Extracellular Domain of IL-10 Receptor Chain-2 (IL-10R2) and Its Arginine-Containing Peptides Are Susceptible Substrates for Human Prostate Kallikrein-2 (KLK2).

The kallikrein-related peptidase KLK2 has restricted expression in the prostate luminal epithelium, and its protein target is unknown. The present work reports the hydrolytic activities of KLK2 on libraries of fluorescence resonance energy-transfer peptides from which the sequence SYRIF was the most susceptible substrate for KLK2. The sequence SYRIF is present at the extracellular N-terminal segment (58SYRIF63Q) of IL-10R2. KLK2 was fully active at pH 8.0-8.2, found only in prostate inflammatory conditions, and strongly activated by sodium citrate and glycosaminoglycans, the quantities and structures controlled by prostate cells. Bone-marrow-derived macrophages (BMDM) have IL-10R2 expressed on the cell surface, which is significantly reduced after KLK2 treatment, as determined by flow cytometry (FACS analysis). The IL-10 inhibition of the inflammatory response to LPS/IFN-γ in BMDM cells due to decreased nitric oxide, TNF-α, and IL-12 p40 levels is significantly reduced upon treatment of these cells with KLK2. Similar experiments with KLK3 did not show these effects. These observations indicate that KLK2 proteolytic activity plays a role in prostate inflammation and makes KLK2 a promising target for prostatitis treatment.

Macrophages, IL-10, and nitric oxide increase, induced by hyperglycemic conditions, impact the development of murine melanoma B16F10-Nex2.

Epidemiological studies show a strong correlation between diabetes and the increased risk of developing different cancers, including melanoma. In the present study, we investigated the impact of a streptozotocin (STZ)-induced hyperglycemic environment on B16F10-Nex2 murine melanoma development. Hyperglycemic male C57Bl/6 mice showed increased subcutaneous tumor development, partially inhibited by metformin. Tumors showed increased infiltrating macrophages, and augmented IL-10 and nitric oxide (NO) concentrations. In vivo neutralization of IL-10, NO synthase inhibition, and depletion of macrophages reduced tumor development. STZ-treated TLR4 KO animals showed delayed tumor development; the transfer of hyperglycemic C57Bl/6 macrophages to TLR4 KO reversed this effect. Increased concentrations of IL-10 present in tumor homogenates of hyperglycemic mice induced a higher number of pre-angiogenic structures in vitro, and B16F10-Nex2 cells incubated with different glucose concentrations in vitro produced increased levels of IL-10. In summary, our findings show that a hyperglycemic environment stimulates murine melanoma B16F10-Nex2 primary tumor growth, and this effect is dependent on tumor cell stimulation, increased numbers of macrophages, and augmented IL-10 and NO concentrations. These findings show the involvement of tumor cells and other components of the tumor microenvironment in the development of subcutaneous melanoma under hyperglycemic conditions, defining novel targets for melanoma control in diabetic patients.

FTY720 induces apoptosis in B16F10-NEX2 murine melanoma cells, limits metastatic development in vivo, and modulates the immune system

OBJECTIVE: Available chemotherapy presents poor control over the development of metastatic melanoma. FTY720 is a compound already approved by the Food and Drug Administration for the treatment of patients with multiple sclerosis. It has also been observed that FTY720 inhibits tumor growth in vivo (experimental models) and in vitro (animal and human tumor cells). The aim of this study was to evaluate the effects of FTY720 on a metastatic melanoma model and in tumor cell lines. METHODS: We analyzed FTY720 efficacy in vivo in a syngeneic murine metastatic melanoma model, in which we injected tumor cells intravenously into C57BL/6 mice and then treated the mice orally with the compound for 7 days. We also treated mice and human tumor cell lines with FTY720 in vitro, and cell viability and death pathways were analyzed. RESULTS: FTY720 treatment limited metastatic melanoma growth in vivo and promoted a dose-dependent decrease in the viability of murine and human tumor cells in vitro. Melanoma cells treated with FTY720 exhibited characteristics of programmed cell death, reactive oxygen species generation, and increased β-catenin expression. In addition, FTY720 treatment resulted in an immunomodulatory effect in vivo by decreasing the percentage of Foxp3+ cells, without interfering with CD8+ T cells or lymphocyte-producing interferon-gamma. CONCLUSION: Further studies are needed using FTY720 as a monotherapy or in combined therapy, as different types of cancer cells would require a variety of signaling pathways to be extinguished. .

Antifungal and antitumor models of bioactive protective peptides

Peptides are remarkably reactive molecules produced by a great variety of species and able to display a number of functions in uni-and multicellular organisms as mediators, agonists and regulating substances. Some of them exert cytotoxic effects on cells other than those that produced them, and may have a role in controlling subpopulations and protecting certain species or cell types. Presently, we focus on antifungal and antitumor peptides and discuss a few models in which specific sequences and structures exerted direct inhibitory effects or stimulated a protective immune response. The killer peptide, deduced from an antiidiotypic antibody, with several antimicrobial activities and other Ig-derived peptides with cytotoxic activities including antitumor effects, are models studied in vitro and in vivo. Peptide 10 from gp43 of P. brasiliensis (P10) and the vaccine perspective against paracoccidioidomycosis is another topic illustrating the protective effect in vivo against a pathogenic fungus. The cationic antimicrobial peptides with antitumor activities are mostly reviewed here. Local treatment of murine melanoma by the peptide gomesin is another model studied at the Experimental Oncology Unit of UNIFESP.

Peptídeos são moléculas particularmente reativas produzidas por uma grande variedade de espécies, aptos a exercer um número de funções em organismos uni-e multicelulares como mediadores, agonistas e substâncias regulatórias. Alguns deles exercem efeitos citotóxicos em células outras das que os produzem, e podem ter um papel controlando subpopulações e protegendo certas espécies ou tipos celulares. No presente, focalizamos peptídeos antifúngicos e antitumorais e discutimos alguns modelos nos quais seqüências específicas e estruturas exercem efeitos inibitórios diretos ou estimulam uma resposta imune protetora. O peptídeo letal ("killer"), deduzido de um anticorpo anti-idiotípico, com várias atividades antimicrobianas bem como outros peptídeos derivados de imunoglobulinas com atividades citotóxicas incluindo efeitos antitumorais são modelos estudados in vitro e in vivo. O peptídeo P10 da gp43 de P. brasiliensis e a perspectiva de vacina contra a paracoccidioidomicose é outro tópico ilustrando o efeito protetor in vivo contra um fungo patogênico. Peptídeos antimicrobianos catiônicos com atividades antitumorais são os principais revistos aqui. O tratamento local do melanoma murino com o peptídeo gomesina é outro modelo estudado na Unidade de Oncologia Experimental (UNONEX) da UNIFESP.

Preservação de fungos e actinomicetos de interesse médico em água destilada / Preservation of fungi and actinomycetes of medical importance in distilled water

Several methods have been used for the preservation of fungi, all of them presenting advantages and disadvantages. The choice of the methods depends upon the laboratory availabilities, time of preservation, genetic stability of the cultures and other factors. In this work the results obtained through the utilization of Castellani's method (preservation in distilled water) for the maintenance of 174 strains belonging to the "Micoteca do Instituto de Medicina Tropical de São Paulo" are presented. These strains were analyzed after 6, 12, 18 and 24 months, with regard to the percentage of viability taking into consideration the rates of growth and contamination. The smallest percentage of viability occurred in the group of the actinomycetes (50 to 100) and the largest one in the group of the yeasts (near 100). According to other authors, the Castellani's method, besides being simple and economically feasible for small size laboratories, yields good results.

Malaria vaccine development: Various immunization regimens for efficient induction of protective anti-malaria immunity

A malária permanece como a maior causa de morbidade e mortalidade humana em todo o mundo, devido à inexist6encia de medidas de controle eficientes para esta infecçäo. Considera-se que a vacinaçäo pode ser um meio eficaz que complementará outras estratégias de prevençäo e controle desta doença no futuro. Embora a possibilidade de uma vacina contra a malária tenha sido demonstrada nos anos 70, o desenvolvimento de uma facina universalmente eficaz contra esta parasitose tem sido uma difícil tarefa devido a diversos problemas complexos. Um dos aspectos é a complexidade do ciclo de vida do parasita, o qual envolve diferentes estágios que possuem antígenos específicos. Muitos antígenos parasitários têm sido investigados como candidatos potenciais à vacinaçäo, e a busca continua, com antígenos adicionais, sendo recentemente indentificados e caracterizados. Alguns desses antígenos estágio-específicos säo capazes de induzir respostas imunoprotetoras celular e humoral no hospedeiro. Todavia, essas respostas imunoprotetoras säo geralmente restritas geneticamente, adicionando outra dificuldade ao desenvolvimento de uma vacina universalmente eficaz. Por fim, o antígeno estágio-específico deve ser introduzido no hospedeiro utilizando-se um sistema de liberaçäo que possa induzir eficientemente respostas protetoras contra os respectivos estágios. No presente trabalho, revemos as diversas tentativas visando a induçäo de imunidade protetora contra todos os estágios do parasita, levando em consideraçäo os aspectos mencionados acima, que säo os antígenos protetores estágio-específicos, as respostas imunoprotetoras do hospedeiro, e os sistemas de liberaçäo antigênica.

Pesquisa de anticorpos HIV-1 em pacientes com paracoccidioidomicose ativa / HIV-1 antibodies in patients with active paracoccidioidomycosis

A literatura médica, no Brasil, registra rasos casos de paracoccidioidomicose em pacientes aidéticos (Bernard et al., em publicaçäo; Carnaúba et al.; Pedro et al; Goldani et al. e Bakos et al. totalizando 7 (sete) observaçöes, publicadas ou comunicadas em congressos. Esta ocorrência contrasta com a grande freqüência de candidíase (incluindo formas profundas), criptococose e histoplasmose, associadas a infecçäo pelo vírus HIV. Tal fato talves possa ser explicado por ser a AIDS síndrome infecciosa predominante urbana e a paracoccidioidomicose doença essencialmente de zonas rurais. Em soros de 50 pacientes com paracoccidioidomicose, 40 do sexo masculino (80%) e 10 do sexo feminino (20%) foram pesquisados anticorpos HIV-1 pela técnica ELISA e, em 19 casos limítrofes (38%) foi realizado teste confirmatório, pela técnica de Western-blot. Somente em um caso de paracoccidioidomicose foram detectados anticorpos específicos HIV-1 em 5 pacientes (10%) foram revelados anticorpos, provavelmente inespecíficos, anti-p24; gp160 em 3 casos (6%); gpl120 em dois casos (4%); e gp3 em um caso (2%). Dos sete soros de pacientes com bandas consideradas inespecíficas, repetidas as provas sorológicas para AIDS, com amostras colhidas alguns meses após (ver dados anexos), apenas em 1 foram registrados resultados positivos, com faixas de baixa intensidade. Tais pacientes, examinados na época, näo apresentaram nenhuma manifestaçäo clínica de AIDS ou de pré-AIDS