Immune Checkpoint Blockade and Immune Monitoring

The concept of immunological surveillance, a monitoring process in which the immune system detects and destroys by several effector mechanisms, virally infected and neoplastic transformed cells in the body, was developed more than 50 years ago. Based on current research, it is clear that the immune system can recognize and eliminate transformed cells. An increasing number of studies has investigated the immune system in cancer patients and how it is prone to immunosuppression, due in part to the decrease of lymphocyte proliferation and cytotoxic activity. Such weakened immune system is then unable to fully accomplish its role in immunological surveillance, allowing nascent transformed cells to escape the selective pressure of the immune system. The main goal of cancer immunotherapy has been to reawaken the immune system from a suppressive slumber to enable it to attack cancer cells once again. As the results from the last 10 years attest, cancer immunotherapy is the best strategy to restore the activity of the immune system and unleash its potential to destroy cancer cells in cancer patients. This chapter aims to discuss the recent findings on immune monitoring studies and the use of immune checkpoint inhibition in cancer immunotherapy

From yeast killer toxins to antibiobodies and beyond

Antibiobodies are paradigmatic of yeast killer toxin (KT)-like antibodies (KAbs) mimicking the antimicrobial activity of KTs in the frame of the yeast killer phenomenon. Polyclonal, monoclonal and recombinant anti-idiotypic antibiobodies (anti-idiotypic KAbs), internal images of a wide-spectrum KT produced by the yeast Pichia anomala (PaKT), have been produced by immunization with the idiotype of a PaKT-neutralizing monoclonal antibody. Anti-idiotypic KAbs showed microbicidal activity against eukaryotic and prokaryotic pathogenic agents through the interaction with specific KT receptors (KTRs), putatively constituted by beta-glucans. Natural KAbs have been found in animals and humans experimentally or naturally infected by KTR-bearing microorganisms. Recombinant KAb-derived synthetic killer peptides showed further antiviral and immunomodulatory activities. the perspectives of KAbs and killer peptides as potential sources of novel therapeutic agents, and of KTRs and idiotypes as vaccines against infectious diseases are discussed.Istituto Superiore di SanitaUniv Parma, Sez Microbiol, Dipartimento Patol & Med Lab, I-43100 Parma, ItalyUniversidade Federal de São Paulo, Dept Microbiol Imunol & Parasitol, Unidade Oncol Expt, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Microbiol Imunol & Parasitol, Unidade Oncol Expt, São Paulo, BrazilWeb of Scienc

Mortality due to systemic mycoses as a primary cause of death or in association with AIDS in Brazil: a review from 1996 to 2006

Deaths caused by systemic mycoses such as paracoccidioidomycosis, cryptococcosis, histoplasmosis, candidiasis, aspergillosis, coccidioidomycosis and zygomycosis amounted to 3,583 between 1996-2006 in Brazil. When analysed as the underlying cause of death, paracoccidioidomycosis represented the most important cause of deaths among systemic mycoses (~ 51.2%). When considering AIDS as the underlying cause of death and the systemic mycoses as associated conditions, cryptococcosis (50.9%) appeared at the top of the list, followed by candidiasis (30.2%), histoplasmosis (10.1%) and others. This mortality analysis is useful in understanding the real situation of systemic mycoses in Brazil, since there is no mandatory notification of patients diagnosed with systemic mycoses in the official health system.FAPESPCNP

Identification of iGb3 and iGb4 in melanoma B16F10-Nex2 cells and the iNKT cell-mediated antitumor effect of dendritic cells primed with iGb3

<p>Abstract</p> <p>Background</p> <p>CD1d-restricted iNKT cells are protective against murine melanoma B16F10-Nex2 growing subcutaneously in syngeneic C57Bl/6 mice as inferred from the fast tumor development in CD1d-KO in comparison with wild type animals. CD1d glycoproteins are related to the class I MHC molecules, and are involved in the presentation, particularly by dentritic cells (DC), of lipid antigens to iNKT cells. In the present work we attempted to identify the endogenous lipid mediator expressed in melanoma cells inducing such immunesurveillance response and study the possibility of protecting animals challenged with tumor cells with lipid-primed DC.</p> <p>Results</p> <p>Crude cytosolic and membrane fractions from <it>in vivo </it>growing melanoma contained iNKT-stimulating substances. Lipids were then extracted from these cells and one of the fractions (i.e. F3A) was shown to prime bone marrow-derived dendritic cells (BMDC) to stimulate iNKT murine hybridoma (DN32D3) cells to produce IL-2. The active fraction was analyzed by electrospray ionization-mass spectrometry (ESI-LIT-MS) and both iGb3 and iGb4 were identified along with GM3. When iGb3 was incubated with BMDC and tested with DN32D3 cells, IL-2 was equally produced indicating iNKT cell activation. GM3 consistently inhibited this response. To assess the antitumor response-induced by iGb3, a cytotoxicity assay <it>in vitro </it>was used with [³H]-thymidine labeled B16F10-Nex2 cells. At target/effector (iGb3-activated iNKT) cell ratio of 100^-1-100^-4tumor cell lysis was shown. The antitumor activity <it>in vivo </it>was tested in mice challenged i.v. with B16F10-Nex2 cells and treated with iGb3- or α-galactosylceramide-primed DCs. A 4-fold lower tumor load in the lungs was observed with either treatment.</p> <p>Conclusion</p> <p>Our results show the expression of globo and isoglobohexosylceramides in murine melanoma B16F10-Nex2. The expression of iGb3 and its precursor, iGb4, on tumor cells may prime an effective iNKT cell-dependent antitumor response, modulated negatively by GM3 which is also produced in these cells. iGb3-primed BMDC exerted a significant iNKT cell-mediated anti-tumor activity in mice challenged with melanoma cells.</p

Characterization of thimet oligopeptidase and neurolysin activities in B16F10-Nex2 tumor cells and their involvement in angiogenesis and tumor growth

<p>Abstract</p> <p>Background</p> <p>Angiogenesis is a fundamental process that allows tumor growth by providing nutrients and oxygen to the tumor cells. Beyond the oxygen diffusion limit from a capillary blood vessel, tumor cells become apoptotic. Angiogenesis results from a balance of pro- and anti-angiogenic stimuli. Endogenous inhibitors regulate enzyme activities that promote angiogenesis. Tumor cells may express pro-angiogenic factors and hydrolytic enzymes but also kinin-degrading oligopeptidases which have been investigated.</p> <p>Results</p> <p>Angiogenesis induced by B16F10-Nex2 melanoma cells was studied in a co-culture with HUVEC on Matrigel. A stimulating effect on angiogenesis was observed in the presence of B16F10-Nex2 lysate and plasma membrane. In contrast, the B16F10-Nex2 culture supernatant inhibited angiogenesis in a dose-dependent manner. This effect was abolished by the endo-oligopeptidase inhibitor, JA-2. Thimet oligopeptidase (TOP) and neurolysin activities were then investigated in B16F10-Nex2 melanoma cells aiming at gene sequencing, enzyme distribution and activity, influence on tumor development, substrate specificity, hydrolytic products and susceptibility to inhibitors. Fluorescence resonance energy transfer (FRET) peptides as well as neurotensin and bradykinin were used as substrates. The hydrolytic activities in B16F10-Nex2 culture supernatant were totally inhibited by <it>o</it>-phenanthrolin, JA-2 and partially by Pro-Ile. Leupeptin, PMSF, E-64, Z-Pro-Prolinal and captopril failed to inhibit these hydrolytic activities. Genes encoding M3A enzymes in melanoma cells were cloned and sequenced being highly similar to mouse genes. A decreased proliferation of B16F10-Nex2 cells was observed in vitro with specific inhibitors of these oligopeptidases. Active rTOP but not the inactive protein inhibited melanoma cell development in vivo increasing significantly the survival of mice challenged with the tumor cells. On Matrigel, rTOP inhibited the bradykinin – induced angiogenesis. A possible regulation of the homologous tumor enzyme in the perivascular microenvironment is suggested based on the observed rTOP inhibition by an S-nitrosothiol NO donor.</p> <p>Conclusion</p> <p>Data show that melanoma cells secrete endo-oligopeptidases which have an important role in tumor proliferation in vitro and in vivo. rTOP inhibited growth of subcutaneously injected B16F10-Nex2 cells in mice. TOP from tumor cells and bradykinin in endothelial cells are two antagonist factors that may control angiogenesis essential for melanoma growth. A regulatory role of NO or S-nitrosothiols is suggested.</p

Psychometric evaluation of the SF-36 (v.2) questionnaire in a probability sample of Brazilian households: results of the survey Pesquisa Dimensões Sociais das Desigualdades (PDSD), Brazil, 2008

<p>Abstract</p> <p>Background</p> <p>In Brazil, despite the growing use of SF-36 in different research environments, most of the psychometric evaluation of the translated questionnaire was from studies with samples of patients. The purpose of this paper is to examine if the Brazilian version of SF-36 satisfies scaling assumptions, reliability and validity required for valid interpretation of the SF-36 summated ratings scales in the general population.</p> <p>Methods</p> <p>12,423 individuals and their spouses living in 8,048 households were selected from a stratified sample of all permanent households along the country to be interviewed using the Brazilian SF-36 (version 2). Psychometric tests were performed to evaluate the scaling assumptions based on IQOLA methodology.</p> <p>Results</p> <p>Data quality was satisfactory with questionnaire completion rate of 100%. The ordering of the item means within scales clustered as hypothesized. All item-scale correlations exceeded the suggested criteria for reliability with success rate of 100% and low floor and ceiling effects. All scales reached the criteria for group comparison and factor analysis identified two principal components that jointly accounted for 67.5% of the total variance. Role emotional and vitality were strongly correlated with physical and mental components, respectively, while social functioning was moderately correlated with both components. Role physical and mental health scales were, respectively, the most valid measures of the physical and mental health component. In the comparisons between groups that differed by the presence or absence of depression, subjects who reported having the disease had lower mean scores in all scales and mental health scale discriminated best between the two groups. Among those healthy and with one, two or three and more chronic illness, the average scores were inverted related to the number of diseases. Body pain, general health and vitality were the most discriminating scales between healthy and diseased groups. Higher scores were associated with individuals of male sex, age below 40 years old and high schooling.</p> <p>Conclusions</p> <p>The Brazilian version of SF-36 performed well and the findings suggested that it is a reliable and valid measure of health related quality of life among the general population as well as a promising measure for research on health inequalities in Brazil.</p

Molecular, Biological and Structural Features of VL CDR-1 Rb44 Peptide, Which Targets the Microtubule Network in Melanoma Cells

Microtubules are important drug targets in tumor cells, owing to their role in supporting and determining the cell shape, organelle movement and cell division. The complementarity-determining regions (CDRs) of immunoglobulins have been reported to be a source of anti-tumor peptide sequences, independently of the original antibody specificity for a given antigen. We found that, the anti-Lewis B mAb light-chain CDR1 synthetic peptide Rb44, interacted with microtubules and induced depolymerization, with subsequent degradation of actin filaments, leading to depolarization of mitochondrial membrane-potential, increase of ROS, cell cycle arrest at G2/M, cleavage of caspase-9, caspase-3 and PARP, upregulation of Bax and downregulation of Bcl-2, altogether resulting in intrinsic apoptosis of melanoma cells. The in vitro inhibition of angiogenesis was also an Rb44 effect. Peritumoral injection of Rb44L1 delayed growth of subcutaneously grafted melanoma cells in a syngeneic mouse model. L1-CDRs from immunoglobulins and their interactions with tubulin-dimers were explored to interpret effects on microtubule stability. The opening motion of tubulin monomers allowed for efficient L1-CDR docking, impairment of dimer formation and microtubule dissociation. We conclude that Rb44 VL-CDR1 is a novel peptide that acts on melanoma microtubule network causing cell apoptosis in vitro and melanoma growth inhibition in vivo

The role of adjuvants in therapeutic protection against paracoccidioidomycosis after immunization with the P10 peptide

Paracoccidioidomycosis (PCM), a common chronic mycosis in Latin America, is a granulomatous systemic disease caused by the thermo-dimorphic fungus Paracoccidioides brasiliensis. The glycoprotein gp43 is the main antigen target of P. brasiliensis and a 15-mer internal peptide (QTLIAIHTLAIRYAN), known as P10, defines a major CD4+-specific T cell epitope. Previous results have indicated that, besides having a preventive role in conventional immunizations prior to challenge with the fungus, protective anti-fungal effects can be induced in P. brasiliensis-infected mice treated with P10 administered with complete Freund’s adjuvant (CFA). The peptide elicits an IFN-γ-dependent Th1 immune response and is the main candidate for effective immunotherapy of patients with PCM, as an adjunctive approach to conventional chemotherapy. In the present study we tested the therapeutic effects of P10 combined with different adjuvants [aluminum hydroxide, CFA, flagellin, and the cationic lipid dioctadecyl-dimethylammonium bromide (DODAB)] in BALB/c mice previously infected with the P. brasiliensis Pb18 strain. Significant reductions in the number of colony forming units of the fungus were detected in lungs of mice immunized with P10 associated with the different adjuvants 52 days after infection. Mice treated with DODAB and P10, followed by mice treated with P10 and flagellin, showed the most prominent effects as demonstrated by the lowest numbers of viable yeast cells as well as reductions in granuloma formation and fibrosis. Concomitantly, secretion of IFN-γ and TNF-α, in contrast to interleukin (IL)-4 and IL-10, was enhanced in the lungs of mice immunized with P10 in combination with the tested adjuvants, with the best results observed in mice treated with P10 and DODAB. In conclusion, the present results demonstrate that the co-administration of the synthetic P10 peptide with several adjuvants, particularly DODAB, have significant therapeutic effects in experimental PCM

Antibodies Against Glycolipids Enhance Antifungal Activity of Macrophages and Reduce Fungal Burden After Infection with Paracoccidioides brasiliensis

Paracoccidioidomycosis is a fungal disease endemic in Latin America. Polyclonal antibodies to acidic glycosphingolipids (GSLs) from Paracoccidioides brasiliensis opsonized yeast forms in vitro increasing phagocytosis and reduced the fungal burden of infected animals. Antibodies to GSL were active in both prophylactic and therapeutic protocols using a murine intratracheal infection model. Pathological examination of the lungs of animals treated with antibodies to GSL showed well-organized granulomas and minimally damaged parenchyma compared to the untreated control. Murine peritoneal macrophages activated by IFN-gamma and incubated with antibodies against acidic GSLs more effectively phagocytosed and killed P brasiliensis yeast cells as well as produced more nitric oxide compared to controls. The present work discloses a novel target of protective antibodies against P brasiliensis adding to other well-studied mediators of the immune response to this fungus.CapesFAPESPUniv Sao Paulo, Dept Microbiol, Inst Biomed Sci, Sao Paulo, BrazilUniv Sao Paulo, Lab Med Mycol IMTSP LIM53, Sao Paulo, BrazilAlbert Einstein Coll Med, Dept Med, New York, NY USAAlbert Einstein Coll Med, Dept Microbiol & Immunol, New York, NY USAUniv Fed Fluminense, Niteroi, RJ, BrazilUniv Fed Sao Paulo, Dept Microbiol Immunol & Parasitol, Sao Paulo, BrazilUniv Fed Sao Paulo, Dept Microbiol Immunol & Parasitol, Sao Paulo, BrazilFAPESP: 2011/17267-4FAPESP: 2013/18655-3Web of Scienc