Inhibition of Human Neutrophil Apoptosis by Paracoccidioides brasiliensis: Role of Interleukin-8

Paracoccidioidomycosis (PCM) is a systemic mycosis caused by Paracoccidiodes brasiliensis that presents a wide spectrum of clinical manifestations . Because of the great number of neutrophils polymorphonuclear neutrophils (PMN) found in the P. brasiliensis granuloma, studies have been done to evaluate the role of these cells during the development of the infection. This fungus is found intracellularly in PMN and monocytes/macrophages, suggesting that it is capable of evading damage and surviving inside these cells. Thus, in the present study, we investigated whether P. brasiliensis can prolong the lifetime of PMN, and if this process would be related with IL-8 levels. PMN apoptosis and intracellular levels of IL-8 were analysed by flow cytometry and culture supernatants IL-8 levels were evaluated by enzyme-linked immunosorbent assay. We found that coincubation with P. brasiliensis yeast cells results in an inhibition of PMN apoptosis, which was associated with increase in IL-8 production by these cells. Cocultures treatment with monoclonal antibody anti-IL-8 reversed the inhibitory effect of P. brasiliensis on PMN apoptosis, besides to increase spontaneous apoptosis of these cells. These data show that, in contrast to other microbial pathogens that drive phagocytes into apoptosis to escape killing, P. brasiliensis can extend the lifetime of normal human PMN by inducing autocrine IL-8 production.     

Toll样受体2和Toll样受体4在免疫应答衣原体感染中的作用

衣原体是重要的人类病原体,其能够导致多种疾病的发生.由衣原体引起的许多人类疾病被认为是免疫病理学介导的.已经证明Toll样受体(TLRs)是多种病原体感染的主要模式识别受体( PRRs),在起始固有免疫应答,建立适应性免疫应答中发挥着重要作用.在TLR家族中,TLR2和TLR4与衣原体感染的相关性研究备受关注,在识别衣原体感染、调节宿主的早期免疫应答、炎症反应和病理形成中执行着关键性的作用.研究TLR2和TLR4在免疫应答衣原体感染中的作用可以更好地理解TLRs介导的分子免疫机制,可能有助于研发免疫治疗的分子靶标,最终有效预防、控制衣原体感染引起的疾病.     

Human neutrophils susceptibility to Paracoccidioides brasiliensis: an ultrastructural and cytochemical assay.

Paracoccidioidomycosis, caused by the dimorphic fungus Paracoccidioides brasiliensis, is the most prevalent systemic mycosis of Latin America, with Brazil accounting for 80% of the reported cases. The great number of neutrophils found in P. brasiliensis granulomas demonstrates the importance of polymorphonuclear neutrophils (PMN) cells during this mycotic infection. It has been found that neutrophils from healthy human donors can ingest and kill the fungus through a typical phagocytic process. The present work tests the phagocytic ability of neutrophils collected from patients that had had and were considered cured of paracoccidioidomycosis. Transmission electron microscopy and cytochemical studies indicate that patients' neutrophils eventually degenerate during phagocytosis of P. brasiliensis. Endogen peroxidase and NAD(P)H-oxidase are activated during the process showing that the respiratory burst and the neutrophil degranulation are triggered by the attachment of the yeast cells. Apparently these processes are not enough to kill P. brasiliensis. Although fungicidal activity can be determined by colony forming unit (CFU) counting, qualitative data suggest, as noted, that neutrophils from patients with treated paracoccidioidomycosis degenerate during the phagocytosis process. Hence, this work demonstrates the existence of a functional neutrophil deficiency against P. brasiliensis in susceptible individuals. The exact origin of this susceptibility is still to be determined in further studies.     

Molecular detection and identification of Paracoccidioides brasiliensis.

Nearly 800 nucleotides from the 5' terminus of the 28S ribosomal gene of Paracoccidioides brasiliensis were sequenced, and a 14-base DNA probe specific for this species was identified. Hybridization results showed that the probe identified P. brasiliensis ribosomal DNA in a panel of ribosomal DNAs representing a total of 48 species of fungi.     

Primers for clinical detection of Paracoccidioides brasiliensis

From a 0.72-kb fragment universally generated in Paracoccidioides brasiliensis strains, primers were designed and tested on genomic DNA of this and other pathogenic fungi. They were specific and highly sensitive for P. brasiliensis DNA. Positive results were obtained when these were tested in clinical samples.     

Random sequencing of Paracoccidioides brasiliensis genes.

Paracoccidioides brasiliensis genome has been reported as having a size of about 30 Mb. By digestion of genomic DNA from strain IVICPb 73 (ATCC 32071), we have constructed a DNA library with an insert size average of 8 kb in Escherichia coli XL1 Blue. We have fully sequenced 7 clones comprising 51,022 bp which represent 20 putative protein-coding sequences (seven of them, partial) and one tRNA. The 20 coding sequences cover 46% of the total 51,022 bp with introns present in 10 out of the 20 sequences. Database similarity analysis reveals the presence of genes conserved in other fungal species and higher organisms, including humans.     

Agrobacterium tumefaciens-mediated transformation of Paracoccidioides brasiliensis.

Paracoccidioides brasiliensis is the causative agent of paracoccidioidomycosis, an important mycosis endemic to Latin America. As the tools to study gene function in P. brasiliensis are only in the early stage of development, there is presently no system that allows for both the delivery and integration of exogenous nucleic acids into its genome. We report in this paper the transformation of the yeast phase of P. brasiliensis (ATCC-60855) with Agrobacterium tumefaciens (GV3101) carrying the vector pAD1625. The microorganisms were co-cultivated for 2 days and then incubated for 10 days at 35 degrees C on selective media. PCR and dot-blot targeted at a fragment of 222 bp from the hph (hygromycin phosphotransferase) gene which confers Hygr confirmed the transformation of P. brasiliensis.     

Susceptibility and resistance of inbred mice to Paracoccidioides brasiliensis

Nine different inbred strains of mice inoculated intraperitoneally with yeast cells of Paracoccidioides brasiliensis showed significantly varying patterns of susceptibility. The A/SN strain was found to be the most resistant, while BIOD2/nSn, BIO.A and BIOD2/oSn the most susceptible strains. These susceptibility differences were not dependent on the size of challenge inocula and sex of animals. All strains studied showed a mean survival time proportional to the size of inocula used. Although almost all infected male mice presented a shorter survival time when compared with females, significant mortality differences between sexes were found only in two of the strains studied, namely BALB/c and BIOD2/nSn. The H-2 region did not influence the susceptibility pattern since the A/SN and BIO.A strains share the same H-2 haplotype and were respectively highly resistant and susceptible to P. brasiliensis. Furthermore, the presence of C5 and unresponsiveness to lipopolysaccharide had no influence on the mortality data observed. Specific antibodies were detected only in a small number of animals and titres were consistently low, appearing later in the resistant (A/SN) than in a susceptible strain (BIO.A). Omentum, spleen and liver were the most affected organs in both strains, but the susceptible mice had more granulomatous lesions and earlier dissemination of the fungus.     

Comparison of the cell walls of Paracoccidioides loboi and Paracoccidioides brasiliensis by using polysaccharide-binding dyes

Biopsies from patients with Jorge Lobo's disease (keloid blastomycosis) and paracoccidioidomycosis (South American blastomycosis), caused respectively by Paracoccidioides loboi a non-cultivated fungus and Paracoccidioides brasiliensis, were stained with six different dyes having the property of forming complexes with some polysaccharides and oligosaccharides. The cell-walls of both fungi showed similar staining characteristics, suggesting a similar chemical structure. The role of the fungal cell-walls in evasion mechanisms is discussed.     

The role of chlamydospores of Paracoccidioides brasiliensis.

The role of chlamydospores in the conversion process from a mycelial-to-yeast form using the slide culture method was studied. Three clinical isolates and two other isolates from armadillo, belonging to the fungal species Paracoccidioides brasiliensis, were cultured on Sabouraud dextrose agar (SDA), potato dextrose agar (PDA) and brain heart infusion dextrose agar (BHIDA). Initially, the mycelial forms of each isolate were grown at 25 degrees C for 7, 14, 30 or 60 days on slide cultures and then the temperature was shifted to 35 degrees C. Interestingly, the slide cultures of all the isolates at 25 degrees C formed chlamydospores on either SDA or BHIDA, whereas, on PDA medium, aleurioconidia were formed. If the slide cultures on BHIDA were incubated at 35 degrees C for 7 to 14 days, multiple budding forms could be observed. This phenomenon was not evident in the slide cultures of SDA or PDA. The results of this morphological study indicate that in P. brasiliensis, chlamydospores may play an important role in the conversion process from a mycelial-to-yeast form.     

Microsatellite analysis of three phylogenetic species of Paracoccidioides brasiliensis

Paracoccidioides brasiliensis is the etiological agent of paracoccidioidomycosis, an important human systemic mycosis in Latin America. Recently, the existence of three different phylogenetic species (S1, PS2, and PS3) of P. brasiliensis was demonstrated. Despite being genetically isolated, all three species were capable of inducing disease in both humans and animals, although lower virulence has been found with the PS2 species. The available molecular methods developed to characterize and type strains have not been useful for assigning isolates to the described species, creating the need for molecular markers capable of distinguishing genetically isolated groups. Here, we describe a PCR and sequencing-based microsatellite marker system that is stable, easy to assay, adaptable to large series of isolates, and discriminatory enough to be used as a typing system in identifying the three proposed species of P. brasiliensis. In addition, this system provides an unambiguous tool for strain discrimination between two (S1 and PS2) of the three phylogenetic species.     

A 32-Kilodalton Hydrolase Plays an Important Role in Paracoccidioides brasiliensis Adherence to Host Cells and Influences Pathogenicity

One of the most crucial events during infection with the dimorphic fungus Paracoccidioides brasiliensis is adhesion to pulmonary epithelial cells, a pivotal step in the establishment of disease. In this study, we have evaluated the relevance of a 32-kDa protein, a putative adhesion member of the haloacid dehalogenase (HAD) superfamily of hydrolases, in the virulence of this fungus. Protein sequence analyses have supported the inclusion of PbHad32p as a hydrolase and have revealed a conserved protein only among fungal dimorphic and filamentous pathogens that are closely phylogenetically related. To evaluate its role during the host-pathogen interaction, we have generated mitotically stable P. brasiliensis HAD32 (PbHAD32) antisense RNA (aRNA) strains with consistently reduced gene expression. Knockdown of PbHAD32 did not alter cell vitality or viability but induced morphological alterations in yeast cells. Moreover, yeast cells with reduced PbHAD32 expression were significantly affected in their capacity to adhere to human epithelial cells and presented decreased virulence in a mouse model of infection. These data support the hypothesis that PbHad32p binds to extracellular matrix (ECM) proteins and modulates the initial immune response for evasion of host defenses. Our findings point to PbHAD32 as a novel virulence factor active during the initial interaction with host cells in P. brasiliensis.The adherence of pathogenic microorganisms to host tissues is considered indispensable for their initial colonization and successful infection and dissemination (38). The internalization process has been shown to depend greatly upon the adherence to the host cell surface that generates a cytoplasmic uptake signal (24). In the case of dimorphic fungal pathogens, of which the site of primary infection is generally the lung, the ability to adhere to epithelial cells represents a mechanism by which the infecting agent avoids the entrapment by respiratory tract mucus and, later on, elimination by the action of mucigen ciliary cells (24). Paracoccidioides brasiliensis is the etiological agent of paracoccidioidomycosis (PCM), one of the most common endemic systemic mycoses in Latin America (30, 32). As in other thermodimorphic fungi, P. brasiliensis mycelial fragments and microconidia act as the infectious propagules, reaching the lung alveoli, where, at the temperature of the host''s tissues (37°C), it shifts to the parasitic yeast form (6). During this process, adherence of P. brasiliensis to pulmonary epithelial cells is considered an essential event.Extracellular matrix (ECM) proteins have been shown to play an important role during the initial interaction and adherence between host cells and clinically relevant dimorphic fungi, such as P. brasiliensis, Penicillium marneffei, and Histoplasma capsulatum (17, 18, 22, 25). In P. brasiliensis, the major immunogenic antigen, Gp43 (a 43-kDa glycoprotein), detected in the cell wall and as an exocellular compound of both the yeast and mycelial phases, was proven to bind to laminin, a major ECM protein (27, 37, 39). More recently, Gonzalez and coworkers identified a 32-kDa protein in cell wall protein extracts of both forms of P. brasiliensis that was capable of binding to various ECM proteins, including laminin, fibronectin, and fibrinogen (14). Additionally, they demonstrated that this 32-kDa protein is involved in the initial conidial adherence to pulmonary epithelial cells that express ECM proteins on the surface, acting as a bridge between the two cell types (13).The main goal of this work was to further characterize this 32-kDa protein and its true role during the P. brasiliensis infectious process. Protein sequence analysis revealed a putative adhesion member of the haloacid dehalogenase (HAD) superfamily of hydrolases, P. brasiliensis Had32p (PbHad32p). Using antisense RNA (aRNA) technology and Agrobacterium tumefaciens-mediated transformation (ATMT), we constructed a mitotically stable P. brasiliensis PbHAD32 aRNA strain with consistently reduced gene expression (1, 2). Yeast cells with reduced PbHAD32 expression were significantly affected in their capacity to adhere to epithelial cells. Moreover, the knockdown strain presented decreased virulence in a mouse model of infection, pointing to PbHAD32 as a novel virulence factor during the initial interaction with host cells.     

Down-regulation of dendritic cell activation induced by Paracoccidioides brasiliensis

Paracoccidioidomycosis (PCM) endemic in Latin America, is a progressive systemic mycosis caused by Paracoccidioides brasiliensis (Pb). The infection can evolve to different clinical forms that are associated to various degrees of suppressed cell-mediated immunity. With the recognition that DCs are able to initiate response in na?ve T cells and that they also participate in Th cell education, the present study was undertaken to check whether DCs interact with P. brasiliensis, as well as to elucidate possible mechanisms and consequences of this interaction. Our results indicate that P. brasiliensis infection and purified gp43, its main antigenic component, lead to down-regulation of MHC-II and adhesion properties of immature DCs. The down-regulation was also observed in LPS-induced DC maturation. In addition, an inhibition of IL-12 and TNF-alpha production by both P. brasileinsis or gp43, was observed in LPS-induced DC maturation. These results suggest that protein, released in great amounts by the fungus, might be used, to reduce the effectiveness of the immune response.     

Susceptibility and resistance of inbred mice to Paracoccidioides brasiliensis.

Nine different inbred strains of mice inoculated intraperitoneally with yeast cells of Paracoccidioides brasiliensis showed significantly varying patterns of susceptibility. The A/SN strain was found to be the most resistant, while BIOD2/nSn, BIO.A and BIOD2/oSn the most susceptible strains. These susceptibility differences were not dependent on the size of challenge inocula and sex of animals. All strains studied showed a mean survival time proportional to the size of inocula used. Although almost all infected male mice presented a shorter survival time when compared with females, significant mortality differences between sexes were found only in two of the strains studied, namely BALB/c and BIOD2/nSn. The H-2 region did not influence the susceptibility pattern since the A/SN and BIO.A strains share the same H-2 haplotype and were respectively highly resistant and susceptible to P. brasiliensis. Furthermore, the presence of C5 and unresponsiveness to lipopolysaccharide had no influence on the mortality data observed. Specific antibodies were detected only in a small number of animals and titres were consistently low, appearing later in the resistant (A/SN) than in a susceptible strain (BIO.A). Omentum, spleen and liver were the most affected organs in both strains, but the susceptible mice had more granulomatous lesions and earlier dissemination of the fungus.     

Molecular detection of Paracoccidioides brasiliensis in road-killed wild animals.

Paracoccidioides brasiliensis infections have been little studied in wild and/or domestic animals, which may represent an important indicator of the presence of the pathogen in nature. Road-killed wild animals have been used for surveillance of vectors of zoonotic pathogens and may offer new opportunities for eco-epidemiological studies of paracoccidiodomycosis (PCM). The presence of P. brasiliensis infection was evaluated by Nested-PCR in tissue samples collected from 19 road-killed animals; 3 Cavia aperea (guinea pig), 5 Cerdocyon thous (crab-eating-fox), 1 Dasypus novemcinctus (nine-banded armadillo), 1 Dasypus septemcinctus (seven-banded armadillo), 2 Didelphis albiventris (white-eared opossum), 1 Eira barbara (tayra), 2 Gallictis vittata (grison), 2 Procyon cancrivorus (raccoon) and 2 Sphiggurus spinosus (porcupine). Specific P. brasiliensis amplicons were detected in (a) several organs of the two armadillos and one guinea pig, (b) the lung and liver of the porcupine, and (c) the lungs of raccoons and grisons. P. brasiliensis infection in wild animals from endemic areas might be more common than initially postulated. Molecular techniques can be used for detecting new hosts and mapping 'hot spot' areas of PCM.     

TLR2和TLR4在原发性肝癌中的表达

目的：本研究通过检测原发性肝癌Toll样受体2（TLR2）和Toll样受体4（TLR4）的表达，分析其与临床病理生理因素的关系，探讨TLR2，TLR4在原发性肝癌疾病过程中的可能作用。方法：采用免疫组化法和实时荧光定量PCR分别在蛋白水平及mRNA水平检测原发性肝癌组织和配对癌旁组织TLR2、TLR4的表达。结果：原发性肝癌组织在蛋白水平和mRNA水平TLR2和TLR4的表达均明显低于癌旁组织（P<0.01）。但免疫组化结果显示不论是肝癌组织还是癌旁组织，TLR2和TLR4的表达均明显高于正常肝组织（P<0.01，P<0.05），有门静脉分支癌栓的患者癌组织TLR4的表达强度低于无门静脉分支癌栓的患者（P<0.05）。结论：肝癌组织TLR2和TLR4的表达与癌旁组织相比受到相对抑制，但高于正常肝组织，TLR2和TLR4信号途径可能参与了原发性肝癌的病理过程。     

Electrophoretic karyotype of environmental isolates of Paracoccidioides brasiliensis.

Five of the 12 environmental isolates of the dimorphic fungus Paracoccidioides brasiliensis known to date, were analysed by contour-clamped homogeneous electric field gel electrophoresis (CHEF). The electrophoretic pattern was shown to consist of five bands, with molecular size ranging from 3.2 to 10 Mb, a model quite similar to the one found in the clinical isolates previously tested and used here as controls. However, one of the bands in the environmental isolates had a lesser weight (7.2 Mb), than the one corresponding to the clinical counterparts (8.8 Mb). This resulted in a smaller genome, approximately 29.7 Mb. The small differences that were found indicate the presence of chromosome polymorphism in this fungus.     

Production of Paracoccidioides brasiliensis exoantigens for immunodiffusion tests.

Growth curves of the yeast form of Paracoccidioides brasiliensis B-339 based on total and viable cell counts were determined. Crude culture filtrate antigens were obtained after 7, 10, 15, 20, 25, and 30 days of incubation. Different patterns of proteins were obtained by affinity chromatography on Sepharose 4B-immunoglobulin G complex made with immunoglobulin G from patients with paracoccidioidomycosis, with subsequent analyses by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and scanning densitometry. Three major proteins were excreted during the time course of a 30-day culture: a doublet at 20 to 21 kilodaltons (kDa) and molecules of 43 and 52 kDa. The 43-kDa antigen was present throughout the growth period, and its level reached a peak on days 15 to 20 and then decreased considerably toward day 30. The antigenic preparations collected on days 7, 10, 15, and 20 gave better reactions in immunodiffusion tests than those collected on days 25 and 30. The 7-day exoantigen gave a sensitivity of 97.1% and specificity of 100% on immunodiffusion. The main line of precipitation had a very high intensity, showing a total identity with that of a previously purified glycoprotein of 43 kDa. A 7-day crude exoantigen displayed a high level of sensitivity and specificity, being reproducible from batch to batch and retaining its activity for years when kept lyophilized. A protocol is recommended for the production of a stable diagnostic antigen to be used in immunodiffusion tests for paracoccidioidomycosis.     

Separation of Paracoccidioides brasiliensis conidia through percoll gradients.

The conidia of Paracoccidioides brasiliensis are the structures most likely to serve as the infectious propagules of this fungus. This study describes our attempts to purify conidia by eliminating mycelial fragments. Purification was attempted using discontinuous 95% and 60% Percoll gradients with densities of 1.167 and 1.107, respectively, prepared either in 0.15 mol/L PBS or 0.25 mol/L sucrose. The best results were observed with the 95% and 90% gradients in sucrose; with the former, conidial purity ranged from 70.6 to 100%, with a mean of 82.3% and a coefficient of variation (VC) of 11.7. With 90% gradients, purity was achieved between 70.4 and 92.5%. The mean in this case was 80.6% and the VC was 9.2%. The use of two consecutive 95% Percoll gradients in sucrose was tested. The recovery efficiency per plate, which averaged 2.5 x 10(6) conidia per plate with one gradient, increased to 5.1 +/- 1.3 x 10(6) conidia with two gradients. The use of Percoll did not affect the viability of the conidia, which was always > or = 90%. This method allows the preparation of a conidial sample almost free from contamination with mycelial fragments, thus facilitating quantitative determination of cause and effect in in-vivo interactions between P brasiliensis and its hosts.