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    Number of colony forming units (CFU)/g of lung tissue from Swiss mice infected with Paracoccidioides brasiliensis. *P < 0.05 compared with week 1 after infection. Mean ± SEM, N = 8.

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    Photomicrographs of the lungs of Swiss mice infected with Paracoccidioides brasiliensis. (A) One week after inoculation: start of the infection and organization of the granuloma. (B) Production of reticular fibers (arrow). (C and D) Two weeks after inoculation: amorphous and fragmented cells, budding yeasts, and reticular fibers impregnated by silver. (E and F) Four weeks after inoculation: an intensification of the infection was observed, with giant cells (arrow) with a tendency of centralization and more peripheral plasmocytes. (G) Eight weeks after infection: the presence of organized granuloma, with compact aggregates of macrophages, epithelial cells and yeasts in the central portion, and intense peripheral basophilia. (H) A greater quantity of reticular fibers encircling groups of yeast. Staining was carried out with hematoxylin and eosin (H&E) in (A, C, E, and G), and with Gomori–Grocott in (B, D, F, and H). This figure appears in color at www.ajtmh.org.

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    (A, B, C, D, and F) Photomicrographs of the lung parenchyma of Swiss mice infected with Paracoccidioides brasiliensis. (E) Non-infected lung stained with HE. In the infected lungs an increase in the amount of type I and III collagen fibers was observed in the granulomas stained by picrosirius, over time, as can be seen in figures (A, B, C, and D), which correspond to the first, second, fourth, and eighth weeks after inoculation, respectively. (F) Image stained with picrosirius without polarized light, corresponding to the sequential section in image (D), where a granuloma with yeast foci around the bronchus can be seen. This figure appears in color at www.ajtmh.org.

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    Photomicrographs of the lungs of Swiss mice infected with Paracoccidioides brasiliensis, and stained by the picrosirius technique, as modified by Dolber and Spach, 24 showing a granuloma 8 weeks after inoculation. A 63× objective NA1.2. Collagen fibers can be seen encircling the granuloma. This figure appears in color at www.ajtmh.org.

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    Photomicrographs of the lungs of Swiss mice infected with Paracoccidioides brasiliensis. Pycnotic structures (arrow) with the characteristics of apoptotic bodies in the granulomas of mice infected for (A) 1 week and (B) 8 weeks, using HE stain. This figure appears in color at www.ajtmh.org.

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Morphologic Organization of Pulmonary Granulomas in Mice Infected with Paracoccidioides brasiliensis

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  • 1 Post-graduate Program in Clinical Analyses, Laboratory of Medical Mycology, Laboratory of Immunology, Department of Clinical Analyses, Laboratory of Histology, Department of Morphophysiological Sciences, Universidade Estadual de Maringá, Maringá, Paraná, Brazil

A morphologic study of the lungs was carried out in Swiss mice infected with yeast isolated from Paracoccidioides brasiliensis (Pb18). The lung was processed 1, 2, 4, and 8 weeks after inoculation for histologic staining with hematoxylin and eosin (H&E), methenamine silver nitrate (Gomori–Grocott), and picrosirius to qualitative and quantitative analyses of the granulomas and the presence of fungal lesions. The numbers of CFUs/g counted in the lungs were 189.8 ± 20.64, 353.6 ± 46.21, 547.2 ± 108.1, and 295.2 ± 89.17 in the first, second, fourth, and eighth weeks, respectively. One week after infection, inflammatory cells and reticular and collagens fibers, the latest typical of fibrosis, were detected. After 2 and 4 weeks, a progressive intensification of the infection and fibrosis was observed, but in week 8 a more organized granuloma was evident, with macrophages, epithelioid cells, and yeasts in the central portion, and intense peripheral basophilia. Pycnotic structures typical of apoptotic bodies were observed in weeks 1 and 8. The different histologic staining used acted as a fundamental tool for the study of the morphologic organization of granuloma formation.

INTRODUCTION

Paracoccidioidomycosis (PCM) is a systemic mycosis of granulomatous nature, autochthonous to Latin America,1 caused by the thermally dimorphic fungus Paracoccidioides brasiliensis (Pb).2 In nature, the fungus exists in the filamentous form, which produces conidia, the structures responsible for the human infection.3

The respiratory tract is considered to be the main entry route of the infection, and from an initial focus in the lung, lymphatic or hematogenous dissemination to other regions of the organism occurs.3 A definitive diagnosis is obtained by the demonstration of the etiologic agent in biological fluids or tissues, and its identification is achieved through fresh culture or histopathologic microscopic examination.4 Its importance for public health is linked to the social and economic costs derived not only from the activity of the disease, but also from the frequent secondary lesions caused by this mycosis, which are a common cause of worker incapacitation and generally lead to death if not treated.5

Clinical manifestations occur in a very insidious manner, including a dry cough that later increases secretions and causes difficulty in breathing when doing physical activities. However, the most limiting consequence of this mycosis is the occurrence of chronic pulmonary insufficiency6 resulting from the development of fibrosis. 7,8 Pulmonary manifestations are present in 90% of patients,6 and for this reason, the virulence of Pb needs to be studied through the experimental infection of animals that are susceptible to the fungus and capable of showing the same general disease pattern and chronicity that is observed in humans.810

Depending on fungal-host relationship, changes with regard to pattern of granuloma formation and the occurrence of fibrosis in the organs involved may occur. 11 This fibrosis seems to be a result of a substantial increase in the production of cytokines, such as tumor necrosis factor (TNF)-α and transforming growth factor (TGF)-β, which are capable of inducing an accumulation of collagen, sometimes causing severe hemodynamic alterations, especially in the lungs, and resulting in the lungs becoming anatomically compromised. 1214

Because paracoccidioidomycosis develops slowly, it is asymptomatic and sometimes has a long incubation time. In general, when the patient seeks medical help, the lesions are already established in various evolutionary stages. 15 Experimental studies provide important information about various aspects of the disease, starting from its initial stage and enabling the establishment of systematic correlations between lesion patterns and their clinical forms.8

Pulmonary fibrosis is an incapacitating consequence of the disease that can severely disrupt respiratory function and compromise the well being of the patient. Its development is characterized by a proliferation of fibroblasts and an enlargement of the extracellular matrix. 12 Pulmonary fibrosis occurs simultaneously with the inflammatory process and leukocyte infiltration. This picture progresses with time and consolidates to form a series of chronic granulomatous processes. 7,1417

The inoculation of P. brasiliensis yeast through the lateral caudal vein of mice enables the lungs to be affected by a standard diffusion of the fungus, 18 and the experimental model is therefore capable of reproducing the characteristics of the chronic pulmonary form of the disease in humans; 19 offering the advantages of being able to standardize the quantity of the inoculate and of being reproducible. 20,21

The histopathology of organs infected by Pb was studied through conventional staining methods to show the alterations caused by infection with this microorganism. The techniques of staining with hematoxylin and eosin (HE) and Gomori’s technique of methenamine silver nitrate (Gomori–Grocott) 22 are the most frequently used methods for the histopathologic evaluation of organs infected by P. brasiliensis.

Microbiologic techniques for the isolation and identification of the agent in infectious diseases offer a quick and specific determination of the etiologic agent. However, the ability to use an experimental model in mice, associated to histopathologic analysis techniques, has the advantage of adding important information about the evolution of paracoccidioidomycosis, and in some circumstances is capable of providing data for the establishment of a diagnosis. Tissue analyses enable the evolution of the microorganism in the host to be followed, as well as the evaluation of tissue damage, and tissue response to the infection. The use of cytopathologic and histopathologic methods enables clinical microbiologists and pathologists to make early and more precise patient diagnoses. The objective of this study is to demonstrate the morphologic organization of pulmonary granulomas in mice infected with P. brasiliensis, after 1, 2, 4, and 8 weeks, using as tools conventional techniques for histologic staining.

MATERIALS AND METHODS

Fungal isolate.

An isolate of P. brasiliensis (Pb18) from the mycology collection of the Department of Immunology of the Universidade Federal de São Paulo (UNIFESP), cultivated in Fava Neto medium and kept at 35°C in the Laboratory of Medical Mycology of the Universidade Estadual de Maringá (UEM) was used in this study.

For the preparation of the inoculum, 4 mL of sodium phosphate buffer (PBS) (pH 7.2) was added to a tube containing the fungus, which had been cultivated for 5 days in yeast form. After homogenization, the suspension was transferred to a tube containing glass beads and shaken for several seconds in a vortex. The concentration of the cells was adjusted to 2 × 106 fungi/mL in a Neubauer chamber. Cell viability was determined by the Janus green dye exclusion method and was more than 95%.

Animals.

All of the procedures involving the use of animals were approved by the Ethics Committee for Animal Experimentation (CEEA) of UEM, under protocol 037/2004. Groups of eight male mice of Swiss lineage aged between 4 and 5 weeks and with a mean weight of 30 grams, originating from the Central Animal Laboratory of UEM were used. During the experiments, the mice were kept in the Animal Laboratory Section of the Laboratory for Experimentation on Paracoccidioidomycosis of the Department of Clinical Analyses, UEM, under controlled environmental conditions, with a temperature of 23 to 24°C and a 12-hour light/dark cycle, receiving balanced feed and water ad libitum.

Infection of the animals with the P. brasiliensis fungal isolate.

The animals were distributed into four groups of eight, and endovenously inoculated (lateral caudal vein) with a volume of 0.1 mL of fungal suspension containing 2 × 105 yeast-form cells of the Pb18 isolate. Each of the control groups was composed of six mice, which received 0.1 mL of PBS.

Sacrifice and histologic processing.

Both infected and control animals were killed by an overdose of anesthetic (Ketamine and Xylazine) 1, 2, 4, and 8 weeks after inoculation. The whole of the lungs were removed and the lower right lobe was collected for histologic processing. The remaining parts were weighed, macerated, and processed for the counting of colony-forming units per gram (CFUs/g) of tissue.

The segments destined for histopathologic processing were fixed in Bouin’s solution for 24 hours and processed for embedding in paraffin. They were then serially cut into 7-μm-thick sections and sequentially stained using the following methods: HE, for cellular characterization and the granuloma count; Gomori–Grocott for the localization and identification of the fungus and reticular fibers. This staining technique is based on methenamine silver impregnation, and it is frequently used in the diagnosis of fungal lesions, because it is able to detect polysaccharides present in the cellular wall of the fungus by the process of oxidation. Furthermore, it enables the identification of reticular fibers, rich in type III collagen, present in the granulomas. 7,17

The three-dimensional arrangement and the orientation of the collagen fibers can be evidenced by staining with sirius-red, by the Picrosirius (PSR) technique, and analyzed in a polarizing microscope to identify the collagen and classify it into type I and III. Through this method, type I collagen appears strongly birefringent, with yellow or red fibers, whereas type III collagen appears weakly birefringent, with green fibers. 23 Pre-treatment of the tissue sections with phosphomolybidic acid (PMA) followed by the PSR staining technique reduces the background, making the method more sensitive and enabling its use in a confocal microscope. 24

The lungs of the mice infected for 8 weeks were also observed and recorded in a confocal microscope (LSM 5/Pascal/Zeiss), for which the segments were cut into 30-μm sections and stained according to the Picrosirius technique, as modified by Dolber and Sapach. 24 The images were visualized using Zeiss LSM Image Browser software. The optical sections were made using an argon laser with a wavelength of 488 nm.

Determination of the CFUs.

The lung fragments were weighed, placed in sterile Petri dishes with 5 mL of PBS, and macerated until a homogeneous suspension was obtained. Then, 300 μL were retrieved from the suspensions, placed into Petri dishes containing brain heart infusion (BHI) agar medium, and incubated at 35°C for 15 days. The counts of CFUs were carried out on the seventh and fifteenth days of incubation, and the numbers of CFU/g of tissue were calculated.

Granuloma count.

A histologic section of lung was outlined in a light chamber (Olympus BX41, Olympus Corp., Tokio, Japan) to create a chart that plotted the positions of the granulomas found in all of the sections analyzed, thereby avoiding repeated counts of the same granuloma. The counts were carried out under a 20× objective on an Olympus BX41 microscope by two observers.

To determine the approximate area in which the counts were carried out, a histologic section of the lungs of each animal was scanned and its area determined using an image analysis program (Image Pro Plus, version 4.5, Media Cybernetics, Silver Spring, MD). A mean area of 22.42 mm2 ± 0.73 was calculated and used as a standard for the area in which the granulomas were counted. The numbers of granulomas were therefore expressed as number of granulomas/22.42 mm2.

Statistical analysis.

Analysis of variance (ANOVA) was used for the comparisons of the numbers of granulomas and CFUs in the lungs from the different groups. Parametric Tukey test was used as the post-test, with a 5% level of significance.

RESULTS

Number of CFUs and granulomas in lungs infected with Pb.

From the first to the fourth week after inoculation, there was a progressive increase in the number of CFUs in the lungs of the infected mice, followed by a reduction in the eighth week (Figure 1). The increase was significantly larger in the fourth week compared with that in the first week (P < 0.05). The mean ± SE of CFUs/g in the lungs were 189.8 ± 20.64, 353.6 ± 46.21, 547.2 ± 108.1, and 295.2 ± 89.17 in the first, second, fourth, and eighth weeks, respectively. Table 1. shows the mean number of granulomas for the weeks studied.

Histopathology.

In the histopathologic study using HE, after the first week of infection, the parenchyma showed infiltration of plasmocytes, neutrophils and, predominantly, macrophages around the yeast-form foci, characterizing the start of the infection and the organization of granulomas. In the granuloma stained by the Gomori–Grocott method, the fungi in both budding and fragmented yeast (Figures 2A), and 2B), were observed diffusely distributed through the pulmonary parenchyma. The first signs of fibrosis were detected in the first week by the Grocott and Picrosirius methods, which enabled the visualization of reticular fibers encircling groups of yeast and type I and type III collagen fibers (Figure 3A), in small initial granulomas.

In the second week (Figures 2C and 2D), the granulomas were already better delineated. The infiltrate was predominantly made up of agglomerates of isolated or non-isolated macrophages and plasmocytes. There were many amorphous, fragmented and contracted cells in their central regions, around the fungi that presented little budding.

An increase in the quantity of, and an apparent thickening of, the reticular fibers was evident when using the Gomori–Grocott method. Although there was no statistical difference, there was a smaller number of granuloma in the second week compared with the first, fourth, and eighth weeks. When using the Picrosirius method, the collagen fibers were only weakly visualized.

In the fourth week (Figures 2E and 2F), there was an intensification of the infectious process. More intense inflammatory infiltrate was observed, composed of giant cells, neutrophils, and plasmocytes. There was also an apparent structural evolution in the granulomas, which had a more organized arrangement of fibers. The establishment of two distinct areas, the center and the periphery, was observed. In the central area, phagocytic cells ordered around the yeasts, delimiting the pathogen, were seen. Numerous macrophages formed aggregates of epithelioid cells. It was also possible to observe giant multi-nucleated cells enveloping the fungi and various neutrophils around the yeasts. In the peripheral region, intense basophilia characterized the presence of plasmocyte nuclei and possibly of fibroblasts. When using the Grocott stain, this area appeared more clearly, interspersed with reticular fibers and enveloping groups of yeasts. The granulomas, large and well developed, showed a large number of budding yeasts. An increase in the production of the reticular (Figure 2F) and collagen (Figure 3C) fibers was observed.

In the eighth week, the defensive process appeared to be more intense, with a greater number of granulomas of various sizes and organization levels. The granulomas were constituted by compact aggregates of macrophages, epithelioid cells, giant cells, and numerous parasites in the central portion. Their organization into a central zone with yeasts and a peripheral zone became more evident (Figures 2G and 2H). In this period, there was a very evident evolution in the process of fibrosis (Figures 2H and 3D). A large number of yeasts were found to be completely delimited by an extensive fibrous network in each granuloma. Under polarization, the granulomas showed a greater number of immature green fibers and thicker yellow-red fibers. An increase in the vascularization of the pulmonary parenchyma was observed around the granuloma when using HE.

In the eighth week, the granulomas were observed under a confocal microscope (Figure 4). The images showed the three-dimensional arrangement of the granulomas, with surface cells and fibers arranged around it. It was not possible to identify what type of fiber encircled the structure.

In the first and eighth weeks of infection, pycnotic structures with the characteristics of apoptotic bodies were observed (Figures 5A and 5B). In the control groups, no alterations with regard to the number or type of cells were observed at any time.

DISCUSSION

In this study, the evolution of pulmonary paracoccidioidomycosis in Swiss mice after endovenous inoculation with a highly virulent isolate of Pb18 25 was evaluated. The evolution of the infection was observed for 8 weeks and a pattern of granuloma formation was reported using conventional staining methods.

Experimental studies with animals provide important information about aspects of the disease from its initial stage, thereby enabling correlations to be established in a systematic manner between lesion patterns and their clinical forms. 10

The pulmonary alterations resulting from infection with Pb, described in this study, correspond to those observed by other authors that have studied the progressive form of experimental paracoccidioidomycosis, although with different species of animals, infection times, organs studied, and manner of inoculation. 7,15,17 The inoculation path used in this study (lateral caudal vein) is capable of infecting the lungs with a standard diffusion of the agent 18 and has the further advantage of enabling standardization of the quantity of the inoculate and its reproducibility. 20,21

The recovery of P. brasiliensis through its CFUs during the 8 weeks of infection clearly indicated that the infection was established, and the number of colonies suggested the progression of the infection in the first 4 weeks, and an apparent remission detected in the eighth week. Experiments carried out with Balb/c mice inoculated with conidia via the intranasal route, resulted in a decrease in the number of CFUs, after the first week of infection, followed by a progressive increase up to the sixteenth week.7

The number of granuloma did not reflect the number of CFUs. Their count suggests that there was a defensive response from the animal at the beginning of the infectious process that manifested itself as a decrease in the number of granulomas in the second week. However, in the subsequent weeks, the number of granulomas began to increase again, which shows that the pathogen proliferated, thereby stimulating the defensive response. The animals did not show alterations at the macroscopic level, although the histopathologic study revealed the evolution of an infectious process, involving the formation of inflammatory infiltrate and granulomas in the pulmonary parenchyma.

The response of the host against P. brasiliensis depends more on the immune response mediated by cells than by the humoral response. The macrophages are the defense cells most involved against P. brasiliensis.26 These cells locate themselves around the yeast and, through a process of maturation, undergo structural and functional modifications to form aggregates with the appearance of an epithelial cell, creating a structure called an epithelioid granuloma, 27 which is the most typical form of inflammatory tissue reaction against P. brasiliensis.28,29 In this state, the macrophage increases its volume, having an enlarged nucleus of loose chromatin, resembling the nucleus of a fibroblast. However, contrary to fibroblasts, epithelioid cells have abundant pinkish cytoplasm, although with non-defined borders. The macrophages are free, whereas the epithelioid cells are bound to each other in the granuloma. 27

When staining with HE, granulomas with the cytologic characteristics of a cell-type defense response were detected, with a predominance of macrophages either isolated or formed into giant cells, plasmocytes and neutrophils, with a large accumulation of double profile yeast-form cells. The Gomori–Grocott method clearly demonstrated the presence of the yeast in all periods analyzed and, most of the time, it showed multiple budding.

In the first week after inoculation, the lungs had alterations characteristic of an acute infection; however, in the second week a chronic infectious process had already begun. In the first 2 weeks after lung infection, a large number of cellular fragments and degenerated fungi were stained by HE and Gomori–Grocott, respectively. Some pycnotic nuclear fragments indicated the occurrence of programmed cell death, especially in the first and eighth weeks of infection. The ability of the pathogens to induce phagocyte apoptosis could be an important virulence factor to reduce the defense mechanisms of the host. 30

The increase in the number of neutrophils became more evident in the fourth week, when it was also possible to observe giant multinuclear cells phagocytizing the fungi and various neutrophils around the yeasts. Various authors have debated over the importance of the fungicidal and fungistatic role of neutrophils against P. brasiliensis, and their importance for the defense of the host in the initial stages of infection. 31 Neutrophils have a high capacity to ingest the yeast forms of P. brasiliensis, and the initial contact of the fungi with the neutrophils unchains mechanisms capable of killing the yeasts located inside the neutrophils or in their extracellular spaces. 31

An evolution in the organization of the granuloma was observed by the Gomori–Grocott and Picrosirius methods, through the formation of two distinct regions, one central containing the isolated fungi, and one peripheral with a tendency of fibrosis.

When staining with sirus-red under polarization, the production of type I and III collagen fibers in a mesh arrangement was observed, with a predominance of red fibers in some granulomas and green fibers in others, suggesting there were differences in the degree of maturation of these structures. When staining with the Gomori–Grocott method, an initial concentric arrangement of reticular fibers was found. This organization shows the attempt of the host organism to form an organized barrier to prevent the dissemination of the fungus. These characteristics were more evident in the eighth week of infection, when greater birefringence was also observed in the granuloma-free regions of the pulmonary parenchyma.

Furthermore, in the eighth week a process of angiogenesis, forming capillaries in the pulmonary parenchyma, was observed around some of the granulomas. Increases in vascularization have also been observed, fourteen weeks after infection, in peritoneal granulomas. 17 The proliferation of blood vessels in the periphery of the granuloma has the objective of nourishing the granulomatous structure, as with time and the growth of the granuloma, its central portion can suffer caseous necrosis resulting from a lack of nutrition, thereby forming a necrotic center. 27 The emergence of a greater number of granulomas of varied sizes and different degrees of maturity, as observed in this study, implies that the granulomatous process has a limited ability to control the dissemination of the fungus after a long period of infection. 15

To understand the structural organization of granulomas induced by intraperitoneal inoculation with Pb, Kerr and others 17 studied the omentum, diaphragm, liver, and lungs of albino rats 4 months after infection. In all of the organs, the granulomas had a central zone with a predominance of fine type III collagen fibers arranged into a mesh. In the periphery of the granulomas, there was a predominance of thick type I fibers in a concentric arrangement. This demonstrated that the quantity of collagen gradually increased from the center to the periphery of the granulomas, suggesting a dynamic process of sclerosis.

Tuder and others 32 found intense fibrosis in the lungs of patients with paracoccidioidomycosis and suggested the possibility that the fungus itself induces the highest production of reticular fibers, and that the production of the collagen may be stimulated by the macrophage-fibroblast interaction.

The accumulation of collagen in the more advanced stages of the inflammatory response could be related to a substantial increase in the production of cytokines such as TNF-α and TGF-β by the defense cells. Intense fibrosis sometimes causes severe hemodynamic alterations, especially in the lungs, which can result in the lungs becoming anatomically and functionally compromised. 13,14

Modified picrosirius stain (PMA-PSR) was used with 30-μm sections of granulomas, 8 weeks after infection. The slides were viewed in a confocal microscope and images obtained in sequential planes, which enabled the three-dimensional collagen fibers to be visualized. However, it was not possible to differentiate the fibers into type I and type II. In this study, the evolution of the process of granuloma formation in the pulmonary parenchyma of Swiss mice infected with P. brasiliensis for a period of 8 weeks was demonstrated using different staining techniques that are commonly used in histopathology laboratories.

Even though granulomas are structures that develop to isolate the infecting agent and avoid its dissemination throughout the organism, they have important consequences for the functioning of the infected organ. In the specific case of the lungs, the fibrosis it causes can result in the occurrence of chronic pulmonary insufficiency,6 which incapacitates the patient in their daily life.

Modern science has already developed medicines capable of combating Pb; however, the pulmonary fibrosis persists as a consequence of the disease for the rest of the life of the patient. In this study, histopathologic methods served as a fundamental tool for the study of the evolution of granuloma formation caused by this important systemic mycosis. This study supports other studies that have evaluated these mechanisms with the aim of resolving the fibrotic side effects resulting from infections caused by microorganisms such as P. brasiliensis.

Table 1

Number of granulomas/22.42 mm2 of lung from Swiss mice after 1, 2, 4, and 8 weeks of infection with an isolate of Paracoccidioides brasiliensis

Table 1
Figure 1.
Figure 1.

Number of colony forming units (CFU)/g of lung tissue from Swiss mice infected with Paracoccidioides brasiliensis. *P < 0.05 compared with week 1 after infection. Mean ± SEM, N = 8.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 80, 5; 10.4269/ajtmh.2009.80.798

Figure 2.
Figure 2.

Photomicrographs of the lungs of Swiss mice infected with Paracoccidioides brasiliensis. (A) One week after inoculation: start of the infection and organization of the granuloma. (B) Production of reticular fibers (arrow). (C and D) Two weeks after inoculation: amorphous and fragmented cells, budding yeasts, and reticular fibers impregnated by silver. (E and F) Four weeks after inoculation: an intensification of the infection was observed, with giant cells (arrow) with a tendency of centralization and more peripheral plasmocytes. (G) Eight weeks after infection: the presence of organized granuloma, with compact aggregates of macrophages, epithelial cells and yeasts in the central portion, and intense peripheral basophilia. (H) A greater quantity of reticular fibers encircling groups of yeast. Staining was carried out with hematoxylin and eosin (H&E) in (A, C, E, and G), and with Gomori–Grocott in (B, D, F, and H). This figure appears in color at www.ajtmh.org.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 80, 5; 10.4269/ajtmh.2009.80.798

Figure 3.
Figure 3.

(A, B, C, D, and F) Photomicrographs of the lung parenchyma of Swiss mice infected with Paracoccidioides brasiliensis. (E) Non-infected lung stained with HE. In the infected lungs an increase in the amount of type I and III collagen fibers was observed in the granulomas stained by picrosirius, over time, as can be seen in figures (A, B, C, and D), which correspond to the first, second, fourth, and eighth weeks after inoculation, respectively. (F) Image stained with picrosirius without polarized light, corresponding to the sequential section in image (D), where a granuloma with yeast foci around the bronchus can be seen. This figure appears in color at www.ajtmh.org.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 80, 5; 10.4269/ajtmh.2009.80.798

Figure 4.
Figure 4.

Photomicrographs of the lungs of Swiss mice infected with Paracoccidioides brasiliensis, and stained by the picrosirius technique, as modified by Dolber and Spach, 24 showing a granuloma 8 weeks after inoculation. A 63× objective NA1.2. Collagen fibers can be seen encircling the granuloma. This figure appears in color at www.ajtmh.org.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 80, 5; 10.4269/ajtmh.2009.80.798

Figure 5.
Figure 5.

Photomicrographs of the lungs of Swiss mice infected with Paracoccidioides brasiliensis. Pycnotic structures (arrow) with the characteristics of apoptotic bodies in the granulomas of mice infected for (A) 1 week and (B) 8 weeks, using HE stain. This figure appears in color at www.ajtmh.org.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 80, 5; 10.4269/ajtmh.2009.80.798

*

Address correspondence to Luzmarina Hernandes, Laboratory of Histology, Departamento de Ciências Morfofisiológicas, Universidade Estadual de Maringá, Avenida Colombo 5790, cep 87020 900, Maringá, Paraná, Brazil. E-mail: lhernandes@uem.br

Authors’ addresses: Fernanda C. Da Silva and Luzmarina Hernandes, Laboratory of Histology, Departamento de Ciências Morfofisiológicas, Universidade Estadual de Maringá, Avenida Colombo 5790, cep 87020 900, Maringá, Paraná, Brazil, E-mails: lhernandes@uem.br and nandacristie@gmail.com. Terezinha I. E. Svidzinski and Eliana V. Patussi, Laboratory of Medical Mycology, Universidade Estadual de Maringá, Avenida Colombo 5790, cep 87020 900, Maringá, Paraná, Brazil, E-mails: tiesvidzinski@uem.br and evpatussi@gmail.com. Cristina P. Cardoso and Márcia M. De Oliveira Dalalio, Laboratory of Immunology, Departamento de Análises Clínicas. Universidade Estadual de Maringá, Avenida Colombo 5790, cep 87020 900, Maringá, Paraná, Brazil, E-mails: crispadre@yahoo.com.br and mmodalalio@uem.br.

Acknowledgments: We thank UEM/CNPq for financial support for this research, and Maria Euride Carlo Cancino, Maria dos Anjos Fortunato, Ana Paula Santi, and Tânia Pereira Salci for technical support. The manuscript was translated by Peter Grimshaw. The American Society of Tropical Medicine and Hygiene (ASTMH) assisted with publication expenses.

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