Identification of Potentially Therapeutic Immunogenic Peptides From Paracoccidioides lutzii Species.

Paracoccidioidomycosis (PCM) is an endemic mycosis in Latin America caused by the thermodimorphic fungi of the genus Paracoccidioides spp. Paracoccidioides lutzii (PL) is one of the 5 species that constitute the Paracoccidioides genus. PL expresses low amounts of glycoprotein (Gp) 43 (PLGp43) and PLGp43 displays few epitopes in common with the P. brasiliensis (PB) immunodominant antigen PBGp43, which is commonly used for serological diagnosis of PCM. This difference in structure between the glycoproteins markedly reduces the efficiency of serological diagnosis in patients infected with PL. We previously demonstrated that peptide 10 (P10) from the PBGp43 induces protective immune responses in in vitro and in vivo models of PB PCM. Since, P10 has proven to be a promising therapeutic to combat PB, we sought to identify peptides in PL that could similarly be applied for the treatment of PCM. PL yeast cell proteins were isolated from PL: dendritic cell co-cultures and subjected to immunoproteomics. This approach identified 18 PL peptides that demonstrated in silico predictions for immunogenicity. Eight of the most promising peptides were synthesized and applied to lymphocytes obtained from peptide-immunized or PL-infected mice as well as to in vitro cultures with peptides or dendritic cells pulsed the peptides. The peptides LBR5, LBR6 and LBR8 efficiently promoted CD4+ and CD8+ T cell proliferation and dendritic cells pulsed with LBR1, LBR3, LBR7 or LBR8 stimulated CD4+ T cell proliferation. We observed increases of IFN-γ in the supernatants from primed T cells for the conditions with peptides without or with dendritic cells, although IL-2 levels only increased in response to LBR8. These novel immunogenic peptides derived from PL will be employed to develop new peptide vaccine approaches and the proteins from which they are derived can be used to develop new diagnostic assays for PL and possibly other Paracoccidioides spp. These findings identify and characterize new peptides with a promising therapeutic profile for future against this important neglected systemic mycosis.

Virulence factors of Paracoccidioides brasiliensis as therapeutic targets: a review.

Paracoccidiodomycosis (PCM) is a systemic mycosis caused by the fungus Paracoccidioides brasiliensis and Paracoccidioides lutzii. The disease requires long and complicated treatment. The aim of this review is to address the fungal virulence factors that could be the target of the development of new drugs for PCM treatment. Virulence factors favoring the process of fungal infection and pathogenicity are considered as a microbial attribute associated with host susceptibility. P. brasiliensis has some known virulence factors which are 43 kDa glycoprotein (gp 43) which is an important fungal antigen, 70 kDa glycoprotein (gp 70), the carbohydrates constituting the fungal cell wall α-1,3, glucan and ß-1,3-glucan, cell adhesion molecules and the presence of melanin pigments. The discovery and development of drugs that interact with these factors, such as inhibitors of ß-1,3-glucan, reduced synthesis of gp 43, inhibitors of melanin production, is of great importance for the treatment of PCM. The study of virulence factors favors the understanding of pathogen-host relationships, aiming to evaluate the possibility of developing new therapeutic targets and mechanisms that these molecules play in the infectious process, favoring the design of a more specific treatment for this disease.

Low-level laser therapy to the mouse femur enhances the fungicidal response of neutrophils against Paracoccidioides brasiliensis.

Neutrophils (PMN) play a central role in host defense against the neglected fungal infection paracoccidioidomycosis (PCM), which is caused by the dimorphic fungus Paracoccidioides brasiliensis (Pb). PCM is of major importance, especially in Latin America, and its treatment relies on the use of antifungal drugs. However, the course of treatment is lengthy, leading to side effects and even development of fungal resistance. The goal of the study was to use low-level laser therapy (LLLT) to stimulate PMN to fight Pb in vivo. Swiss mice with subcutaneous air pouches were inoculated with a virulent strain of Pb or fungal cell wall components (Zymosan), and then received LLLT (780 nm; 50 mW; 12.5 J/cm2; 30 seconds per point, giving a total energy of 0.5 J per point) on alternate days at two points on each hind leg. The aim was to reach the bone marrow in the femur with light. Non-irradiated animals were used as controls. The number and viability of the PMN that migrated to the inoculation site was assessed, as well as their ability to synthesize proteins, produce reactive oxygen species (ROS) and their fungicidal activity. The highly pure PMN populations obtained after 10 days of infection were also subsequently cultured in the presence of Pb for trials of protein production, evaluation of mitochondrial activity, ROS production and quantification of viable fungi growth. PMN from mice that received LLLT were more active metabolically, had higher fungicidal activity against Pb in vivo and also in vitro. The kinetics of neutrophil protein production also correlated with a more activated state. LLLT may be a safe and non-invasive approach to deal with PCM infection.

Dendritic cells transfected with scFv from Mab 7.B12 mimicking original antigen gp43 induces protection against experimental Paracoccidioidomycosis.

Paracoccidioidomycosis (PCM), endemic in Latin America, is a progressive systemic mycosis caused by Paracoccidioides brasiliensis (P. brasiliensis), which primarily attacks lung tissue. Dendritic cells (DCs) are able to initiate a response in naïve T cells, and they also participate in Th-cell education. Furthermore, these cells have been used for therapy in several disease models. Here we transfected DCs with a plasmid (pMAC/PS-scFv) encoding a single chain variable fragment (scFv) of an anti-Id antibody that is capable of mimicking gp43, the main antigenic component of P. brasiliensis. First, Balb/c mice were immunized subcutaneously with pMAC/PS-scFv and, after seven days, scFv protein was presented to the regional lymph nodes cells. Moreover, we showed that the DCs transfected with scFv were capable of efficiently activating proliferation of total lymph node cells and inducing a decrease in lung infection. Therefore, our results suggested that the use of scFv-transfected DCs may be a promising therapy in the paracoccidioidomycosis (PCM) model.

Peptide immunization as an adjuvant to chemotherapy in mice challenged intratracheally with virulent yeast cells of Paracoccidioides brasiliensis.

Immunization with peptide P10, derived from gp43, and chemotherapy were used together in an attempt to improve treatment of paracoccidioidomycosis and prevent relapses. The combined treatment showed an additive protective effect when administered at 48 h or 30 days after intratracheal challenge. Its use is recommended to improve regular chemotherapy and reduce the duration of treatment.

Avaliação da atividade antifúngica do ajoene derivado do Allium sativum (Liliaceaea) no tratamento da paracoccidioidomicose experimental / Evaluation of the antifungal activity of ajoene derived from the Allium sativum (Liliaceaea) in the treatment of paracoccidioidomycosis experimental

O objetivo deste estudo foi avaliar, pela primeira vez, a atividade do AJOENE, produto natural isolado do extrato do alho (Allium sativum), in vivo na infecção experimental com Paracoccidioides brasiliensis. Grupos de camundongos Swiss (n=40) foram inoculados por via intraperitonial com 5x106 células leveduriformes com a cepa Pb18. Quatro semanas após a infeccão, um dos grupos foi tratado com Itraconazol, droga de referência (20mg/kg, 1x dia, vo), outro grupo recebeu AJOENE (20mg/kg, dias alternados, ip), o terceiro grupo não recebeu tratamento, sendo considerado controle positivo e o quarto grupo foi constituído de animais não infectado e não tratado e foi considerado controle negativo. Após 2, 6, 10 e 13 semanas de tratamento, foi realizada a coleta do sangue e alguns parâmetros foram determinados para a avaliação da infecção e do tratamento. O sangue total foi utilizado para testes hematológicos como: contagem global e diferencial de leucócitos. O soro foi utilizado para avaliação do perfil bioquímico através da dosagem das enzimas Aspartato Aminotransferase (AST), Fosfatase Alcalina (FAL), Amilase e também a avaliação dos níveis de anticorpos anti-Pb através do teste de ELISA clássico. Também, através de análise histopatológica houve demonstração do fungo nos tecidos. Os resultados sugerem que o AJOENE apresentou atividade antifúngica para P. brasilensis, pois reduziu significativamente os níveis de anticorpos anti-Paracoccidioides brasiliensis a partir de 10ª semana de tratamento (AU)

Paracoccidioidomicose subaguda progressiva: tratamento de um doente com anfotericina B e nutriçäo parenteral / Progressive subacute paracoccidioidomycosis: treatment of a patient with amphotericin B and parenteral feeding

Os Autores descrevem um caso de paracoccidioidomicose subaguda progressiva, com quadro clínico sugestivo de síndrome de má absorçäo, em que o doente näo se beneficiara apenas com o tratamento antifúngico convencional. Ao se introduzir como medida auxiliar a nutriçäo parenteral houve evidente melhora clínica e laboratorial. Desta maneira os Autores propöem o uso associado da nutriçäo parenteral no tratamento de doentes com esta forma clínica de paracoccidioidomicose

Açäo da anfotericina B e Levamisole em camundongos albinos inoculados com Paracoccidioidomicose Brasiliensis / Study on anphotericin B and levamisole in albino mice inoculated with paracoccidioides brasiliensis

Paracoccidioidomicose experimental, em camundongos albinos de 3-4 semanas de idade. Inoculaçäo de 0,5 ml de suspensäo, contendo 1x10(devada a sexta potência) células/ml de Paracoccidioides brasiliensis e tratamento com anfotericina B (1 mg/Kg) em doses diárias; com levamisole (3 mg/Kg) administrada em intervalos de 15 dias e em séries de uma dose por dia, em três dias consecutivos; e com anfotericina B e levamisole associadas, de maneira a cobrir o período de 50, 80 e 95 dias para necrópsia. Estudo comparativo do quadro histológico de fígado e baço, com ênfase em granulomas, parasitas, células de Küpffer, células gigantes, reaçäo linforreticular, folículos e amioloidose. O animal revela-se satisftório, como modelo experimetnal, porém de uso limitado, devido ao fenômeno de auto-cura. Isoladamente, anfotericina B é menos eficiente do que quando em associaçöes com levamisole, para a reduçäo do número de parasitas. Animais com amiloidose esplênica apresentam alto número de parasitas. O quadro reativo revela qe a açäo de levamisole, isolada ou combinadamente com anforicina B, induz aumento de defesa com notável incremento da açäo de células fagocitárias