Therapeutic treatment with scFv-PLGA nanoparticles decreases pulmonary fungal load in a murine model of paracoccidioidomycosis.

Paracoccidioidomycosis (PCM) is a systemic mycosis with lymphatic dissemination that is caused by Paracoccidioides species. Treatment of PCM consists of chemotherapeutics such as itraconazole, trimethoprim, sulfamethoxazole or amphotericin B. However, several studies are aiming to develop therapeutic alternatives for the treatment of fungal infection using new molecules as adjuvants. The single-chain variable fragments (scFv) from an antibody that mimics the main fungal component incorporated within poly(lactide-co-glycolic) acid (PLGA) nanoparticles helped treat the fungal disease. After expressing the scFv in Picchia pastoris (P. pastoris), the recombinant molecules were coupled with PLGA, and the BALB/c mice were immunized before or after infection with yeast Paracoccidioides brasiliensis (P. brasiliensis). Our results showed decreased disease progression and decreased fungal burden. Taken together, our results showed an increased of IFN-γ and IL-12 cytokine production and an increased number of macrophages and dendritic cells in the pulmonary tissue of BALB/c mice treated with a high concentration of our molecule. Our data further confirm that the scFv plays an important role in the treatment of experimental PCM.

Involvement of a 1-Cys peroxiredoxin in bacterial virulence.

The killing of bacterial pathogens by macrophages occurs via the oxidative burst and bacteria have evolved to overcome this challenge and survive, using several virulence and defense strategies, including antioxidant mechanisms. We show here that the 1-Cys peroxiredoxin LsfA from the opportunistic pathogen Pseudomonas aeruginosa is endowed with thiol-dependent peroxidase activity that protects the bacteria from H(2)O(2) and that this protein is implicated in pathogenicity. LsfA belongs to the poorly studied Prx6 subfamily of peroxiredoxins. The function of these peroxiredoxins has not been characterized in bacteria, and their contribution to host-pathogen interactions remains unknown. Infection of macrophages with the lsfA mutant strains resulted in higher levels of the cytokine TNF-α production due to the activation of the NF-kB and MAPK pathways, that are partially inhibited by the wild-type P. aeruginosa strain. A redox fluorescent probe was more oxidized in the lsfA mutant-infected macrophages than it was in the macrophages infected with the wild-type strain, suggesting that the oxidative burst was overstimulated in the absence of LsfA. Although no differences in the phagocytosis rates were observed when macrophages were infected with wild-type and mutant bacteria in a gentamicin exclusion assay, a higher number of wild-type bacterial cells was found in the supernatant. This difference was not observed when macrophages were pre-treated with a NADPH oxidase inhibitor, confirming the role of LsfA in the bacterial resistance to ROS generated via NADPH oxidase. In an acute pneumonia model, mice infected with the mutant strains presented higher cytokine release in the lungs and increased activated neutrophil recruitment, with reduced bacterial burden and improved survival rates compared to mice infected with the wild-type bacteria. LsfA is the first bacterial 1-Cys Prx shown to modulate host immune responses and its characterization will allow a better understanding of the role of redox signaling in host-pathogen interactions.

Recognition of enteroinvasive Escherichia coli and Shigella flexneri by dendritic cells: distinct dendritic cell activation states.

The innate and adaptive immune responses of dendritic cells (DCs) to enteroinvasive Escherichia coli (EIEC) infection were compared with DC responses to Shigella flexneri infection. EIEC triggered DCs to produce interleukin (IL)-10, IL-12 and tumour necrosis factor (TNF)-α, whereas S. flexneri induced only the production of TNF-α. Unlike S. flexneri, EIEC strongly increased the expression of toll like receptor (TLR)-4 and TLR-5 in DCs and diminished the expression of co-stimulatory molecules that may cooperate to inhibit CD4(+) T-lymphocyte proliferation. The inflammation elicited by EIEC seems to be related to innate immunity both because of the aforementioned results and because only EIEC were able to stimulate DC transmigration across polarised Caco-2 cell monolayers, a mechanism likely to be associated with the secretion of CC chemokine ligands (CCL)20 and TNF-α. Understanding intestinal DC biology is critical to unravelling the infection strategies of EIEC and may aid in the design of treatments for infectious diseases.

Recognition of enteroinvasive Escherichia coli and Shigella flexneri by dendritic cells: distinct dendritic cell activation states

The innate and adaptive immune responses of dendritic cells (DCs) to enteroinvasive Escherichia coli (EIEC) infection were compared with DC responses to Shigella flexneri infection. EIEC triggered DCs to produce interleukin (IL)-10, IL-12 and tumour necrosis factor (TNF)-α, whereas S. flexneri induced only the production of TNF-α. Unlike S. flexneri, EIEC strongly increased the expression of toll like receptor (TLR)-4 and TLR-5 in DCs and diminished the expression of co-stimulatory molecules that may cooperate to inhibit CD4+ T-lymphocyte proliferation. The inflammation elicited by EIEC seems to be related to innate immunity both because of the aforementioned results and because only EIEC were able to stimulate DC transmigration across polarised Caco-2 cell monolayers, a mechanism likely to be associated with the secretion of CC chemokine ligands (CCL)20 and TNF-α. Understanding intestinal DC biology is critical to unravelling the infection strategies of EIEC and may aid in the design of treatments for infectious diseases.

B-1 cells modulate oral tolerance in mice.

Although the origin and functions of B-1 cells are controversial, they are considered as a cellular element of innate immunity due to their ability to produce natural autoantibodies of the IgM type. These antibodies are encoded by a relatively limited repertoire of V genes, and their resulting diversity is smaller than that produced by conventional B cells. B-1 cells constitute the larger fraction of B cells in the peritoneal cavity and migrate to non-specific inflammation sites. In addition, they contribute to the production of IgA antibodies in the intestinal lamina propria. It has been demonstrated that they participate in the induction and maintenance of peripheral tolerance. Herein, the participation of B-1 cells in inducing oral tolerance is evaluated. Unexpectedly, BALB/Xid mice, the animals deficient in B-1 cells, are not tolerized to OVA but instead are responsive to oral immunization. Conversely, BALB/c mice respond to oral tolerance to this antigen. We used these biological characteristics of these animals to investigate whether B-1 cells are involved in the induction of oral tolerance to OVA. Results show that B-1 cells from BALB/c mice, treated orally with OVA and adoptively transferred to BALB/Xid mice were able to suppress local hypersensitivity reaction and lymphoproliferative cellular response observed in BALB/Xid mice. These data demonstrate that B-1 cells have regulatory properties and are involved in the induction of oral tolerance.

Importância de células apresentadoras de antígeno em determinar padrão TH1/TH2 em camundongos resistentes e suscetíveis à paracoccidioidomicose / Importance of antigen-presenting cells in determining the Th1/Th2 pattern in experimental paracoccidioidomycosis

Paracoccidioidomicose é uma micose sistêmica, endêmica na América Latina, cusada pelo fungo dimórfico térmico Paracoccidioides brasiliensis (Pb). A infecçao pode levar a diferentes formas clínicas que estao associados com vários graus de supressao da resposta imune celular. No modelo murino foram determinadas duas principais linhagens de camundongos isogênicos, A/Sn e BLO.A, que sao resistentes e suscetíveis, respectivamente à infecçao pelo o fungo. Assumindo que a resposta imune efetora é mediada pela ativaçao preferencial de células do tipo Th1, e nao de células Th2, no presente trabalho avaliou-se a importância de diferentes células apresentadoras de antígeno (macrófagos, linfócitos B e células dendríticas) no desenvolvimento de uma resposta imune contra Pb. Em camundongos resistentes, gp43, o mais importante componente antigênico de Pb, é preferencialmente apresentado por macrófagos e células dendríticas, que estimulam produçao de citocinas do tipo Th1 por linfócitos T. Por outro lado, em animais suscetíveis, gp43 foi preferencialmente apresentada por células B que induziu a ativaçao de células do tipo Th2. Além disso, células T de camundongos resistentes responderam como se fossem de animais suscetíveis quando estimulados por gp43 apresentada por macrófagos e linfócitos B de camundongos suscetíveis e vice-versa, indicando que nao existe diferença significante no repertório de linfócitos T de camundongos A/Sn e BLO.A. Quando células T de camundongos F1 (A/Sn x BLO.A) foram estimulados por gp43 apresentada por macrófagos e linfócitos B de camundongos A/Sn e BIO.A, observou-se que macrófagos de BLO.A estimularam com baixa eficiência a ativaçao de células Th1 e células B de camundongos BLO.A estimularam linfócitos T a secretarem elevados níveis de IL-10. Portanto, nossos resultados sugerem que células apresentadoras de antígeno podem estar envolvidas na evoluçao da infecçao por Pb