Elaine G. Rodrigues

The use of cell-free antigens of Paracoccidioides brasiliensis in serological tests

A simple and rapid method for extracting specific cell-free antigens (CFA) from the yeast form of Paracoccidioides brasiliensis grown on agar slants was developed. Extracts were analysed mainly by immunodiffusion (ID) tests, for the immunodiagnosis of paracoccidioidomycosis. Extracts were obtained from 2 to 7-day-old cultures. The extract obtained after the third day, which contained 200-300 micrograms of protein/ml, gave the best ID reactions, with 100% sensitivity and specificity. The main precipitation band was intense and showed total identity with that formed by the specific 43 kDa glycoprotein. Other tests such as counterimmunoelectrophoresis and immunoelectrophoresis were also used successfully with CFA. The 3-day CFA extract showed a pattern of polypeptides in the 10-110 kDa range after SDS-polyacrylamide gel electrophoresis. Five components of 20, 43, 45, 55 and 70 kDa reacted on immunoblots with sera from patients with active chronic paracoccidioidomycosis. The specific diagnostic antigen of 43 kDa always predominated in all CFA preparations. The present method is thus very useful for the rapid production of an antigenic extract which can be readily characterized and used in the serodiagnosis of paracoccidioidomycosis.

Cyclopalladated compounds as chemotherapeutic agents: antitumor activity against a murine melanoma cell line.

Palladacycle compounds obtained from N, N‐dimethyl‐1‐phenethylamine (dmpa), phenyl‐2‐pyridinyl‐acetylene and 1‐phenyl‐3‐N, N‐dimethylamine‐propine, respectively, were complexed to 1, 2 ethanebis (diphenylphosphine) (dppe) ligand to synthesize antitumor cyclopalladated complexes that were tested in vitro and in vivo against syngeneic B16F10‐Nex2 murine melanoma cells of low immunogenicity implanted subcutaneously in mice. Complexes were not toxic to mice injected 3 times i.p. with as much as 60 μM/animal/week. Of 3 cyclopalladated complexes that were inhibitory in vitro at low concentrations (<1.25 μM), complex 7a was the most active in vivo, delaying tumor growth and prolonging animal survival. In vitro, binucleate complex 7a caused a collapse of respiratory activity with an abrupt decrease of extracellular acidification on short incubation (up to 100 min), followed by DNA degradation after 24 hr. The apoptosis‐like reaction to this Pd‐complex was not accompanied by increased levels of caspases 1 and 3. Complex 7a bound to a bacterial plasmid DNA, causing late conformational changes after 24 hr. Two other complexes with different C, N‐cycles were also apoptotic and 2 binucleated ones were inactive. These results introduce the palladacycle‐dppe complexes as promising antitumor drugs with exquisite structural specificities and for action in vivo and in vitro.

Monoclonal Antibody to Fungal Glucosylceramide Protects Mice against Lethal Cryptococcus neoformans Infection

ABSTRACT Glucosylceramides (GlcCer) are involved in the regulation of Cryptococcus neoformans virulence. In the present study, we demonstrate that passive immunization with a monoclonal antibody to GlcCer significantly reduces host inflammation and prolongs the survival of mice lethally infected with C. neoformans, revealing a potential therapeutic strategy to control cryptococcosis.

Antibody complementarity-determining regions (CDRs) can display differential antimicrobial, antiviral and antitumor activities.

Background Complementarity-determining regions (CDRs) are immunoglobulin (Ig) hypervariable domains that determine specific antibody (Ab) binding. We have shown that synthetic CDR-related peptides and many decapeptides spanning the variable region of a recombinant yeast killer toxin-like antiidiotypic Ab are candidacidal in vitro. An alanine-substituted decapeptide from the variable region of this Ab displayed increased cytotoxicity in vitro and/or therapeutic effects in vivo against various bacteria, fungi, protozoa and viruses. The possibility that isolated CDRs, represented by short synthetic peptides, may display antimicrobial, antiviral and antitumor activities irrespective of Ab specificity for a given antigen is addressed here. Methodology/Principal Findings CDR-based synthetic peptides of murine and human monoclonal Abs directed to: a) a protein epitope of Candida albicans cell wall stress mannoprotein; b) a synthetic peptide containing well-characterized B-cell and T-cell epitopes; c) a carbohydrate blood group A substance, showed differential inhibitory activities in vitro, ex vivo and/or in vivo against C. albicans, HIV-1 and B16F10-Nex2 melanoma cells, conceivably involving different mechanisms of action. Antitumor activities involved peptide-induced caspase-dependent apoptosis. Engineered peptides, obtained by alanine substitution of Ig CDR sequences, and used as surrogates of natural point mutations, showed further differential increased/unaltered/decreased antimicrobial, antiviral and/or antitumor activities. The inhibitory effects observed were largely independent of the specificity of the native Ab and involved chiefly germline encoded CDR1 and CDR2 of light and heavy chains. Conclusions/Significance The high frequency of bioactive peptides based on CDRs suggests that Ig molecules are sources of an unlimited number of sequences potentially active against infectious agents and tumor cells. The easy production and low cost of small sized synthetic peptides representing Ig CDRs and the possibility of peptide engineering and chemical optimization associated to new delivery mechanisms are expected to give rise to a new generation of therapeutic agents.

A cyclopalladated complex interacts with mitochondrial membrane thiol-groups and induces the apoptotic intrinsic pathway in murine and cisplatin-resistant human tumor cells

BackgroundSystemic therapy for cancer metastatic lesions is difficult and generally renders a poor clinical response. Structural analogs of cisplatin, the most widely used synthetic metal complexes, show toxic side-effects and tumor cell resistance. Recently, palladium complexes with increased stability are being investigated to circumvent these limitations, and a biphosphinic cyclopalladated complex {Pd2 [S(-) C2, N-dmpa]2 (μ-dppe)Cl2} named C7a efficiently controls the subcutaneous development of B16F10-Nex2 murine melanoma in syngeneic mice. Presently, we investigated the melanoma cell killing mechanism induced by C7a, and extended preclinical studies.MethodsB16F10-Nex2 cells were treated in vitro with C7a in the presence/absence of DTT, and several parameters related to apoptosis induction were evaluated. Preclinical studies were performed, and mice were endovenously inoculated with B16F10-Nex2 cells, intraperitoneally treated with C7a, and lung metastatic nodules were counted. The cytotoxic effects and the respiratory metabolism were also determined in human tumor cell lines treated in vitro with C7a.ResultsCyclopalladated complex interacts with thiol groups on the mitochondrial membrane proteins, causes dissipation of the mitochondrial membrane potential, and induces Bax translocation from the cytosol to mitochondria, colocalizing with a mitochondrial tracker. C7a also induced an increase in cytosolic calcium concentration, mainly from intracellular compartments, and a significant decrease in the ATP levels. Activation of effector caspases, chromatin condensation and DNA degradation, suggested that C7a activates the apoptotic intrinsic pathway in murine melanoma cells. In the preclinical studies, the C7a complex protected against murine metastatic melanoma and induced death in several human tumor cell lineages in vitro, including cisplatin-resistant ones. The mitochondria-dependent cell death was also induced by C7a in human tumor cells.ConclusionsThe cyclopalladated C7a complex is an effective chemotherapeutic anticancer compound against primary and metastatic murine and human tumors, including cisplatin-resistant cells, inducing apoptotic cell death via the intrinsic pathway.

Synthesis and immunological activity of a branched peptide carrying the T-cell epitope of gp43, the major exocellular antigen of Paracoccidioides brasiliensis

The 43 kDa glycoprotein (gp43) of Paracoccidioides brasiliensis is the major diagnostic antigen of paracoccidioidomycosis (PCM), a prevalent fungal infection in South America. A 15‐mer sequence from gp43, denominated P10, induced T‐CD4+ T helper 1 cellular immune responses in mice of three different haplotypes and protected against intratracheal challenge by a virulent isolate of P. brasiliensis. In an attempt to improve delivery of P10, a promiscuous antigen also presented by human leucocyte antigen‐DR alleles, aiming at immunotherapy, we synthesized a multiple antigen peptide with the protective T‐cell epitope expressed in a tetravalent 13‐mer analog of P10 (M10). M10 induced specific lymph node cell proliferation in mice preimmunized with peptides in complete Freunds adjuvant (CFA). In addition, M10 immunization without CFA significantly protected intratracheally infected mice. We conclude that M10 is a candidate for an anti‐PCM vaccine. In this report we describe: (1) the synthesis of M10; (2) the induction of M10‐elicited T‐cell response and (3) in vivo protection of mice immunized with M10 and challenged by a virulent strain of P. brasiliensis.

Attempts at a peptide vaccine against paracoccidioidomycosis, adjuvant to chemotherapy

Chemotherapy is the basis of treatment of paracoccidioidomycosis in its various forms. Depending on the Paracoccidioides brasiliensis virulence, the status of host immunity, the degree of tissue involvement and fungal dissemination, treatment can be extended for long periods with an alarming frequency of relapses. Association of chemotherapy with a vaccine to boost the cellular immune response seemed a relevant project not only to reduce the time of treatment but also to prevent relapses and improve the prognosis of anergic cases. The candidate immunogen is the gp43 major diagnostic antigen of P.␣brasiliensis and more specifically its derived peptide P10, carrying the CD4+ T-cell epitope. Both gp43 and P10 protected Balb/c mice against intratracheal infections with virulent P. brasiliensis strain. P10 as single peptide or in a multiple-antigen-peptide (MAP) tetravalent construction was protective without adjuvant either by preimmunization and intratracheal challenge or as a therapeutic agent in mice with installed infection. P10 showed additive protective effects in drug-treated mice stimulating a Th-1 type immune response with high IFN-γ and IL-12. P10 and few other peptides in the gp43 were selected by Tepitope algorithm and actually shown to promiscuously bind several prominent HLA-DR molecules suggesting that a peptide vaccine could be devised for a genetically heterogenous population. P10 was protective in animals turned anergic, was effective in a DNA minigene vaccine, and increased the protection by monoclonal antibodies in Balb/c mice. DNA vaccines and peptide vaccines are promising therapeutic tools to be explored in the control of systemic mycoses.

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

BackgroundAngiogenesis 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.ResultsAngiogenesis 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 o-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.ConclusionData 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.

β-Actin-binding Complementarity-determining Region 2 of Variable Heavy Chain from Monoclonal Antibody C7 Induces Apoptosis in Several Human Tumor Cells and Is Protective against Metastatic Melanoma

Background: Immunoglobulin complementarity-determining region (CDR) peptides frequently display antitumor activities. Results: Monoclonal antibody C7 CDR-H2 binds to β-actin and induces polymerization, F-actin stabilization, and tumor cell death. Conclusion: Alterations of actin dynamics trigger reactive oxygen species and tumor cell apoptosis. Significance: The in vitro apoptotic effect and in vivo antimetastatic activity of peptide C7H2 makes it a candidate to be developed as an anticancer drug. Complementarity-determining regions (CDRs) from monoclonal antibodies tested as synthetic peptides display anti-infective and antitumor activities, independent of the specificity of the native antibody. Previously, we have shown that the synthetic peptide C7H2, based on the heavy chain CDR 2 from monoclonal antibody C7, a mAb directed to a mannoprotein of Candida albicans, significantly reduced B16F10 melanoma growth and lung colony formation by triggering tumor apoptosis. The mechanism, however, by which C7H2 induced apoptosis in tumor cells remained unknown. Here, we demonstrate that C7H2 interacts with components of the tumor cells cytoskeleton, being rapidly internalized after binding to the tumor cell surface. Mass spectrometry analysis and in vitro validation revealed that β-actin is the receptor of C7H2 in the tumor cells. C7H2 induces β-actin polymerization and F-actin stabilization, linked with abundant generation of superoxide anions and apoptosis. Major phenotypes following peptide binding were chromatin condensation, DNA fragmentation, annexin V binding, lamin disruption, caspase 8 and 3 activation, and organelle alterations. Finally, we evaluated the cytotoxic efficacy of C7H2 in a panel of human tumor cell lines. All tumor cell lines studied were equally susceptible to C7H2 in vitro. The C7H2 amide without further derivatization significantly reduced lung metastasis of mice endovenously challenged with B16F10-Nex2 melanoma cells. No significant cytotoxicity was observed toward nontumorigenic cell lines on short incubation in vitro or in naïve mice injected with a high dose of the peptide. We believe that C7H2 is a promising peptide to be developed as an anticancer drug.

Endogenous accumulation of IFN-? in IFN-?-R -/- mice increases resistance to B16F10-Nex2 murine melanoma: a model for direct IFN-? anti-tumor cytotoxicity in vitro and in vivo

Syngeneic IFN-γ − / − and IRF-1 − / − mice are very sensitive to B16F10-Nex2 murine melanoma cells implanted subcutaneously. In contrast, IFN-γ-R − / − (GRKO) mice are remarkably resistant to tumor development. Only 0-30% of these animals, challenged with a high dose of melanoma cells (5×105), developed tumors at a late stage. The hypothesis of interferon gamma (IFN-γ) accumulation and consequent cytotoxicity to implanted tumor cells was confirmed in vitro and ex vivo. IFN-γ reduced tumor-cell growth in vitro in 60-81%, added alone or with LPS. Splenocytes and peritoneal macrophages from naive GRKO mice activated with anti-CD3 and interleukin-12 (IL-12), respectively, accumulated IFN-γ at levels 10-fold those of the wild-type. Supernatants of IL-12-activated macrophages from GRKO mice were toxic to B16F10-Nex2 cells, an effect reversible by anti-IFN-γ antibody treatm...