization, the microorganisms known to use mucin as growth substrate, with a specific attention on Akkermansia muciniphila, and the molecular basis of microbial mucin degradation owing to availability of genome sequences.

Fat retention at the tongue and the role of saliva: adhesion and spreading of 'protein-poor' versus 'protein-rich' emulsions.

Fat perception of food emulsions has been found to relate to in-mouth friction. Previously, we have shown that friction under mouth-like conditions strongly depends on the sensitivity of protein-stabilized emulsion droplets to coalescence. Here, we investigated whether this also implies that oral fat retention depends in a similar manner on the stability of the emulsion droplets against coalescence. We investigate the separate contributions of droplet adhesion and droplet spreading to fat retention at the tongue, as well as the role of saliva. We perform ex vivo (Confocal Raman Spectroscopy; Confocal Scanning Laser Microscopy) experiments using pig's tongue surfaces in combination with human in vivo experiments. These reveal that protein-poor (unstable) emulsions are retained more at the tongue than protein-rich (stable) emulsions. Furthermore, the layer formed by adhering protein-poor droplets is more stable against rinsing. Saliva is found to be very efficient in removing fat and emulsion droplets from the oral surface but its role in fat retention needs further research. We relate our results to the colloidal forces governing droplet adhesion and spreading.

Saliva as research material: biochemical, physicochemical and practical aspects.

Whole saliva is a complex mixture of proteins and other molecules which originate from several sources. The biochemical and physicochemical properties of saliva contribute to the numerous functions of saliva in, e.g., speech, maintaining oral and general health, and food processing. Interest in saliva has increased in the last few years for its potential to diagnose viral, bacterial and systemic diseases. The use of saliva as research material may pose particular problems due to its inherent variability and instability. This review describes practical aspects of salivary as research material with emphasis on protein biochemistry and physical chemistry.

Salivary protein/peptide profiling with SELDI-TOF-MS.

In this study, large-scale profiling of salivary proteins and peptides ranging from 2 to 100 kDa was demonstrated using surface-enhanced laser desorption/ionization-time of flight-mass spectrometry (SELDI-TOF-MS). Results show that chip surface type and sample type critically affect the amount and composition of detected salivary proteins. Delayed processing time resulted in both increase and decrease of peak numbers consistent with proteolysis. SELDI-TOF-MS profiles also changed, depending on storage temperature, although sample processing by centrifugation and numbers of freeze-thaw cycles had a minimal impact. In conclusion, SELDI-TOF-MS offers a simple, rapid, high-throughput technique for profiling low-mass (<10 kDa) saliva proteins/peptides. We wish to use this technique to gain insight into the human saliva proteome composition and its changes over time in response to food consumption.

Salivary biomarkers associated with perceived satiety and body mass in humans.

Regulation of food intake and energy homeostasis is controlled by a delicate balancing of numerous central and peripheral factors, including circulating peptide hormones. This study investigated the proteome of saliva using SELDI-TOF-MS in relation to satiety and body mass index (BMI) in humans. Within a longitudinal test session, 18 subjects were exposed to a lunch-induced hunger-satiety shift, while every 15 min collecting their whole saliva and rating their hunger and satiety. Saliva was analysed by SELDI-TOF-MS using IMAC arrays with a chromatographic copper surface (IMAC-Cu). From all subjects and time points measured, peptide and protein profiles showed 190 common peaks. Their interrelationships show that 37% of the variation was accounted for in one dimension. About 30 means had a strong association (0.70<|r|<0.95) with all subjective satiety ratings across time during the test session, and seven peaks were significantly correlated to BMI. Database MS searches indicated characterisation of some relevant metabolic peptide hormones. In conclusion, SELDI-TOF-MS on human saliva provides a valuable and noninvasive way of profiling that enables characterisation of novel and differently expressed peptides and proteins which can be used as biomarkers of satiety and overweight.

SELDI-TOF-MS of saliva: methodology and pre-treatment effects.

Interest in saliva as a diagnostic fluid for monitoring general health and for early diagnosis of disease has increased in the last few years. In particular, efforts have focused on the generation of protein maps of saliva using advanced proteomics technology. Surface-enhanced laser-desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS) is a novel high throughput and extremely sensitive proteomic approach that allows protein expression profiling of large sets of complex biological specimens. In this study, large scale profiling of salivary proteins and peptides, ranging from 2 to 100kDa was demonstrated using SELDI-TOF-MS. Various methodological aspects and pre-analytical variables were analysed with respect to their effects on saliva SELDI-TOF-MS profiling. Results show that chip surface type and sample type (unstimulated versus stimulated) critically affect the amount and composition of detected salivary proteins. Factors that influenced normal saliva protein profiling were matrix composition, sample dilution and binding buffer properties. Delayed processing time experiments show certain new peptides evolving 3h post-saliva donation, and quantitative analyses indicate relative intensity of other proteins and peptides changing with time. The addition of protease inhibitors partly counteracted the destabilization of certain protein/peptide mass spectra over time suggesting that some proteins in saliva are subject to digestion by intrinsic salivary proteases. SELDI-TOF-MS profiles also changed by varying storage time and storage temperature whereas centrifugation speed and freeze-thaw cycles had minimal impact. In conclusion, SELDI-TOF-MS offers a high throughput platform for saliva protein and peptide profiling, however, (pre-)analytical conditions must be taken into account for valid interpretation of the acquired data.

Induction of apoptosis in hematopoietic cells with an antibody against tomoregulin-1.

Tomoregulin-1, a type-I transmembrane protein with two follistatin modules, a unique epidermal growth factor (EGF) domain and a short, highly conserved cytoplasmic tail, was studied. A number of hematopoietic cell lines (L1210, CEM, Jurkat, U937, K562, JY, THP-1 and T2) express tomoregulin-1 endogenously. In these cells, apoptosis was induced by an antiserum (C29) and purified IgG against the follistatin modules, but not by antisera against the EGF-domain or the cytoplasmic tail. Furthermore, C29 induced apoptosis in tomoregulin-1-, but not in mock-transfected cells. Apoptosis was monitored through genomic DNA fragmentation, annexin-V staining and caspase-3 activation. Treatment of the cells with C29 in the presence of H89 (a SerlThr kinase inhibitor) or 8'-bromo-cyclicAMP revealed that apoptosis was mediated by a cAMP-dependent Ser/Thr kinase. Moreover, C29 increased [cAMP]i over 5-fold. Together, these data suggest that the C29 antiserum against tomoregulin-1 induces apoptosis of hematopoietic cells.

Intracellular localization of ornithine decarboxylase and its regulatory protein, antizyme-1.

The enzyme ornithine decarboxylase (ODC) and its regulatory protein antizyme-1 (AZ1) are key regulators in the homeostasis of polyamines. To gain more insight into the exact intracellular distribution of ODC and AZ1, we performed immunocytochemical and Green Fluorescent Protein-fluorocytochemical studies in cultured human cervix carcinoma and human prostatic carcinoma (PC-346C) cells. ODC localization patterns varied from predominantly cytoplasmic to both cytoplasmic and nuclear staining, whereas AZ1 was mostly found in the nucleus. In cells that were synchronized in the mitotic phase, localization of both ODC and AZ1 changed from perinuclear at the beginning of mitosis into nucleoplasmic at close proximity to the chromosomes during meta-, ana- and telophase. Upon completion of mitosis, localization of ODC and AZ1 was reverted back to the cytoplasm, i.e., predominantly perinuclear immediately after cytokinesis. When PC-346C cells were treated with polyamines to induce AZ1-regulated ODC degradation, ODC was predominantly found in the nucleus and colocalized with immunoreactive AZ1. A comparable accumulation of ODC and AZ1 in the nucleus was found in PC-346C cells treated with the polyamine analog SL-11093. The present study suggests that AZ1 is involved in nucleocytoplasmic shuttling of ODC, which may be a prerequisite for ODC regulation and/or function.

Distribution patterns of ornithine decarboxylase in cells and tissues: facts, problems, and postulates.

Ornithine decarboxylase (ODC) is a key enzyme in polyamine biosynthesis. Increased polyamine levels are required for growth, differentiation, and transformation of cells. In situ detection of ODC in cells and tissues has been performed with biochemical, enzyme cytochemical, immunocytochemical, and in situ hybridization techniques. Different localization patterns at the cellular level have been described, depending on the type of cells or tissues studied. These patterns varied from exclusively cytoplasmic to both cytoplasmic and nuclear. These discrepancies can be partially explained by the (lack of) sensitivity and/or specificity of the methods used, but it is more likely that (sub)cellular localization of ODC is cell type-specific and/or depends on the physiological status (growth, differentiation, malignant transformation, apoptosis) of cells. Intracellular translocation of ODC may be a prerequisite for its regulation and function.

Expression of intestinal ornithine decarboxylase during postnatal development in neonatal rats.

Ornithine decarboxylase (ODC) has been shown to play an essential role in intestinal growth and maturation in rats. However, the regulatory mechanisms have not been fully elucidated. We studied the mechanisms of expression of intestinal ODC during postnatal development. Rat small intestinal mucosa was obtained from postnatal days 10, 15, 17, 19, 21, 24 and 30. Intestinal mucosa was assayed for ODC and sucrase activities. In addition, intestinal ODC mRNA, and ODC protein levels were also measured. The results showed that the intestinal sucrase activity was low before postnatal day 19. The sucrase activity then increased steadily from day 19 up to day 30. Intestinal ODC activities remained low from postnatal day 10 to day 17. A sharp increase in ODC activity was noted on day 19, which peaked on day 24 (a 20-fold increase from its low basal level) and declined on day 30. Intestinal ODC proteins followed the same pattern of postnatal expression as that of ODC activity. In contrast, ODC mRNA did not show significant change throughout the study period. The possible mechanisms by which intestinal ODC mRNA levels remain practically unchanged during postnatal development are discussed. We conclude that the ontogenic increase in sucrase activity, a marker for intestinal maturation, occurs at the same time to that of the induction of ODC activity. We also suggest that the induction of intestinal ODC activity during postnatal development is the result of post-transcriptional events or other cellular mechanisms. A better understanding of the regulation of polyamine biosynthesis during postnatal development of the small intestine will provide insights contributing to the maturation of the small intestine.