Immunotherapy for patients with advanced urothelial cancer: Current evidence and future perspectives

In recent years, immunotherapy has produced encouraging results in a rapidly increasing number of solid tumors. The responsiveness of bladder cancer to immunotherapy was first established in nonmuscle invasive disease in 1976 with intravesical instillations of bacillus Calmette-Guérin (BCG). Very recently immune checkpoint inhibitors demonstrated good activity and significant efficacy in metastatic disease. In particular the best results were obtained with programmed death-ligand-1 (PD-L1) and programmed death-1 (PD-1) inhibitors, but many other immune checkpoint inhibitors, including anti-cytotoxic T-lymphocyte-associated protein-4 (CTLA-4) antibodies, are currently under investigation in several trials. Simultaneously other therapeutic strategies which recruit an adaptive immune response against tumoral antigens or employ externally manipulated tumor infiltrating lymphocytes might change the natural history of bladder cancer in the near future. This review describes the rationale for the use of immunotherapy in bladder cancer and discusses recent and ongoing clinical trials with checkpoint inhibitors and other novel immunotherapy agents

MOLECULAR CHARACTERISATION OF A NOVEL ADP-RIBOSYLATING PUTATIVE TOXIN OF NEISSERIA MENINGITIDIS

Molecular characterisation of a novel ADP-ribosylating putative toxin of Neisseria meningitidis VEGGIi D, *BALDUCCI E, MASIGNANI V, DI MARCELLO F, SAVINO S, ARICO’ B, COMANDUCCI M, PIZZA M, RAPPUOLI R IRIS, Chiron SpA, Via Fiorentina 1, 53100 Siena Italy; *Dipartimento Scienze morfologiche e Biochimiche Comparate, Università degli Studi di Camerino, Camerino, Italy Session: Surface antigens Introduction: By computer analysis on the Neisseria meningitidis (serogroup B, MC 58 strain) genome sequence, a protein with a feature similar to known bacterial ADP-ribosylating toxins (CT produced by Vibrio cholerae, LT by Escherichia coli and PT by Bordetella pertussis) has been identified. Enzymatic assay has shown that this protein (NM-ADPRT) possesses both NAD glycohydrolase and ADP-ribosyltransferase activity. In this study we describe the identification of the putative catalytic residues, their site-directed mutagenesis, and the resulting activity of the mutants. Materials and methods: The novel NM-ADPRT and the correspondent mutants, were expressed in E. coli as C-terminus His-tag protein fusions. Site-directed mutagenesis was performed using the Multi Site-Directed Mutagenesis Kit (QuikChange). Recombinant NM-ADPRT forms were purified from E. coli in their soluble form by metal chelate affinity chromatography. Both the wild-type and the mutants were assayed for their ADP-ribosylation and NAD-glycohydolase activites, using [adenine –U-14C] NAD and agmatine as ADP-ribose acceptor. Antisera against NM-ADPRT and the mutant derivatives were obtained by immunization of CD1 mice. 20μg of each recombinant protein were given i.p. together with CFA for the first dose and IFA for the second (day 21) and the third (day 35) booster doses. Blood sample were taken on days 34 and 49. Immune sera were used in western blot and tested in a bactericidal assay. Results and discussion: On the basis of sequence homology of NM-ADPRT with LT, CT and PT we have identified the putative residues involved in enzymatic activity. These residues have been changed by site-directed mutagenesis and the purified mutant toxins have been tested for both ADP-ribosylating and NAD-glycohydrolase activities. Interestingly, some of the mutants show reduced or abolished enzymatic activity indicating that the identified residues play a role in catalysis. Antisera against the wild-type and mutant toxins have bactericidal activity. The titers induced by two mutants were higher than those induced by the wild-type form. These data suggest that the mutations introduced could influence not only the enzymatic activity but also the in vivo stability of the toxin. Conclusion: A novel ADP-ribosyltransferase has been identified in meningococcus B. Catalytic residues have been predicted by sequence homology and their role in catalysis has been confirmed by site-directed mutagenesis. These molecules are also able to induce a bactericidal response

Role of neutron transfer processes on the 6Li+120Sn and 7Li+119Sn fusion reactions

The results concerning the study of 6 Li+ 120 Sn and 7 Li+ 119 Sn systems are presented. These two sistems are characterised by very similar structures of the interacting nuclei and by different Q-value for oneand two- neutron transfer. Our aim is to disentangle the possible effects due to the different n-transfer Q-values, at sub-barriers energies, by comparing the two fusion excitation function. In these experiments the fusion cross section has been measured by using a stack activation technique. No particular differences in the two fusion excitation functions have been observed. The influence of transfer channels on fusion cross-section has been object of investigations in the last years. In particular, the possible dependence of the fusion cross-section on the sign of the neutron transfer Q-value has been much debated in literature. The systematic approach used for the study of the Ca+Zr systems [1] provided relatively clear evidence of the relation between sub-barrier-cross section and the sign of the neutron transfer Q-value, in a model-independent way. According to experimenta

Production of Λ6^{6}_{\Lambda}H and Λ7^{7}_{\Lambda}H with the (Kstop−^{-}_{stop},π+\pi^+) reaction

The production of neutron rich $\Lambda$-hypernuclei via the ($K^-_stop$,$\pi^+$) reaction has been studied using data collected with the FINUDA spectrometer at the DA$\Phi$NE $\phi$-factory (LNF). The analysis of the inclusive $\pi^+$ momentum spectra is presented and an upper limit for the production of $^6_\Lambda$H and $^7_\Lambda$H from $^6$Li and $^7$Li, is assessed for the first time.Comment: 11 pages, 3 figures. Accepted for publication in PL