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Evaluation of the Therapeutic Effect of Radioattenuated Yeast Cells in Experimental Paracoccidioidomycosis
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Paracoccidioides brasiliensis is the agent of paracoccidioidomycosis (PCM), the most prevalent deep mycosis in Latin America. The treatment varies according to the chemotherapeutic drug and the disease severity, and the lack of it can results in high frequency of relapse and sequels. Thus, the search for new therapeutic alternatives is necessary. The aim of this study was to evaluate the therapeutic effect of P. brasiliensis yeast cells attenuated by gamma irradiation (LevRad) in Balb/c mice. Mice immunized with LevRad and/or treated with fluconazole presented a significant decrease in the CFU recovery from the lung, liver and spleen in comparison with non-immunized and non-treated infected mice (60 and 120 days after infection). After 120 days from fungal inoculation, no fungi colonies were obtained from the organs of treated and immunized mice. The tissue structure of these organs was largely preserved. At the same time, anti-Mexo specific IgG antibodies levels and TGF-β, IFN-γ, iNOS transcript levels were high and a decrease on the IL-4, IL-10 and TNF-α transcript levels was also verified. An additive protective effect of immunization with LevRad associated to chemotherapy in a PCM experimental model was verified, providing evidence of the therapeutic effect of attenuated yeast for fungal infections.
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Vaccines play an essential role in keeping humans healthy. Innovative approaches to their use include the utilization of plasmid DNA encoding sequences to express foreign antigens. DNAhsp65 from Mycobacterium leprae is suitable for this purpose due to its ability to elicit a powerful immune response. Controlled release systems represent a promising approach to delivering vaccines. In this work, we used liposomes or PLGA systems to deliver DNAhsp65 to treat the pulmonary fungal infection Paracoccidioidomycosis. Both formulations modulated a protective immune response and reduced the pulmonary fungal burden even in the groups receiving less than four times the amount of the DNAhps65 entrapped within the nanoparticles. Although both systems had the same effective therapeutic results, the advantage of the liposome formulation was that it was administered intranasally, which may be more easily accepted by patients. These systems are a great alternative to be considered as adjuvant vaccine therapy for systemic mycosis.
Paracoccidioides brasiliensis is the agent of paracoccidioidomycosis, the most prevalent mycosis in Latin America. Up to the moment no vaccine has been
reported. The aim of this study was to evaluate the influence of the number of immunizations on the protection elicited by
radioattenuated yeast cells of P. brasiliensis. BALB/c mice were divided into two groups that were immunized once (Group 1) or twice (Group 2), respectively. In each group,
mice were divided into sub-groups that were challenged 30, 45, or 60days after the second immunization. Organ colony-forming
units (CFUs) was determined 90days post-challenge. A significant reduction in CFUs recovery was verified in both groups,
but it was higher in Group 2. Histologic alterations were observed only in Group 1. The cytokines IL-4, IL-10, and IFN-γ were
produced in mice of Group 1. In Group 2, only IFN-γ was significantly detected. IgG2a predominance relative to IgG1 was also
observed in Group 2. Altogether, our results indicated that mice immunized once developed a mixed Th1/Th2 response, which
was less efficient in the infection control, while a trend to a Th1 pattern was obtained with two immunizations, promoting
optimal elimination of P. brasiliensis yeast cells from mice tissues.
Paracoccidioidomycosis (PCM), caused by Paracoccidioides brasiliensis, is the most prevalent invasive fungal disease in South America. Systemic mycoses are the 10th most common cause of death among infectious diseases in Brazil and PCM is responsible for more than 50% of deaths due to fungal infections. PCM is typically treated with sulfonamides, amphotericin B or azoles, although complete eradication of the fungus may not occur and relapsing disease is frequently reported. A 15-mer peptide from the major diagnostic antigen gp43, named P10, can induce a strong T-CD4+ helper-1 immune response in mice. The TEPITOPE algorithm and experimental data have confirmed that most HLA-DR molecules can present P10, which suggests that P10 is a candidate antigen for a PCM vaccine. In the current work, the therapeutic efficacy of plasmid immunization with P10 and/or IL-12 inserts was tested in murine models of PCM. When given prior to or after infection with P. brasiliensis virulent Pb 18 isolate, plasmid-vaccination with P10 and/or IL-12 inserts successfully reduced the fungal burden in lungs of infected mice. In fact, intramuscular administration of a combination of plasmids expressing P10 and IL-12 given weekly for one month, followed by single injections every month for 3 months restored normal lung architecture and eradicated the fungus in mice that were infected one month prior to treatment. The data indicate that immunization with these plasmids is a powerful procedure for prevention and treatment of experimental PCM, with the perspective of being also effective in human patients.
Using the intraperitoneal route of infection, we demonstrated previously that A/Sn mice are resistant and B10.A mice are susceptible to Paracoccidioides brasiliensis infection. Since paracoccidioidomycosis is a deep systemic granulomatous disorder that involves primarily the lungs and then disseminates to other organs and systems, we herein investigated the course of the infection and the resulting immune responses developed by A/Sn and B10.A mice after intratracheal infection with P. brasiliensis yeast cells. It was observed that A/Sn mice develop a chronic benign pulmonary-restricted infection, whereas B10.A mice present a chronic progressive disseminated disease. A/Sn animals were able to restrict fungal infection to the lungs despite the increased fungal load at the beginning of the infection. This behavior was associated with low mortality rates, the presence of adequate and persistent delayed-type hypersensitivity reactions, oxidative burst by bronchoalveolar cells, and production of high levels of specific antibodies in which immunoglobulin G2a (IgG2a) and IgG3 isotype titers were significantly higher than those observed in the susceptible mice. In contrast, B10.A animals showed a constant pulmonary fungal load and dissemination to the liver and spleen. This infection pattern resulted in high mortality rates, discrete delayed-type hypersensitivity reactivity, poorly activated or nonactivated bronchoalveolar cells, and production of specific IgG2b isotype titers significantly higher than those observed in the resistant mice at week 4 of infection. Thus, A/Sn and B10.A mice maintain the same resistance patterns as those observed previously with the intraperitoneal route of infection. Furthermore, the obtained results suggest that resistance to paracoccidioidomycosis is associated with T-cell, macrophage, and B-cell activities that are known to be mediated by gamma interferon.
Paracoccidioidomycosis is the most prevalent systemic mycosis in Latin America, among immunecompetent patients. It's caused by the dimorphic fungus Paracoccidioiddes brasiliensis. Investigations regarding its immunopathogenesis are very important in the understanding of aspects related to natural history, as the protective immunity, and the relationship between host and parasite; also favoring the knowledge about clinical patterns and the elaboration of therapeutic strategies. The disease clinical polymorphism depends, at least, of the immune response profile according to the tissue and blood released citokynes, resulting in tissue damage.
Paracoccidioides brasiliensis is the agent of paracoccidioidomycosis (PCM), the most prevalent deep mycosis in Latin America. The treatment varies according to the chemotherapeutic drug and the disease severity, and the lack of it can results in high frequency of relapse and sequels. Thus, the search for new therapeutic alternatives is necessary. The aim of this study was to evaluate the therapeutic effect of P. brasiliensis yeast cells attenuated by gamma irradiation (LevRad) in Balb/c mice. Mice immunized with LevRad and/or treated with fluconazole presented a significant decrease in the CFU recovery from the lung, liver and spleen in comparison with non-immunized and non-treated infected mice (60 and 120 days after infection). After 120 days from fungal inoculation, no fungi colonies were obtained from the organs of treated and immunized mice. The tissue structure of these organs was largely preserved. At the same time, anti-Mexo specific IgG antibodies levels and TGF-β, IFN-γ, iNOS transcript levels were high and a decrease on the IL-4, IL-10 and TNF-α transcript levels was also verified. An additive protective effect of immunization with LevRad associated to chemotherapy in a PCM experimental model was verified, providing evidence of the therapeutic effect of attenuated yeast for fungal infections.
Paracoccidioidomycosis is a granulomatous systemic mycosis endemic in Brazil and other Latin America countries. A DNA vaccine encoding the immunoprotective peptide 10 (P10) significantly reduced the fungal burden in mice when given prior to or after intratracheal challenge with P. brasiliensis. Presently, the generation/expansion of CD4(+)CD44(hi) memory T cells as well as Foxp3(+) T reg cells in mice immunized with the DNA vaccine (pcDNA3-P10) before and after infection with P. brasiliensis was investigated. Memory CD4(+) CD44(hi) T cells simultaneously with Foxp3(+) T reg cells increased in the spleens and lungs of pcDNA3-P10 immunized mice on day 0, 30, 60 and 120 postinfection. Histopathology of the lung tissue showed minimal inflammation in immunized mice compared with the unimmunized group, suggesting a role for regulatory T cells in controlling the immunopathology. The DNA vaccine shows that the repeated immunization generates memory cells and regulatory T cells that replace the initially protective pro-inflammatory T cells conferring a long term protection while preserving the integrity of the infected tissue.
Soluble antigens of Paracoccidioides brasiliensis yeast cells (PbAg) were fractionated in a fast protein liquid chromatography (FPLC) system, using Q-Sepharose anion-exchange resin, in order to characterize antigenic fractions that could elicit cell reactivity and antibody recognition in human paracoccidioidomycosis (PCM). PbAg fractions were eluted by 20 mM Tris-HCl solution (pH 9.6) with an increasing gradient up to 1 M NaCl. The FPLC system was able to resolve 7 fractions, enumerated from 0 to VI, according to the elution on the NaCl gradient. The analysis of each fraction on SDS-PAGE showed that fractions 0 to V were constituted by multiple protein bands with molecular mass ranging from 18 to 114 kDa. Large amounts of nucleic acids were evidenced in fraction VI, as revealed by agarose gel stained with ethidium bromide. Sera from PCM patients presenting different clinical forms contained antibodies that recognized antigens in all fractions with the exception of fraction VI as detected by ELISA. Further studies were designed to investigate the capacity of these fractions to induce cell proliferation. It was demonstrated that fractions III and V (200 and 450 mM NaCl, respectively) stimulated a significant proliferative response of peripheral blood mononuclear cells, while fraction 0 induced the lowest proliferative response among patients with PCM, in either acute, acute treated, or chronic forms.
The analysis of cytokine profiles helps to clarify functional properties of immune cells, both for research and for clinical diagnosis. The real-time reverse transcription polymerase chain reaction (RT-PCR) is becoming widely used to quantify cytokines from cells, body fluids, tissues, or tissue biopsies. Being a very powerful and sensitive method it can be used to quantify mRNA expression levels of cytokines, which are often very low in the tissues under investigation. The method allows for the direct detection of PCR product during the exponential phase of the reaction, combining amplification and detection in one single step. In this review we discuss the principle of real-time RT-PCR, the different methodologies and chemistries available, the assets, and some of the pitfalls. With the TaqMan chemistry and the 7700 Sequence Detection System (Applied Biosystems), validation for a large panel of murine and human cytokines and other factors playing a role in the immune system is discussed in detail. In summary, the real-time RT-PCR technique is very accurate and sensitive, allows a high throughput, and can be performed on very small samples; therefore it is the method of choice for quantification of cytokine profiles in immune cells or inflamed tissues.