Addition of small-bowel transit scintigraphy to gastric emptying for assessment of patients with upper gastrointestinal symptoms.

BACKGROUND: Dyspeptic symptoms are not well correlated with gastric emptying (GE) results. AIMS: To determine (a) prevalence of delayed SB transit (SBT) in patients undergoing GE scintigraphy for symptoms of gastroparesis; (b) symptoms associated with delayed SBT. METHODS: Patients with symptoms of gastroparesis underwent combined GE and SBT scintigraphy (GES/SBTS). Patients ingested a mixed solid (S)-liquid (L) meal with egg whites labeled with 500 µCi Tc-99 m sulfur colloid and water with 125 µCi In-111 DTPA. Retained S and L gastric activity and percent of L In-111 activity in terminal ileum (TI) and/or cecum/colon at 6 h were determined. Patient Assessment of Gastrointestinal Symptoms (PAGI-SYM) assessed symptoms from 0 (none) to 5 (very severe). KEY RESULTS: Of 363 patients, 174 (47.9%) had delayed S GE, 141 (38.8%) delayed L GE, and 70 (19.3%) delayed SBT. Delayed SBT was seen in 24 (6.6%) with normal S GE and 46 (12.7%) with delayed S GE. Patients with isolated delayed SBT had highest symptom scores for postprandial fullness (3.5), stomach fullness (3.4), nausea (3.2), bloating (3.2), compared to isolated delayed S GE who had highest symptom scores for postprandial fullness (3.7), nausea (3.6), stomach fullness (3.4), and early satiety (3.3). CONCLUSIONS & INFERENCES: Delayed SBT occurred in 19.3% of dyspeptic patients using GES/SBTS. While postprandial and stomach fullness were common to both delayed S GE and delayed SBT, early satiety was associated with delayed S GE whereas bloating was associated with delayed SBT. Thus, SBTS can augment GES to help explain some symptoms associated with dyspepsia and suspected gastroparesis.

New horizons in bladder cancer research.

The 16th Meeting of the International Bladder Cancer Network (IBCN) took place from October 11 to 13, 2018 in Rotterdam, the Netherlands. As in the previous year, muscle-invasive bladder cancer (MIBC) was the main topic of the congress based upon the rapid evolution in this field over the last several years. This year´s meeting was dominated by presentations focusing on genomic subtyping in MIBC and identification of novel therapeutic targets. These topics were complemented by submissions on immunotherapy, a variety of clinical topics, and biomarker research. Based upon the presentations, it may be concluded that the IBCN increasingly serves as an interdisciplinary forum not only for the presentation of work-in-progress covering all facets of bladder cancer research, but also for catalyzing the discussion of discrepant research findings in an effort to find consensus. The 16th Meeting of the IBCN took place from October 11 to 13th, 2018 in Rotterdam, the Netherlands, hosted by Ellen Zwarthoff and Joost Boormans. Approximately 120 participants gathered for another fully packed program displaying recent achievements in basic and clinical research covering the entire spectrum of bladder cancer. This year's meeting was dominated by presentations focusing on genomic subtyping and identification of novel therapeutic targets. These topics were complemented by submissions on immunotherapy, clinical topics, and biomarker research. Keynote lectures were delivered by G. Robertson (Canada) on MIBC genomics and the organizational challenges of the PanCancerAtlas project. Comprehensive information was provided on the Cancer Genome Atlas (TCGA) project including the structure of the consortium and the development and validation of molecular classification of MIBC. Results from the project suggest that regulons (groups of genes controlled by a common regulator) appear to be correlated with prognosis and may replace gene expression analysis in the future. M. Thelen (Switzerland) discussed the role of chemokines in cancer metastasization reporting on his research on the atypical chemokine receptor 3. The inaugural IBCN lecture was presented by M. Ingersoll (France) discussing gender disparities in development of adaptive immunity following Bacillus Calmette-Guerin therapy. In addition to the traditional "Industry meets IBCN" sessions, a format for discussion between industry representatives, researchers, and physicians in break-out groups, new formats were introduced-in the Oxford style of debate, P.J. Goebell (Germany) and W. Stadler (USA) delivered animated discussion on the appropriateness of urologists administering systemic immune checkpoint inhibitors. There was consensus that practice may differ between different health systems and countries, and educational/training background. In a second interesting discussion, D. McConkey (USA) and H. Al-Ahmadie (USA) debated if traditional histology will be replaced by molecular classification of bladder tumors. It was concluded that molecular classification offers valuable additional information but cannot yet replace traditional histology. Furthermore, the growing evidence of tumor heterogeneity in bladder cancer was discussed in a separate topic session. L. Dyrskjot discussed genomic and transcriptomic heterogeneity and their impact on clinical decision making. G. Sjödahl presented data on heterogeneity of molecular subtypes within the primary tumor and between the primary tumor and metastases, that is surprisingly limited. Y. Allory discussed spatial and temporal heterogeneity of bladder cancers particularly focusing on the basal-like phenotype. Due to the continued increase in the number of participants and abstract submissions, a poster session was implemented at this year´s meeting for the first time. Posters were briefly presented in the forum. Two travel awards were presented to H. Yamashita (USA) for his submission on peroxisome proliferator-activated receptor gamma-mediated repression of transcription factor activating protein 2 alfa expression identifying a transcriptional circuit in basal-squamous bladder cancer in a cell line model. S.B. Williams (USA) received his award for his registry-based analysis of outcome and costs in patients with localized MIBC undergoing either bladder sparing trimodal therapy or radical cystectomy. The latter was found to generate improved survival outcomes at lower costs as compared with trimodal therapy. The best presentation awards were presented to A. Kamoun (France) who discussed a consensus molecular classification for MIBC. The international demand for a consensus classification, already raised at previous meetings, has resonated with important players in this field. A potential consensus comprising 6 classes was proposed based upon a thorough analysis of the existing classifications. As an extension of the IBCN meeting a consensus conference on genomic classification of MIBC was held that included all major groups in this field. M. Garige (USA) received his award for a detailed examination of inhibitors of metabolic processes in bladder cancer cells before and after therapy. A take home message of the meeting was that the IBCN increasingly serves as an interdisciplinary forum not for the presentation of work-in-progress covering all facets of bladder cancer research, but also for catalyzing the discussion of discrepant research findings in an effort to find consensus. Exemplified may this also be by the fact that the efforts to unify the subclassification of MIBC had its nidus on the preceding meetings of the IBCN on this topic and brought together the various disciplines which ultimately were able to finalize their consensus work in a last concluding meeting directly following the IBCN. Thus, the IBCN meetings and the close cooperation of IBCN with its official journal, Urological Oncology, continue to provide a unique platform for exchange, discussing and intensifying multidimensional collaborations, thus finally satisfying the increasing need for answers regarding the management of bladder cancer patients. The 17th meeting of the IBCN will take place in Aarhus, Denmark, October 3 to 5, 2019.

Novel PKC-ζ to p47 phox interaction is necessary for transformation from blebbishields.

Cancer stem cells are capable of transformation after apoptosis through the blebbishield emergency program. Reactive oxygen species (ROS) play an essential role in transformation. Understanding how ROS are linked to blebbishield-mediated transformation is necessary to develop efficient therapeutics that target the resurrection of cancer stem cells. Here we demonstrate that a novel PKC-ζ to p47(phox) interaction is required for ROS production in cancer cells. The combined use of the S6K inhibitor BI-D1870 with TNF-α inhibited the PKC-ζ to p47(phox) interaction, inhibited ROS production, degraded PKC-ζ, and activated caspases-3 and -8 to block transformation from blebbishields. BI-D1870 also inhibited transformation from cycloheximide-generated blebbishields. Thus ROS and the PKC-ζ to p47(phox) interaction are valid therapeutic targets to block transformation from blebbishields.

Hydrogen Sulfide Epigenetically Attenuates Homocysteine-Induced Mitochondrial Toxicity Mediated Through NMDA Receptor in Mouse Brain Endothelial (bEnd3) Cells.

Previously we have shown that homocysteine (Hcy) caused oxidative stress and altered mitochondrial function. Hydrogen sulfide (H2S) has potent anti-inflammatory, anti-oxidative, and anti-apoptotic effects. Therefore, in the present study we examined whether H2S ameliorates Hcy-induced mitochondrial toxicity which led to endothelial dysfunction in part, by epigenetic alterations in mouse brain endothelial cells (bEnd3). The bEnd3 cells were exposed to 100 µM Hcy treatment in the presence or absence of 30 µM NaHS (donor of H2S) for 24 h. Hcy-activate NMDA receptor and induced mitochondrial toxicity by increased levels of Ca(2+), NADPH-oxidase-4 (NOX-4) expression, mitochondrial dehydrogenase activity and decreased the level of nitrate, superoxide dismutase (SOD-2) expression, mitochondria membrane potentials, ATP production. To confirm the role of epigenetic, 5'-azacitidine (an epigenetic modulator) treatment was given to the cells. Pretreatment with NaHS (30 µM) attenuated the Hcy-induced increased expression of DNMT1, DNMT3a, Ca(2+), and decreased expression of DNMT3b in bEND3 cells. Furthermore, NaHS treatment also mitigated mitochondrial oxidative stress (NOX4, ROS, and NO) and restored ATP that indicates its protective effects against mitochondrial toxicity. Additional, NaHS significantly alleviated Hcy-induced LC3-I/II, CSE, Atg3/7, and low p62 expression which confirm its effect on mitophagy. Likewise, NaHS also restored level of eNOS, CD31, VE-cadherin and ET-1 and maintains endothelial function in Hcy treated cells. Molecular inhibition of NMDA receptor by using small interfering RNA showed protective effect whereas inhibition of H2S production by propargylglycine (PG) (inhibitor of enzyme CSE) showed mitotoxic effect. Taken together, results demonstrate that, administration of H2S protected the cells from HHcy-induced mitochondrial toxicity and endothelial dysfunction.

Kinetics and mechanism of (•)OH mediated degradation of dimethyl phthalate in aqueous solution: experimental and theoretical studies.

The hydroxyl radical ((•)OH) is one of the main oxidative species in aqueous phase advanced oxidation processes, and its initial reactions with organic pollutants are important to understand the transformation and fate of organics in water environments. Insights into the kinetics and mechanism of (•)OH mediated degradation of the model environmental endocrine disruptor, dimethyl phthalate (DMP), have been obtained using radiolysis experiments and computational methods. The bimolecular rate constant for the (•)OH reaction with DMP was determined to be (3.2 ± 0.1) × 10(9) M(-1)s(-1). The possible reaction mechanisms of radical adduct formation (RAF), hydrogen atom transfer (HAT), and single electron transfer (SET) were considered. By comparing the experimental absorption spectra with the computational results, it was concluded that the RAF and HAT were the dominant reaction pathways, and OH-adducts ((•)DMPOH1, (•)DMPOH2) and methyl type radicals (•)DMP(-H)α were identified as dominated intermediates. Computational results confirmed the identification of transient species with maximum absorption around 260 nm as (•)DMPOH1 and (•)DMP(-H)α, and these radical intermediates then converted to monohydroxylated dimethyl phthalates and monomethyl phthalates. Experimental and computational analyses which elucidated the mechanism of (•)OH-mediated degradation of DMP are discussed in detail.

Commentary on "Phase II trial of cetuximab with or without paclitaxel in patients with advanced urothelial tract carcinoma." Wong YN, Litwin S, Vaughn D, Cohen S, Plimack ER, Lee J, Song W, Dabrow M, Brody M, Tuttle H, Hudes G, University of Pennsylvania, Philadelphia, PA: J Clin Oncol 2012;30(28):3545-51 [Epub 2012 Aug 27].

PURPOSE: The benefit of salvage chemotherapy is modest in metastatic urothelial cancer. We conducted a randomized, noncomparative phase II study to measure the efficacy of cetuximab with or without paclitaxel in patients with previously treated urothelial cancer. PATIENTS AND METHODS: Patients with metastatic urothelial cancer who received one line of chemotherapy in the perioperative or metastatic setting were randomly assigned to 4-week cycles of cetuximab 250 mg/m(2) with or without paclitaxel 80 mg/m(2) per week. We used early progression as an indicator of futility. Either arm would close if seven of the initial 15 patients in that arm progressed at the first disease evaluation at 8 weeks. RESULTS: We enrolled 39 evaluable patients. The single-agent cetuximab arm closed after nine of the first 11 patients progressed by 8 weeks. The combination arm completed the full accrual of 28 patients, of whom 22 patients (78.5%) had visceral disease. Twelve of 28 patients had progression-free survival greater than 16 weeks. The overall response rate was 25% (95% CI, 11% to 45%; three complete responses and four partial responses). The median progression-free survival was 16.4 weeks (95% CI, 12 to 25.1 weeks), and the median overall survival was 42 weeks (95% CI, 30.4 to 78 weeks). Treatment-related grade 3 and 4 adverse events that occurred in at least two patients were rash (six cases), fatigue (five cases), and low magnesium (three cases). CONCLUSION: Although it had limited activity as a single agent, cetuximab appears to augment the antitumor activity of paclitaxel in previously treated urothelial cancers. The cetuximab and paclitaxel combination merits additional study to establish its role in the treatment of urothelial cancers.

The catalase activity of diiron adenine deaminase.

Adenine deaminase (ADE) from the amidohydrolase superfamily (AHS) of enzymes catalyzes the conversion of adenine to hypoxanthine and ammonia. Enzyme isolated from Escherichia coli was largely inactive toward the deamination of adenine. Molecular weight determinations by mass spectrometry provided evidence that multiple histidine and methionine residues were oxygenated. When iron was sequestered with a metal chelator and the growth medium supplemented with Mn(2+) before induction, the post-translational modifications disappeared. Enzyme expressed and purified under these conditions was substantially more active for adenine deamination. Apo-enzyme was prepared and reconstituted with two equivalents of FeSO(4). Inductively coupled plasma mass spectrometry and Mössbauer spectroscopy demonstrated that this protein contained two high-spin ferrous ions per monomer of ADE. In addition to the adenine deaminase activity, [Fe(II) /Fe(II) ]-ADE catalyzed the conversion of H(2)O(2) to O(2) and H(2)O. The values of k(cat) and k(cat)/K(m) for the catalase activity are 200 s(-1) and 2.4 × 10(4) M(-1) s(-1), respectively. [Fe(II)/Fe(II)]-ADE underwent more than 100 turnovers with H(2)O(2) before the enzyme was inactivated due to oxygenation of histidine residues critical for metal binding. The iron in the inactive enzyme was high-spin ferric with g(ave) = 4.3 EPR signal and no evidence of anti-ferromagnetic spin-coupling. A model is proposed for the disproportionation of H(2)O(2) by [Fe(II)/Fe(II)]-ADE that involves the cycling of the binuclear metal center between the di-ferric and di-ferrous oxidation states. Oxygenation of active site residues occurs via release of hydroxyl radicals. These findings represent the first report of redox reaction catalysis by any member of the AHS.

Proton activity in nafion films: probing exchangeable protons with methylene blue.

A simple approach to monitor the H(+) activity of a proton-exchange membrane (Nafion) is introduced by incorporating methylene blue as an indicator dye. The dye exhibits characteristics absorption maxima at 665 and 745 nm corresponding to its singly and doubly protonated forms, respectively. The apparent proton activity of Nafion as monitored from the appearance of doubly protonated methylene blue absorption is equivalent to 1.2 M H2SO4. By monitoring the spectral changes associated with the protonation equilibrium of the dye, it is possible to probe the rate and the exchangeable proton sites within the Nafion film. For the Nafion 117 film, we estimate the total exchangeable proton sites to be 2.5 x 10(19) sites/cm(2) or 4.2 x 10(-5) mols/cm(2). The equilibrium constant for the H(+)/Na(+) exchange for the bound sites is determined to be 2.2. The feasibility of methylene blue as a probe to monitor proton activity during the operation of a direct methanol fuel cell has been explored.

A novel multidrug resistance phenotype of bladder tumor cells grown on Matrigel or SIS gel.

We have previously shown that growth of bladder carcinoma cell lines onto matrices such as Matrigel and small intestinal submucosal (SIS) gel cause distinct changes in cellular morphology and motility. In these studies, we found that bladder cells grown on Matrigel showed increased resistance to either doxorubicin or mitomycin-C whereas growth of cells in SIS gel caused either significant increases or little difference in drug resistance, depending on both the cells and the drug. Finally, it was found that this altered drug sensitivity is reversible with a finite half-life and is likely due to altered drug accumulation and/or cell cycle kinetics.

Antigen-presenting cells (APC) of mice with chronic graft-versus-host disease (GVHD) cause excessive activation-induced death of T helper cells.

Both experimental and clinical forms of chronic GVHD have unique immunological features. The affected animals/individuals suffer from autoimmune disorders such as systemic lupus erythematosus (SLE), and yet they are unable to mount a self MHC-restricted T cell response to foreign antigens. Pathogenesis of the latter phenomenon was investigated in an experimental model of chronic GVHD. Chronic GVHD was induced in 8-10-week-old (B6xC3H)F1 mice by tail vein injection of 5 x 10(7) spleen cells of C3H parental strain. The recipients, when tested 3 months later, were unable to mount a T helper (Th) cell response to a randomly selected immunogen, a vaccine of l0(8) killed Mycobacterium vaccae. The animals showed evidence of generalized lymphoid hyperplasia, as indicated by GVH index >1.34, and also revealed autoantibodies against erythrocytes and dsDNA, indicating establishment of chronic GVHD. However, mice with chronic GVHD of only 3 weeks duration were able to mount the Th cell response to M. vaccae. Three consecutive immunizations of these mice at 1-week intervals, with the same immunogen, resulted in the mice becoming non-responsive to the antigen. All the three responses tested, namely the DTH, lymphoproliferation and the antibody responses, were adversely affected. The non-responsiveness induced was antigen-specific. Mice receiving two immunizations with M. vaccae responded normally to Salmonella enteritidis. Pulse treatment with cyclosporin A 0.5 mg/mouse by the i.p. route, on days 0, 1, 2, 3 and 4 at the time of immunization with M. vaccae on day 1, prevented emergence of non-responsiveness. Based on this evidence, it was concluded that repeated activation of T cells of mice with chronic GVHD induces non-responsiveness. Extent of clonal loss due to activation-induced cell death (AICD) caused by i.p. injection with a superantigen Staphylococcal enterotoxin B (SEB) was investigated in F1 mice with chronic GVHD. I.p. injection of 25 microg/mouse of SEB induced loss of SEB responding clones in both normal F1 mice and those having chronic GVHD; however, the extent of loss was much greater in the latter. In vitro antigen-specific proliferation of primed splenic T cells of normal F1 mice was observed to be quite poor when antigen was presented by APC of mice with chronic GVHD of 3 weeks duration. Proliferation profiles of T cells of normal F1 mice, in response to stimulation with concanavalin A (Con A) or SEB, were studied, using as APC irradiated spleen cells of normal F1 mice or of F1 mice with chronic GVHD of 3 weeks duration. With Con A and APC of normal F1 mice, peak proliferation was observed at 48 h, which remained at the same level up to 72 h and declined thereafter, possibly due to AICD. With SEB and the normal APC, proliferation progressively peaked at 72 h and declined thereafter. With APC of mice with chronic GVHD, the 48 h proliferative responses of both Con A and SEB were comparable to those caused by APC of normal F1 mice; however, thereafter the responses declined steeply, suggesting greater AICD. Based on these results, it was concluded that APC of mice with chronic GVHD are functionally altered to induce greater AICD.

Immunomodulatory properties of porins of some members of the family Enterobacteriaceae.

The outer membrane protein (OMP) preparation of Salmonella typhi was observed to have several immunomodulatory properties. Treatment of mice with an intraperitoneal injection of the OMP preparation enhanced both cellular and humoral responses of the mice to an unrelated antigen, a killed vaccine of Mycobacterium vaccae; both the delayed-type hypersensitivity (DTH) response and the antibody titers were enhanced. The predominant isotype of the antibody shifted from immunoglobulin G1 (IgG1) to IgG2a upon treatment with OMP. Treatment of mice with the OMP preparation improved the efficiency of in vitro antigen presentation by the peritoneal cells and also induced the cells to secrete interleukin-1. Treatment with the lipopolysaccharide (LPS) preparation of S. typhi had the opposite effect; i.e., the DTH response to M. vaccae was suppressed. Treatment with OMP neutralized the suppressive effects of LPS. The OMP preparation also had an enhancing effect on the innate immune mechanisms of the mice. Intraperitoneal injection of the OMP preparation enhanced the microbicidal activity of the peritoneal cells, and production of nitric oxide intermediates was stimulated. Injection of the OMP preparation into footpads of naive nonimmune mice induced a sustained hypersensitivity response that peaked at 24 h. Purified porins of the OMP preparation could induce both immunomodulation and hypersensitivity. Porins prepared from five different Salmonella strains and a strain of normal fecal Escherichia coli also exhibited immunomodulatory and hypersensitivity-inducing activities.

Murine CD8+ T suppressors against mycobacterial 65-kDa antigen compete for IL-2 and show lack of major histocompatibility complex-imposed restriction specificity in antigen recognition.

The mechanism of antigen-specific suppression and reasons for aberrant major histocompatibility complex (MHC) class II restriction mediated by CD8+ T cells was investigated in a previously reported murine model of immunosuppression, generated by intraperitoneal priming with Mycobacterium vaccae. Both the CD4+ T helper cells (Th) and CD8+ T suppressor cell (Ts) of M. vaccae-primed mice recognized the 65-kDa antigen of the bacillus, presented by I-A and I-E, respectively. The CD8+ Ts could inhibit non-antigen-specific proliferation of primed CD4+ T cells induced by the exogenously added interleukin (IL)-2 (concanavalin A-stimulated culture supernatant). For inhibition, the Ts had to be activated by the 65-kDa antigen. The degree of inhibition was dependent upon the amount of added IL-2 and the relative numbers of primed CD8+ and CD4+ T cells. On incubation with antigen-presenting cells, and the 65-kDa antigen, the primed CD8+ T cells absorbed IL-2 as efficiently as primed CD4+ T cells. Based on this, it was concluded that the primed CD8+ T cells induced suppression by competition for IL-2. Employing the same model, the MHC restriction of recognition of the suppressor epitope of the 65-kDa antigen by the CD8+ Ts was investigated. The epitopes presented by diverse MHC class II molecules, such as self I-A, I-E and even allogeneic I-E were similar, because they were recognized by the same population of primed CD8+ Ts. Further, immunization of C57BL/6 mice with Ltk-cells expressing H-2 DkKk alloantigens, stimulated CD8+ T cells capable of recognizing M.vaccae 65-kDa antigen. Based on these data, it was proposed that recognition of the suppressor epitope of the 65-kDa antigen by the primed CD8+ Ts exhibits lack of restriction specificity imposed by MHC diversity.

Major histocompatibility complex restriction of T-cell suppression of immune response to mycobacteria.

In earlier work, intraperitoneal (i.p.) immunization with Mycobacterium vaccae was shown to generate a T-suppressor (Ts) response but intradermal (i.d.) immunization did not. We have now studied the major histocompatibility complex (MHC) restriction of this Ts response. The ability of C57BL/6 (H-2b), BALB/c (H-2d), and the (C57BL/6 x BALB/c) F1 mice to generate suppression after i.p. immunization with 10(8) killed M. vaccae was investigated. The BALB/c and the F1 mice generated suppression, but the C57BL/6 mice failed to do so. The suppression could be ascribed to Lyt-2+, L3T4- antigen-specific T cells. The F1 suppressors generated after i.p. immunization could suppress the generation of T-cell responses to i.d. immunization with M. vaccae in the parental BALB/c but not in the C57BL/6 mice. Monoclonal anti-I-A antibody could suppress the antigen-induced proliferative response of mice primed i.d. with M. vaccae. In contrast, monoclonal anti-I-E antibody enhanced antigen-specific proliferation of spleen cells primed i.p. with M. vaccae. The suppressors generated by i.p. priming of mice with M. vaccae could also suppress the in vitro antigen-induced proliferative response of i.d.-primed spleen cells; the suppression could be blocked by anti-I-E antibody. Thus, the T-cell-mediated suppression in the above experimental model was I-E restricted. The inability of the C57BL/6 mice to generate suppression after i.p. immunization with M. vaccae was ascribed to the lack of I-E expression by mice of H-2b strain.